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Noncanonical Amino Acids in BiocatalysisClick to copy article linkArticle link copied!
- Zachary Birch-PriceZachary Birch-PriceManchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester M1 7DN, U.K.More by Zachary Birch-Price
- Florence J. HardyFlorence J. HardyManchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester M1 7DN, U.K.More by Florence J. Hardy
- Thomas M. ListerThomas M. ListerManchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester M1 7DN, U.K.More by Thomas M. Lister
- Anna R. KohnAnna R. KohnManchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester M1 7DN, U.K.More by Anna R. Kohn
- Anthony P. Green*Anthony P. Green*Email: [email protected]Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester M1 7DN, U.K.More by Anthony P. Green
Chemical Reviews
Cite this: Chem. Rev. 2024, 124, 14, 8740–8786
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Abstract
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In recent years, powerful genetic code reprogramming methods have emerged that allow new functional components to be embedded into proteins as noncanonical amino acid (ncAA) side chains. In this review, we will illustrate how the availability of an expanded set of amino acid building blocks has opened a wealth of new opportunities in enzymology and biocatalysis research. Genetic code reprogramming has provided new insights into enzyme mechanisms by allowing introduction of new spectroscopic probes and the targeted replacement of individual atoms or functional groups. NcAAs have also been used to develop engineered biocatalysts with improved activity, selectivity, and stability, as well as enzymes with artificial regulatory elements that are responsive to external stimuli. Perhaps most ambitiously, the combination of genetic code reprogramming and laboratory evolution has given rise to new classes of enzymes that use ncAAs as key catalytic elements. With the framework for developing ncAA-containing biocatalysts now firmly established, we are optimistic that genetic code reprogramming will become a progressively more powerful tool in the armory of enzyme designers and engineers in the coming years.
This publication is licensed under
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License Summary*
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Attribution (BY): Credit must be given to the creator.
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License Summary*
You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:
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Copyright © 2024 The Authors. Published by American Chemical Society
Special Issue
Published as part of Chemical Reviews virtual special issue “Noncanonical Amino Acids”.
1. Introduction
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Nature has evolved proteins with a diverse array of structures and functions using a standard set of twenty amino acid building blocks, as defined by the universal genetic code. Enzymes are a subset of proteins that accelerate the biochemical processes needed to sustain life. These biological catalysts use complex networks of active site residues and well-defined substrate binding pockets to process challenging transformations with unrivalled efficiencies and specificities. (1) In some cases, enzymes recruit additional functional components such as metal ions or organic cofactors to expand upon the limited chemical diversity contained within the standard amino acid side chains. (2)
Over the past decades the fields of biocatalysis and enzymology have benefitted greatly from increasingly sophisticated methods for protein engineering, which have given us unprecedented control over protein sequence, structure and function. For example, site directed mutagenesis has been extensively used to study the roles of individual amino acids in catalysis, advancing in our understanding of enzyme mechanisms. (3) More extensive engineering of proteins has been enabled by methods such as directed evolution (4,5) and computational (re)design, (6−8) leading to the development of biocatalysts with improved stability, selectivity, efficiency and substrate range. (9,10) These approaches have even allowed access to enzymes with mechanisms and catalytic functions that are unknown in nature. (11,12)
Although powerful, most enzyme engineering approaches only make use of nature’s standard alphabet of twenty canonical amino acids (cAAs). These standard amino acids are limited in their chemical diversity, which ultimately restricts our control of protein structure and function. To address this limitation, recent years have seen the emergence of powerful genetic code reprogramming methods that allow introduction of new functional elements into proteins as noncanonical amino acid (ncAA) side chains. (13,14) These methods can be divided into global replacement strategies, where one of the twenty standard amino acids is replaced by a noncanonical structural analogue, or site selective genetic code expansion (GCE) strategies where proteins are synthesized from 21 or more amino acids. To date, these methods have been used to encode hundreds of structurally diverse ncAAs into proteins for diverse applications (Figure 1). (15,16) Genetic code reprogramming has allowed introduction of new spectroscopic handles (17−19) and targeted replacement of individual atoms or functional groups, (20,21) providing new insights into how enzymes operate. These methods have also been used to develop engineered biocatalysts with augmented properties. (22−26) By combining genetic code reprogramming with directed evolution, it has also been possible to embed new modes of catalysis into proteins that would be difficult to access within the constraints of the genetic code. (27,28)
Figure 1
Figure 1. NcAAs discussed in this review. (A) NcAAs incorporated via selective pressure incorporation (SPI), expressed protein ligation (EPL), or solid-phase peptide synthesis (SPPS). (B) NcAAs incorporated by GCE. The orthogonal translation system(s) used to incorporate each ncAA are listed. For several ncAAs, multiple incorporation techniques are discussed in this review, and these are also listed. †DAP is incorporated as a precursor featuring a photocleavable group, which matures to DAP upon irradiation at 365 nm. ‡4-NH2Phe is incorporated as 4-AzPhe, which is then chemically reduced in situ to form 4-NH2Phe.
In this review article, we discuss the different approaches available to incorporate ncAAs into proteins and illustrate how these amino acids have been used for diverse applications in enzyme design, engineering, and characterization.
2. Enabling Technologies
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2.1. Post-Translational Protein Modifications
New functionality can be introduced into proteins post-translationally using a variety of chemical or enzymatic methods. (29,30) The thiol group of Cys is commonly targeted for protein modification, usually via alkylation or disulphide bond exchange reactions, due to its high nucleophilicity and relative scarcity in proteins. (31) Although achieving specific and selective modification is often challenging, post-translational protein functionalization is highly versatile and has allowed incorporation of diverse nonproteogenic elements. (32−35) Covalent modification of Cys residues to introduce new catalytic elements has provided artificial enzymes for a range of chemical transformations. For example, flavin analogues have been installed into the active site pocket of the cysteine protease, papain, to generate biocatalysts with oxidoreductase activity. (36−38) Ruthenium and rhodium complexes have also been introduced into protein scaffolds via Cys functionalization, to provide enzymes for ring closing metathesis (39) and hydroformylations, (40−42) respectively.
2.2. Solid-Phase Peptide Synthesis
Functionalized peptides and proteins can be produced using solid-phase peptide synthesis (SPPS), involving sequential coupling of protected amino acids on a solid support. (43) This approach is technically challenging and time-consuming but offers flexibility, allowing synthesis of user-defined peptide sequences containing a variety of canonical and artificial building blocks, including those that are toxic to cells or poorly compatible with the cellular translation machinery (e.g., D-amino acids). (44−47) Peptides synthesized using SPPS are typically restricted to sequences of less than 100 residues; however larger proteins can be constructed by combining SPPS with a variety of ligation strategies including native chemical ligation (NCL) and expressed protein ligation (EPL). (48−54) NCL offers greater flexibility with respect to the number and distribution of ncAAs that can be introduced into the polypeptide chain; however, producing large proteins is both costly and technically challenging. In contrast, EPL allows for more facile production of large modified proteins but is typically limited to the introduction of ncAA-containing synthetic sequences at the C-terminus. These synthetic or semisynthetic methods have been used to create polymer-modified glycoprotein mimetics, (55−58) proteins with modified backbones (59−61) and defined patterns of posttranslational modifications, (62−64) split-proteins, (65) proteins containing fluorescent probes (66−68) and proteins with altered catalytic properties. (47,69,70)
2.3. Selective Pressure Incorporation
Selective pressure incorporation (SPI) exploits the promiscuity of native aminoacyl tRNA synthetases (aaRSs) to aminoacylate a canonical tRNA with a close structural analogue of its cognate cAA, leading to sense codon reassignment (Figure 2). This method results in global replacement of one of the twenty standard amino acids by a ncAA. SPI relies on the use of natural or engineered auxotrophic expression strains that are deficient in a target amino acid. (71−73) A seminal study by Cowie and Cohen reported the quantitative substitution of Met for selenomethionine (SeMet) in proteins produced by the Met auxotroph Escherichia coli (E. coli) ML304d. (74) In this case, the auxotrophic strain displayed normal exponential growth when Met was swapped for SeMet in the growth media, installing SeMet throughout every protein in the bacterial proteome. Other auxotrophic strains have also been developed, enabling the global replacement of Trp with 4-fluorotryptophan (75) (4-FTrp) and valine with α-aminobutyric acid. (76) However, for many ncAAs, global incorporation throughout the host proteome is detrimental to cell growth. To address this challenge, auxotrophic strains are often cultured in defined media containing a low concentration of the cAA, which becomes depleted during cell growth. The ncAA is then supplemented into the media at an appropriate growth state when protein expression is induced. Alternatively, the cells can be harvested prior to induction and resuspended in fresh media containing ncAA but lacking the cAA. To further expand the range of ncAAs that can be incorporated using SPI, the substrate promiscuity of canonical aaRSs has been extended using protein engineering. (76−79)
Figure 2
Figure 2. SPI of ncAAs. SPI employs an auxotrophic expression system to globally replace a target canonical amino acid (cAA) with a close structural analogue. An endogenous aaRS loads its cognate tRNA with the ncAA which is incorporated into proteins. Created with BioRender.com.
SPI has enabled the successful incorporation of a wide range of ncAAs into recombinant proteins for diverse applications (Figure 1A). For example, proteins containing heavy atom analogues of cAAs are routinely used in protein X-ray crystallography to aid experimental phasing. (80−84) Isosteric amino acid analogues have been used to investigate protein folding, stability and activity. (85−90) SPI has also been used to install spectroscopic probes (91−95) and biorthogonal handles for protein conjugations. (77,79,96−101) As detailed in the sections below, this approach has also been widely used to improve enzyme stability and activity.
2.4. Genetic Code Expansion
GCE enables the site selective incorporation of ncAAs in vivo or in vitro, in response to a reassigned codon. In this way proteins containing 21 or more amino acids can be biosynthesized.
2.4.1. In Vitro Genetic Code Expansion
Peptides and proteins containing ncAAs have been ribosomally synthesized in vitro using cell free extracts (e.g., E. coli lysates) or reconstituted protein synthesis systems (e.g., PURExpress). (104−106) Here, tRNAs precharged with the target ncAA are supplemented into an in vitro expression system and decoded by the ribosome in response to a repurposed codon (Figure 3). Early work on in vitro genetic code reprogramming focused on chemically modifying aminoacyl-tRNAs. (107,108) For example, conversion of a Phe-loaded tRNA to the corresponding α-hydroxy acid enabled production of proteins with polyester backbones. (108)
Figure 3
Figure 3. Strategies for the generation of ncAA-loaded tRNAs employ either chemoenzymatic methods (top left, PDB: 2C5U (102)) or Flexizymes (bottom left, PDB: 3CUN (103)). These ncAA-tRNAs can then be incorporated into a polypeptide chain using cell-free expression (CFE) systems (right). Created with BioRender.com.
The development of chemo-enzymatic strategies for synthesizing aminoacyl-tRNAs has provided a more general approach that has expanded the range of ncAAs that can be incorporated into proteins. Here, an N-protected amino acid is first chemically coupled to di(adenosine 5′-)diphosphate or a 5′-phospho-2′-deoxyribocytidylriboadenosine (pdCpA) dinucleotide at the 3′-hydroxyl group. The resulting aminoacylated nucleotide fragment is then ligated to a truncated tRNA using T4 RNA ligase and deprotected to yield the final aminoacyl tRNA. (104,109) Alternatively, aminoacylation ribozymes, which are RNA-based catalysts, can be used to directly charge tRNAs with ncAAs. (110) In particular, Flexizymes have proven to be valuable catalysts for mediating transacylation of activated amino acid esters onto the 3′-hydroxyl group of tRNAs. (111,112) By mixing Flexizymes with tRNA and the respective amino acid ester, nearly any tRNA can be loaded with almost any desired ncAA (Figure 3). (105) To date, efficient and precise ribosomal translation of proteins containing diverse ncAAs has been achieved. (113) Combining Flexizyme genetic code reprogramming technology with mRNA display has proven especially valuable for discovery of macrocyclic peptide binders in drug development. (114) Although powerful, one drawback of these in vitro methods is that they cannot be easily scaled due to the requirement to supply super stoichiometric quantities of aminoacyl-tRNAs as well as the high costs associated with reconstituted in vitro translation systems. These challenges have so far limited the use of these technologies in biocatalysis due to the relatively large quantities of protein required for most applications.
2.4.2. In Vivo Genetic Code Expansion
In vivo genetic expansion methods provide a more scalable approach to the synthesis of ncAA-containing proteins. These methods rely on an orthogonal aminoacyl-tRNA synthetase–tRNA pair to direct the incorporation of an ncAA in response to a repurposed codon introduced into the gene of interest (Figure 4). (115) Of the codons available, the amber codon (UAG) is most commonly used, although reassigned sense codons and quadruplet codons have also been exploited. (116,117) The aaRS/tRNA pair used for ncAA incorporation must be orthogonal in the context of all endogenous aaRS/tRNA pairs within the host organism. This orthogonality is most commonly achieved by importing a heterologous aaRS/tRNA pair from a different domain of life. (118) If necessary, orthogonality can be improved through aaRS and/or tRNA engineering. Finally, the substrate specificity of the heterologous aaRS is engineered so that it uniquely recognizes the ncAA of interest. (118) Although several methods have been reported to engineer aaRSs, (119−123) this is most often achieved by subjecting large active site libraries to a two-stage selection protocol that links cell viability to aaRS activity and specificity. (124) A positive selection step, in which cell survival is dependent upon suppression of an amber codon introduced into the chloramphenicol acetyl transferase (CAT) gene, selects for aaRSs that aminoacylate the suppressor tRNA with the target amino acid. A subsequent negative selection step then removes aaRSs that incorporate cAAs. The positively selected clones are transformed into cells containing a gene encoding a toxic barnase protein with amber mutations at permissive sites. The cells are then grown in the absence of the ncAA, and the presence of aaRS variants that are able to utilize cAAs will result in cell death. The activity and specificity of surviving clones can be quantified using a fluorescence screening assay which relies on suppression of an amber codon introduced into green fluorescent protein (GFP). (125)
Figure 4
Figure 4. Positive and negative selection processes can be used to engineer orthogonal aaRS-tRNA pairs to improve incorporation efficiency and/or specificity. The engineered aaRS catalyzes an aminoacylation reaction between its cognate tRNA and ncAA, with the ncAA added to the growing polypeptide chain during translation in response to a repurposed codon (e.g., the amber stop codon, UAG). Created with BioRender.com.
Using this approach, hundreds of structurally and functionally diverse ncAAs can now be incorporated into proteins (Figure 1B). (126,127) For example, a large number of aromatic ncAAs have been incorporated into proteins produced in E. coli using engineered TyrRS-tRNATyr pairs derived from Methanocaldococcus jannaschii (MjTyrRS-tRNATyr). (128) The structural diversity of encodable ncAAs has been further expanded following the discovery of PylRS-tRNAPyl pairs, which naturally suppress amber codons to incorporate ‘the 22nd amino acid’ pyrrolysine (Pyl, O) in methanogenic archaea. (127,129,130) Due to a high degree of active site plasticity and orthogonality in E. coli, yeast and mammalian cells, PylRS systems have emerged as the most versatile platform for GCE in vivo. (127,131)
3. Probing Enzyme Mechanisms with Noncanonical Amino Acids
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Site directed mutagenesis has proven to be an invaluable tool in enzymology, providing a versatile strategy for identifying catalytically important residues. However, standard mutations of active site residues can sometimes lead to unintended structural changes that can complicate interpretation of structure–activity relationships. Furthermore, mutation of key catalytic residues often dramatically reduces activity, which provides important but limited mechanistic insights. The incorporation of ncAAs can open new avenues to study complex biochemical mechanisms by allowing more nuanced perturbations to enzyme structure and function. (14,20,22,132) Such modifications can be used to introduce spectroscopic probes, enable covalent trapping of intermediates and fine-tune active site environments by replacement of specific atoms or functional groups.
3.1. Incorporation of Spectroscopic Handles and Biophysical Probes
Strategic incorporation of ncAAs with distinct spectroscopic and biophysical properties has been used to study enzyme function and structure through a range of techniques, including NMR, (133,134) EPR, (135) IR, (136) and X-ray crystallography. (137) Additionally, ncAAs with fluorescent sidechains such as dansyl-Ala (138) or (7-hydroxycoumarin-4-yl)ethylglycine (Hco), (20) or with biorthogonal handles to which fluorescent molecules can be conjugated, (139−141) have been introduced into peptides and proteins to monitor folding and conformational changes, (142−144) for example in superoxide dismutase, (138) myoglobin (Mb), (145) deubiquitinases, (146) and T4 lysozyme. (147)
3.1.1. Noncanonical Amino Acids to Facilitate Protein Structure Determination
Global incorporation of SeMet through SPI has enabled great advances in protein structure determination by X-ray crystallography, through additional anomalous signals derived from the presence of heavy atoms within the crystal lattice. (148) Genetic incorporation of 4-iodophenylalanine (4-IPhe) (137) or 3-iodotyrosine (3-ITyr) (149) gives a site selective way to introduce an alternative heavy atom to facilitate such single-wavelength anomalous diffraction (SAD) experiments and allow structure determination with less diffraction data. Additionally, a metal-chelating ncAA, (8-hydroxyquinolin-3-yl)alanine (3-HqAla), provided a binding site for Zn(II) within the crystal lattice of O-acetylserine sulfhydrylase, which enabled SAD phasing to determine protein structure. (150)
Recently, X-ray free-electron lasers (XFELs) have allowed structural characterization of transient reaction intermediates using time-resolved serial femtosecond crystallography. Light is a convenient trigger for time-resolved experiments, and several natural photoenzymes have been studied with XFELs. (151−157) Although not yet applied to catalysis, GCE can be used to introduce artificial phototriggers into proteins, such as photocaged AAs or triplet sensitizers. The Wang laboratory used GCE to encode (S)-2-amino-3-(7-fluoro-9-oxo-9H-xanthen-2-yl)propanoic acid (FXO), an ncAA that has a strongly absorbing xanthone side chain that undergoes a photocrosslinking when placed in the hydrophobic pocket of a human liver fatty acid binding protein. (158) Time-resolved crystallography experiments were used to monitor conformational changes in the xanthone side chain between 10 and 300 ns after irradiation. In principle, this approach could be extendable to studying mechanisms of designed enzymes with triplet photosensitizers (vide infra). (159)
3.1.2. Nuclear Magnetic Resonance Studies with Noncanonical Amino Acids
Protein nuclear magnetic resonance (NMR) spectroscopy is a widely used technique that detects signals from spin-active nuclei, and can provide information about reaction kinetics or conformational changes upon ligand binding. To study proteins via NMR, proteins can be expressed in 15N- and/or 13C-enriched media, resulting in uniform labeling of the entire protein. NcAAs can be used to simplify the interpretation of protein NMR data by enabling site-specific isotope labeling or by introducing NMR-active nuclei into proteins, which can be used as sensitive probes that can report on changes to the local conformation and/or electronic environment upon, for example, ligand binding. (160,161) This approach can prove especially valuable for analysis of large proteins, where interpretation of spectra can be complicated. NcAA incorporation has been widely used to selectively install isotopic labels and new spin active nuclei to facilitate protein NMR analysis. In this section we present illustrative examples of where ncAAs have been used to study enzymes through NMR. The reader is directed to excellent reviews for a more comprehensive overview. (133,162)
One early report from 1968 studied staphylococcal nuclease by replacing 14 of the amino acids with their deuterated analogues. (160) The simplified NMR spectra facilitated analysis of the effects of metal ion and inhibitor binding. Site-specific isotopic enrichment was first achieved using a chemically aminoacylated suppressor tRNA to introduce a single 13C-labeled Ala82 into T4 lysozyme in response to a TAG codon. (134) This enabled isolation of the signal from Ala82 in 13C NMR spectra and allowed for observation of changes in shift dispersion upon denaturation. More recently, an engineered tRNAPyl/G1PylRS pair was used to encode 15N-/13C-labeled 7-azatryptophan (7-AzaTrp) into a protease from the Zika virus, establishing a method for achieving site-specific peak assignments in Heteronuclear Single Quantum Coherence (HSQC) spectra. (163)
Three different ncAAs (O-trifluoromethoxytyrosine (O-CF3Tyr), 13C-/15N-labeled O-MeTyr, and 15N-labeled O-nitrobenzyltyrosine (O-NBTyr)) were introduced into eleven positions of the active site of the thioesterase domain of human fatty acid synthase. The differential chemical shifts in HSQC and 19F NMR spectra in response to ligand binding confirmed the ligand binding site, and gave information on protein motion. (164) Similarly, 13C-O-MeTyr was used to provide evidence for the existence of multiple, ligand-dependent conformational states of the cytochrome P450 CYP119. (165)
NcAAs can also be used to introduce spin-active nuclei that do not naturally occur in proteins. Due to the intrinsic NMR sensitivity of 19F and the lack of fluorine-containing cAAs, ncAAs such as 5-fluorotryptophan (5-FTrp), 6-FTrp, 3-FTyr, and 4-FPhe have provided useful spectroscopic handles for studying protein structure and function. (166−175)
SPI was used to introduce trifluoromethionine (F3Met) and difluoromethionine (F2Met) at positions 1, 14 and 107 of lysozyme from bacteriophage λ (LaL). (174,176) Of three sites of ncAA incorporation, the F2Met14 residue in the core of the protein showed the largest chemical shift difference between the two fluorine signals, likely due to restricted side chain movement. Upon binding of an oligosaccharide inhibitor, the two F2Met14 signals diverge even further, suggesting a conformational change to the enzyme in a region closer to one of the two fluorine atoms. (177) In a similar manner, replacement of Met with F3Met in DNA polymerase I from Thermus aquativus (KlenTaq) enabled 19F-NMR studies to study enzyme dynamics. (178) Individual 19F resonances could be resolved and the spectra could be used to distinguish between the free enzyme, the binary complex in the presence of a DNA primer, and the ternary complex with a ddNTP bound. This method has also been used to study conformational changes upon ligand binding at the dimeric interface of nitroreductase and histidinol dehydrogenase. (168) The 19F spectra of nitroreductase containing 4-CF3Phe at position 124 showed a single peak corresponding to the ncAA, which shifted upfield upon binding of an inhibitor due to conformational changes.
Fluorine NMR signals show scalar 19F-19F coupling when in proximity. Orton et al. demonstrated these effects are observable within the hydrophobic core of the E. coli protein cis–trans prolyl-isomerase B by positioning multiple fluorinated ncAAs. (179) Genetic incorporation of two 4-CF3Phe or O-CF3Tyr residues gave observable 19F-19F Total Correlation Spectroscopy (TOCSY) and Double Quantum Filtered Correlation Spectroscopy (DQF-COSY) cross peaks with high sensitivity. These studies suggest that through space 19F-19F couplings between fluorine substituted ncAAs can offer a sensitive tool for studying protein structure and function.
NcAAs can also be used to introduce functional groups that occupy distinct chemical shift regions within 1H NMR spectra. For example, Ekanayake et al. developed an engineered aminoacyl-tRNA synthetase to incorporate Νε-(((trimethylsilyl)methyl)-carbamoyl)lysine (TMSNLys) for study of the dimeric SARS-CoV-2 main protease. (133) 1H NMR signals from the trimethylsilyl (TMS) group of TMSNLys and a second ncAA, Nε-(((trimethylsilyl)-methoxy)carbonyl)lysine (TMSLys) appear at ∼ 0 ppm, a clear spectral region with few features from aqueous buffer or protein environments. Distinct peak shifts were observed upon binding of two ligands, a cyclic peptide inhibitor and Calpeptin. Interestingly, the expected perturbations were not observed with one putative allosteric ligand, pelitinib, suggesting that this ligand does not bind in the region previously proposed in the literature.
3.1.3. Incorporation of Noncanonical Amino Acids for Electron Paramagnetic Resonance Spectroscopy
Electron paramagnetic resonance (EPR) spectroscopy is a powerful biophysical technique for characterizing species with unpaired electrons, such as transition metals complexes or organic radicals. (180) Site-selective spin labeling has been achieved by reacting Cys residues with nitroxide-labeled reagents, although this method relies on the introduction and/or removal of cysteine residues that might have functional significance. (181) As an alternative, GCE can be used to directly encode paramagnetic moieties as amino acid side chains (135) or ncAAs with bioorthogonal handles to which spin labels can be efficiently conjugated. (182−185)
Direct genetic incorporation of a spin-labeled amino acid ((S)-2-amino-6-(1-oxy-2,2,5,5-tetramethylpyrroline-3-carboxamido)hexanoic acid, OPCLys) was achieved using an engineered tRNAPyl/PylRS pair. (135) Purified GFP with the spin-active ncAA at position 39 gives a nitroxide-characteristic EPR signal with a spectral shape indicative for low N–O• mobility, in contrast to that of the free amino acid that shows a spectrum characteristic to that of a fast-tumbling small molecule. Interestingly, quantification of this signal reveals that the intensity is approximately 50% of that expected, likely due to some instability of nitroxide radicals in vivo.
A higher degree of spin-labeling is achievable through post-translational derivatization of ncAAs. Genetic incorporation of 4-acetylphenylalanine (4-AcPhe) has been used to introduce nitroxide spin labels into proteins by acid-catalyzed derivatization of the ncAA side chain with a hydroxylamine nitroxide, first in the model protein T4 lysozyme. (186) This approach has been extended to include alternative ncAA-spin probe combinations. By incorporating ncAAs with either azide or alkyne functional groups, Cu(I)-catalyzed azide-alkyne cycloadditions (CuAACs) or strain-promoted azide-alkyne cycloadditions (SPAACs) can be used to introduce spin labels into proteins. (183,187−191) For example, Gd(III) moieties were conjugated to two sites in the Zika virus NS2B-NS3 protease using 4-azidophenylalanine (4-AzPhe). (192) Double electron–electron resonance (DEER) measurements in the presence and absence of a known inhibitor for this protease revealed similar distance distributions, suggesting the commonly used linked construct of this protease remains in the closed conformation, irrespective of ligand binding.
3.1.4. Noncanonical Amino Acids for Protein Infrared Spectroscopy
Infrared spectroscopy (IR) is a well-established method of study protein structure and function. (193,194) The IR spectra of proteins have a transparent window between 1800 and 2500 cm–1, which can be exploited by incorporating CD (carbon-deuterium), CN, (195−199) SCN, (200) N3, (201−204) and NO2 (205) bonds into proteins to introduce distinct spectroscopic features. In this way, deuterated Met has been used as a vibrational probe to study a range of proteins including myeloperoxidase, (206) dihydrofolate reductase, (207) and plastocyanin. (208)
The addition of ncAAs containing cyano- (19) and nitro- (205) groups to the genetic code affords a method for site specific introduction of vibrational probes, (209) that are sensitive to hydrogen bonding, packing interactions, and protein conformation. (210) The Boxer laboratory introduced 4-cyanophenylalanine (4-CNPhe), 3-cyanophenylalanine (3-CNPhe), and S-cyanohomocysteine (S-CNHcs) into the active site of Ribonuclease S (RNaseS) through SPPS of peptide fragments (residues 1–20) that, when combined with a recombinantly expressed protein fragment (residues 21–124), gave active RNase S variants with no significant changes to enzyme structure or activity. (136) Vibrational Stark Effect (VSE) experiments using each ncAA were used to calibrate the sensitivity of the nitrile stretch to external electric fields, allowing a quantitative interpretation of the frequency shifts, providing a measure of the local electrostatic fields (see section 3.3.2). An expansion of this method used 13C-labeled 4-CNPhe to assess solvatochromic effects and account for hydrogen bonding to the aromatic nitriles, allowing an estimate of the average total electrostatic field within an enzyme active site. (211)
Genetically encoded 3-azido-Tyr (3-N3Tyr) served as a 2-dimensional IR probe in the study of the metalloenzyme DddK, an iron-dependent enzyme that catalyzes the conversion of dimethylsulfoniopropionate to dimethylsulfide. (212) Standard substitutions of Tyr64 abolished catalytic function, however substitution to 3-N3Tyr was well-tolerated and allowed measurement of fs–ps time scale water dynamics. The addition of even low concentrations of the denaturation reagent guanidinium hydrochloride was shown to decrease water confinement in the active site and substantially reduce catalytic activity without affecting the wider protein structure. The authors proposed that Tyr64 is activated as a catalytic base through deprotonation by a neighboring ordered water molecule, and disruption of this process by addition of the denaturation reagent causes loss of catalytic activity.
3.2. Covalent Trapping of Reactive Intermediates and Transient Complexes
NcAAs have been used to covalently trap close analogues of catalytic intermediates and catalytically relevant protein–protein complexes to facilitate their characterization. This approach has provided new structural insights into transient reaction intermediates and can be used to interrogate biosynthetic pathways in vivo.
3.2.1. Capturing Acyl-Enzyme Intermediates
Many enzymes such as Ser hydrolases, Cys proteases, and ubiquitinases proceed via the formation of transient acyl-enzyme (ester or thioester) intermediates. Replacement of the catalytic serine/cysteine residue with 2,3-diaminopropionic acid (DAP) gave a powerful new method of trapping these intermediates with nonhydrolyzable amide bonds (Figure 5A). (213) The structural similarity between DAP and Cys/Ser complicates its direct incorporation through cellular translation. Instead, the Chin lab developed an engineered tRNAPyl/PylRS pair to incorporate a photocaged derivative of DAP that can be deprotected post-translation upon irradiation with light. This approach was used for structural analysis of valinomycin synthetase (Figure 5B,C), a member of the nonribosomal peptide synthetase family. Structural characterization of a covalently linked substrate-enzyme complex by X-ray crystallography revealed a large conformational change to a ‘lid’ region upon substrate acyl-enzyme complex formation.
Figure 5
Figure 5. DAP incorporation into Valinomycin synthetase. (A) Genetically encoded (2S)-2-amino-3-([(2-[1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl]thio)ethoxy)carbonyl] ncAA is photodeprotected by irradiation at 365 nm to give DAP, which forms stable acyl-enzyme intermediates with an amide bond that is resistant to hydrolysis. (B) The active site of Valinomycin synthetase (protein shown as a gray cartoon, PDB: 6ECE (213)) with a noncanonical DAP nucleophile in position 2463 (atom-colored sticks, brown carbons) bound to a dodecadepsipeptide substrate (atom colored sticks, blue carbons). (C) Large structural differences are observed in the lid region of Valinomycin synthetase when bound to a dodecadepsipeptidyl intermediate (gray cartoon, PDB: 6ECE (213)) in comparison to a tetradepsipeptidyl intermediate (blue cartoon, PDB: 6ECD (213)).
Subsequent work from the Chin laboratory described the use of DAP incorporation to create ‘substrate traps’ in mammalian cells. (214) Proteases and other hydrolases equipped with DAP nucleophiles were purified from live cells with substrate fragments bound via stable amide bonds, thus allowing the annotation of proteins of previously unknown function.
3.2.2. Photo-Cross-Linking to Map Protein–Protein Interactions
Benzophenones are powerful photocross-linking agents that have been widely used as photophysical probes. (215) The addition of 4-benzoylphenylalanine (BpAla) to the genetic code using an engineered tRNATyr/MjTyrRS pair allowed introduction of this functional motif into proteins with unparalleled site selectivity. (216) This approach has enabled mapping of transient protein–protein interactions through covalent photocrosslinking, (217) including complexes between glutathione S-transferase (GST) dimers, (218) SecA Atpase and the SecYEG translocon, (219) the transcriptional activator Gal4 and the inhibitor protein Gal80, (220) the large subunit of rubisco and its chaperone RbcX2, (221) acyl carrier proteins and ketosynthases, (222,223) and vaccinia H1-related (VHR) phosphatase dimers. (224) In one notable example, this approach was used to identify a new preinitiation complex in the mechanism of a human mitochondrial RNA polymerase (mtRNAP). (225) Mammalian transcription apparatus contains mtRNAP, a transcription initiation factor-like protein TFB2M, and a major nucleoid protein TFAM. Upon incorporation of BpAla into the C-terminal region of TFAM, photoinduced cross-linking to mtRNAP was observed, dependent on DNA-binding, independent of the major nucleoid protein TFB2M. These data suggest the mtRNAP mechanism involves formation of an initial complex involving mtRNAP, TFAM and promoter DNA.
3.3. Modulating Noncovalent Interactions and Intermediate Lifetimes
GCE allows for selective and targeted substitution of individual functional groups or atoms within an enzyme active site. These molecular edits can then be correlated with changes in catalytic activity and reaction mechanism, to build detailed structure–activity relationships that are not possible to achieve with standard mutagenesis methods. (226,227) In this section, we illustrate examples of how ncAAs have be used to tune the pKa and/or redox potential of selected residues, (228) modulate hydrogen bonding networks (229) or other noncovalent interactions, (230) and to perturb the lifetimes of reactive intermediates. (231)
3.3.1. Tuning the pKa and/or Reduction Potential of Key Residues
Using ncAAs to modulate the pKa and/or reduction potential of key functional residues has provided new insights into the mechanisms of a diverse set of enzymes.
Variants of RNase A with the catalytic His, His12 and His119, replaced by 4-fluorohistidine (4-FHis) were synthesized by stepwise peptide ligation, to give single and double mutants of the enzyme. (48) The pH-rate profiles of each variant for the cleavage of uridyl-3′,5′-adenosine were different to that of the wild type (WT) due to the lower pKa of 4-FHis cf. His. RNase A His12(4-FHis) has a broader pH profile and is able to effect catalysis at ∼ 2 pH units lower than the WT, consistent with its proposed role as a catalytic base. In contrast, replacement of His119, which is thought to serve as a catalytic acid, reduced activity substantially, likely due to the more acidic 4-FHis existing in an inactive nonprotonated state.
Replacement of an active site His of a porcine pancreatic phospholipase A2 (PLA) by 1,2,4-triazole-3-alanine (Taz) using SPI yields a catalytically active enzyme with an altered pH-rate profile to the WT. (232) WT PLA has a pH optimum of 6, with no activity at pH 3. However, at both pH 6 and pH 3 PLA His48Taz is active, at approximately a 5-fold lower rate in comparison to the WT maximum. The mechanism of phospholipase relies on water activation by basic His48. The lower pKa of Taz compared with His ensures that it remains in its active, nonprotonated state at lower pH and is able to activate the nucleophilic water. In contrast, replacement of the only remaining His in a His31Asn/His137Asn double mutant of LaL by Taz had limited effect on the pH-rate profile. (233)
To tune the pKa of an active site tyrosine in the DNA polymerase KlenTaq, 2,3,5-trifluorotyrosine (2,3,5-F3Tyr) was introduced at position 671 using GCE. (234) Structural analysis of KlenTaq in complex with a DNA template containing an abasic site shows Tyr671 occupies the position of the missing base in the DNA template, forming a hydrogen bond to the N3 of purine bases to guide incorporation of dATP and dGTP. (235) Replacement of Tyr671 with 2,3,5-F3Tyr671 using GCE dramatically reduced activity toward single nucleotide incorporations at abasic sites, showing how even subtle perturbations to hydrogen bonding interactions can impact enzyme activity.
Class I ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to 2′-deoxynucleotides through a mechanism that involves generation of a transient thiyl radical and long-range, reversible radical transfer between subunits α and β (Figure 6A,B). (236) Early docking studies suggested the diferric-Tyr122 radical cofactor in the β-subunit is ∼ 35 Å from the oxidized residue Cys439 in the α subunit, with the proposed radical transfer pathway following Tyr122β → W49β → Tyr365β → Tyr731α → Tyr730α → Cys439α. (237) To study the RNR mechanism, the oligomeric state of the enzyme, and to conclusively identify the redox active residues involved in radical transfer, the Stubbe laboratory has used ncAA incorporation to great effect (Figure 6). (236) Mutation to canonical residues along the electron transfer pathway abolishes radical transfer, however, site specific introduction of fluorinated tyrosines (FnTyr, n = 2–4), (238,239) 3-aminotyrosine (3-NH2Tyr), (240,241) and 3,4-dihydroxyphenylalanine (DOPhe, introduced by EPL methods) (242,243) yields redox-competent variants of the two subunits (α and β). Replacing a Tyr residue along the radical pathway with 3-NH2Tyr or DOPhe traps the radical at that position, as it is unable to oxidize the next amino acid in the chain. (240) UV–vis and EPR spectroscopies were used to quantify the presence of trapped DOPhe• or 3-NH2Tyr• radical signals. The stoichiometry of radical generation gave evidence for the active form of RNR requiring two α/β pairs (i.e., one radical equivalent per α2β2).
Figure 6
Figure 6. Mechanistic studies on RNRs using ncAAs have shed light on the electron transfer pathway and enabled structural characterization of the active form of the multimer. (A) A cryogenic electron microscopy structure of RNR (PDB: 6W4X (231)) in its active α2β2 form was captured using a 2,3,5-F3Tyr122 mutation. The protein chains are shown as cartoons, and GDP and TPP are shown as red and gray spheres, respectively. (B) The mechanism of RNRs, which catalyze the conversion of nucleoside di- and triphosphates to deoxynucleotides. (236) TR = thioredoxin. (C) DEER experiments provided information on the relative distances between the Tyr122 radical in the unreacted α/β pair and radicals on an N3NDP mechanistic inhibitor or radicals trapped on 3-NH2Tyr.
DEER spectroscopy has been used to measure the distance between a trapped 3-NH2Tyr• radical (generated at positions 356 in the β subunit or 730 and 731 in the α subunit) and a Tyr122 radical in the adjacent subunit pair (Figure 6C). (238) Similarly, inter-radical distances have been derived between 3-NO2Tyr122• and Tyr356•, 3-NO2Tyr122•, or Tyr731• in adjacent subunit pairs. (244) Additionally, the distance between Tyr122 and a nitrogen-centered radical covalently attached to Cys439 was measured using a mechanistic inhibitor, 2-azido-2-deoxycytidine diphosphate. The distances derived from DEER experiments are shown with red arrows in Figure 6C. Replacement of Tyr122 with 2,3,5-F3-Tyr or 3-NO2Tyr similarly generates 0.5 equivalents of radicals per α/β pair, further suggesting that RNR activity is reliant on formation of an α2β2 complex. (244) The incorporation of 2,3,5-F3-Tyr at position 122, in combination with a Glu52Gln mutation, enabled structural characterization of the active α2β2 form by cryo-electron microscopy. (231) The 2,3,5-F3-Tyr122 substitution allowed trapping of the radical at Tyr356 along the radical transfer pathway leading to tighter subunit affinity.
Noncanonical Tyr analogues were also used to investigate the role of a key catalytic residue in the Fe(II) and 2-oxoglutarate-dependent enzyme, Verruculogen synthase (FtmOx1). (245) FtmOx1 installs an endoperoxide between C21 and C27 of fumitremorgin B using a high energy ferryl intermediate to break an allylic C21–H bond, followed by dioxygen capture, addition of the C21-O–O• radical to the C27 alkene, and hydrogen atom transfer (HAT) to the resulting C26• radical (Figure 7). Replacement of both active site Tyr residues (68 and 244) with ncAA analogue 3-FTyr facilitated unambiguous identification of Tyr86 as the hydrogen donor to C26•, with Tyr224 playing no essential role. Substitution of Tyr68 caused a blue shift in the sharp Tyr• absorption feature observed by stopped-flow absorption spectroscopy (maximum signal intensity observed at 0.35 s). X-band EPR spectra of samples freeze-quenched after 0.35 s exhibit large hyperfine coupling from 19F ortho to the phenolic oxygen radical that are not seen with the WT enzyme. These effects were not replicated in Tyr244 variants, confirming the role of Tyr68. Substitution of Tyr68 or Tyr244 with both 3-NH2Phe or 3-ClTyr preserved catalytic function. The radical species observed with 4-NH2Phe has a UV–vis absorbance spectrum in agreement with previously observed 4-methylaniline radicals and an EPR signal with additional hyperfine splittings from both 14N and the remaining proton.
Figure 7
Figure 7. Mechanistic proposal for the FtmOx1-catalyzed hydrogen atom transfer from Tyr68 to C26•.
The photoenzyme protochlorophyllide oxidoreductase (POR) catalyzes the reduction of protochlorophyllide to chlorophyllide. (246) Recently, Taylor et al. replaced a key conserved tyrosine in the POR active site, which had previously been proposed to act as a proton donor in POR photocatalysis, with fluorinated Tyr analogues. (247) Tyr fluorination led to a reduction in catalytic activity and impaired substrate binding. However, time-resolved laser spectroscopy reveals that the rate of proton transfer is almost identical in the WT and 3-FTyr193 variants. These data suggest that Tyr193 is unlikely to serve as a proton donor but instead is important for substrate binding.
S-adenosylmethionine (SAM)-dependent methyltransferases are known to form strong carbon-oxygen hydrogen bonds (CH···O) from an active site Tyr residue to their substrate SAM. To understand the importance of this interaction, Horowitz et al. incorporated 4-NH2Phe in place of Tyr335 in the active site of lysine methyltransferase SET7/9. (248) This mutation was shown to be structurally conservative by X-ray crystallography, but reduced SAM binding affinity by ∼ 10,000 fold (to ∼ 1 mΜ). In contrast, SET7/9 Tyr355(4-NH2Phe) was able to bind the coproduct S-adenosyl-L-homocysteine with a similar affinity to that of the WT methyltransferase (∼900 μM), suggesting that the Tyr hydroxyl plays an important role in discriminating substrate vs product binding. The kcat of SET7/9 Tyr355(4-NH2Phe) is 35-fold lower than that of the WT enzyme.
3.3.2. Modulating Electric Fields
Enzymes can apply strong electric fields to activate substrates in their active sites. VSE spectroscopy can be used to measure such effects, provided a suitable spectroscopic handle is available in the substrate or the protein. (249−251) Ketosteroid isomerase (KSI) catalyzes a rate-limiting double bond migration of steroid substrates, which contain an IR-visible carbonyl bond (Figure 8A). (252) The Boxer laboratory used VSE spectroscopy to observe the large electric field applied to the carbonyl of a product analogue (Figure 8C) in the KSI active site. (229) This active site has an oxyanion hole consisting of Asp(H)103 and Tyr16, which forms part of an extended hydrogen-bonding network with Tyr32 and Tyr57 (Figure 8B). (253) Using GCE, each of the three active site Tyr residues were substituted with 3-chlorotyrosine (3-ClTyr). The impact of these substitutions on the local electric field was measured using VSE spectroscopy and correlated with the changes in catalytic activity compared to the WT enzyme. (254) The results show a linear relationship between the electric field applied to the carbonyl and the ΔG⧧, suggesting that the role of the Tyr16 hydroxyl and the adjacent hydrogen bonding network is to tune the electrostatic potential for efficient KSI catalysis.
Figure 8
Figure 8. 3-ClTyr incorporation into Ketosteroid Isomerase (KSI) to tune the active site electric field. (A) The mechanism of KSI. (B) The active site of KSI (PDB: 5KP1 (254)) with the ncAA 3-ClTyr in the active site, shown with orange carbons. The protein backbone is shown as a gray cartoon. Active site residues and the substrate and transition state analogue equilenin are shown as atom-colored sticks, with gray and blue carbons, respectively. (C) The product analogue 19-nortestosterone used for VSE experiments.
3.3.3. Noncanonical Amino Acids to Modulate Cation−π Interactions
Terpene synthases catalyze complex carbocation-based cyclization and rearrangement cascades to give a diverse set of terpenoid natural products. Aromatic ncAAs have been used to modulate cation−π interactions in intermediates in these enzymes.
NcAAs with a range of electron-withdrawing substituents were used to tune cation intermediate stabilization in aristolochene synthase. (255) Replacement of a key active site residue Trp344 by Phe, 4-ClPhe, 4-CF3Phe, and 4-NO2Phe revealed a correlation between the electronic properties of the aromatic side chains and catalytic activity, with more electron-rich substituents leading to higher total turnovers, implicating residue 344 in stabilization of a key cationic intermediate.
A combination of canonical and noncanonical substitutions were used to interrogate cation-π interactions formed by two active site Phe residues in prokaryotic squalene-hopene cyclase (SHC), which mediates the conversion of squalene to hopene via a C22-hopanyl cation intermediate. (256) Interestingly, substitution of Phe365 or Phe605 with the more electron-rich aromatics O-MeTyr, Tyr, and Trp, intended to increase the strength cation−π interactions, increases the catalytic rate at low temperature but leads to a rate reduction at higher temperatures, plausibly due to increased conformational disorder upon introduction of larger aromatic sidechains. To further investigate the effect of the cation-π binding energies on catalytic rates, Phe365 and Phe605 were systematically replaced by 4-FPhe, 3,4-F2Phe, and 3,4,5-F3Phe, which are of similar van der Waals radii to Phe. The catalytic rate decreases with increasing fluorination at either position in a manner proportional to the predicted cation-π binding energies of the aromatic side chains.
3.3.4. Tuning Metal Coordination Environments
Introduction of noncanonical metal coordinating residues into proteins allows the electronic and steric properties of catalytic metal centers to be fine-tuned, providing new structure–activity relationships that cannot be obtained with standard amino acid substitutions.
Nickel-dependent superoxide dismutases (NiSODs) have a metal coordination environment comprised of two Cys ligands, the N-terminal amine, the imidazole of His1, and the backbone amidate of Cys2 (Figure 9). (257) The catalytic nickel center can exist as a five-coordinate pyramidal Ni(III) or a four-coordinate planar Ni(II) species, which lacks the imidazole ligand of His1. To investigate the role of the backbone amidate ligand, a semisynthetic variant of NiSOD with the His1-Cys2 amide replaced by a secondary amine was prepared by NCL (Figure 9). (258) This backbone modification leads to a ∼ 100 fold reduction in the catalytic rate. X-ray absorption near edge structure (XANES) and EPR spectroscopies reveal that the modified variant exists almost exclusively in the four-coordinate Ni(II) state, ca. 50:50 Ni(II)/Ni(III) mixture observed with WT preparations. These experiments suggest that the redox potential of the WT enzyme is finely tuned by the coordination environment to allow facile access to catalytically important Ni(II) and Ni(III) states.
Figure 9
Figure 9. Active site of WT NiSOD (left) and a variant with a secondary amine backbone substitution (right).
The metal coordination environment of [NiFe]-dependent hydrogenase was modified by systematically replacing each of the four coordinating Cys ligands by Sec using allo-tRNAs and selenocysteine synthases. (259,260) In all cases, the catalytic rate of Sec-containing hydrogenases were lower than the WT, retaining between 3 and 14% of the WT activity. However, replacement of the nonbridging Cys576 ligand, which coordinates the nickel cofactor at a position cis to the putative H2 binding site, led to increased oxygen tolerance. The authors suggest that the increased size of the Se atom may prevent O2 binding to the vacant metal coordination site, or alternatively that the more nucleophilic Sec attacks reactive oxygen intermediates to generate a selenoxide that is easily reduced to release H2O. Interestingly, Sec-containing thioredoxin (TR) has also been shown to be more oxygen tolerant than its Cys-containing counterpart. (261) In another study, Wu et al. capitalized on the redox properties of selenocysteine to develop an artificial glutathione peroxidase by replacing the catalytic serine of subtilisin with Sec using a two-step chemical method. Interestingly this Sec modified subtilisin also displays acyl transferase activity. (262)
The identity of the metal-coordinating axial ligand varies across heme enzyme families and is known to play a critical role in controlling catalytic function. (263,264) As such, there is considerable interest in understanding the relationships between proximal ligand electron donation, the structures and reactivities of the Fe(IV)═O intermediates compounds I and II, and overall catalytic function (Figure 10). (265) Natural cytochrome P450s contain Cys-ligated heme iron centers that can perform challenging oxidations of unactivated C–H bonds, chemistry which is inaccessible with histidine-ligated heme peroxidases, even though both enzyme classes use similar Fe(IV)═O intermediates.
Figure 10
Figure 10. Electron donation to the iron center affects ferryl reactivity. (top) Cytochrome P450s are capable of hydrogen atom abstraction by the intermediate Compound I. Increased electron donation through an ncAA selenolate ligand increases the rate compared to WT P450. (bottom) Heme peroxidase compound II is reduced through proton coupled electron transfer. His to MeHis substitution decreases the electron donation to the ferryl intermediate and reduces its proton affinity, slowing the rate of compound II reduction.
The role of the cysteine axial ligand has been explored through its targeted replacement by selenocysteine in a variety of cytochrome P450 enzymes, including P450cam, CYP125, and CYP119. (266−270) Upon mutating the axial Cys to Sec, spectroscopic features and spin states of P450s are similar to the WT enzymes, with minor red-shifts in the Soret maxima and changes in the Raman spectra that demonstrate that Sec is a more electron donating ligand than Cys. It was subsequently shown that the compound I state of Sec-ligated CYP119 was able to cleave C–H bonds more rapidly than the thiolate-ligated WT, providing a direct link between ligand strength and ferryl reactivity. (269) This increased reactivity can be attributed to generation of a more basic compound I state in the Sec-ligated enzyme, increasing the driving force for H-atom abstraction.
A similar approach has been used to derive relationships between ligand strength and ferryl heme reactivity in heme peroxidases. These enzymes have an “imidazolate-like” axial ligand, that is more electron donating than a typical histidine, due to a hydrogen bond between the noncoordinating Nδ atom and a conserved aspartate in the proximal pocket. GCE was used to replace the axial His175 of cytochrome c peroxidase (CcP) by a less-electron donating MeHis, which lacks the proximal hydrogen bonding interaction to Asp235. (271,272) This substitution was shown to be structurally conservative by X-ray crystallography and resulted in a ∼ 20-fold reduction in kcat for ferrous cytc oxidation with negligible changes in KM. In contrast to the situation encountered in P450s, ligand replacement in CcP had minimal impact on the reactivity of compound I (an oxidized neutral ferryl heme and a Trp191 radical cation). Instead, His175MeHis substitution caused a 10-fold reduction in the rate of proton-coupled electron transfer to compound II. This trend can be attributed to weaker electron donation from the axial MeHis ligand affording an electron deficient ferryl-oxygen with reduced proton affinity.
In a subsequent study, Trp51 in the distal pocket of CcP was replaced by a noncanonical 3-(benzothienyl)alanine (STrp), which is structurally similar to Trp but lacks the N–H moiety that forms a hydrogen bond to the ferryl oxygen in compounds I and II. (230) Removal of this hydrogen bond stabilized compound I, but leads to a more basic and reactive compound II state. This increased compound II reactivity manifests in a > 60-fold increase in peroxidase activity toward the small molecule substrate guaiacol (2-methoxyphenol), but interestingly has minimal effect on the rate of oxidation of the biological redox partner, cytc.
3.4. Mimics of Post-Translational Modifications
The structures and functions of native proteins are often tailored through PTMs. In many cases, these covalent modifications cannot easily be recapitulated in recombinant proteins, and as a result their functional significance can be difficult to elucidate. (273) GCE offers a powerful approach to probe the role of PTMs, through selective introduction of ncAAs that are structural mimics of post-translationally modified residues. (274−276)
3.4.1. Lysine and Tyrosine Modifications
Lys residues are subject to a range of reversible PTMs for regulating enzyme function, protein–protein interactions, and cellular localization. (277) The genetic encoding of Nε-lysine derivatives using engineered tRNAPyl/Pyl-tRNA synthetase pairs (278−283) have been used to great effect in the study of individual PTM function in histones (284−289) and enzymes throughout the citric acid cycle. (290−293) For example, acetylation of Lys295 decreases citrate synthase activity by 10-fold, whereas acetylation of Lys238 leads to a 2-fold activity increase. (294) GCE was also used to incorporate Nε-acetylLys (AcLys) into the selenoprotein thioredoxin reductase, to investigate the effect of the PTM on enzyme activity and regulation. (295) A series of thioredoxin reductase variants with acetylated Lys at positions 141, 200, and 307 were all shown to have increased activity compared with the parent enzyme. The authors propose that AcLys incorporation destabilizes inactive multimeric states and favors the active dimeric form of the enzyme.
Dual incorporation of phosphoserine (Sep) (296) and AcLys was used to mimic the coexisting acetylation and phosphorylation PTMs present in native malate dehydrogenase (MLDH). (297) Lysine acetylation at position 140 increased enzyme activity by 3.5-fold, while a single Ser280Sep mutation reduced activity to ca. 30% of the WT enzyme. Simultaneous incorporation of both ncAAs restored the MLDH activity to WT levels, suggesting the two PTMs work in synergy to moderate the activity. Structural modeling of the homotetramer reveals positions 140 and 280 are in proximity at the dimeric interface, opposite a hydrophobic surface region of the protein, suggesting that the PTMs serve to modulate surface charge and stabilize the oligomeric state.
Stop codon suppression (SCS) was used to incorporate Nε-threonyllysine (ThrLys) to study the effect of this recently discovered PTM on Aurora Kinase A activity. (298) Threonylation of Lys162 completely inhibits kinase activity toward a synthetic heptapeptide, with computational modeling suggesting that the modified side chain occupies the ATP binding site. This inhibitory effect can be reversed by the deacetylase Sirtuin 3, which removes the threonylated group from the Lys162.
Human glycolysis enzymes have been found to be heavily lactylated, (299) with lactylation of fructose-bisphosphate aldolase A at the active site Lys147 being a particularly prevalent protein modification. GCE was used to replace Lys147 by a noncanonical lactyllysine (LacLys), which resulted in inhibition of enzyme activity. These results led the authors to propose a lactylation-dependent negative feedback loop in glycolysis, whereby enzymes upstream of an overactivated glycolysis pathway are inhibited leading to reduced glycolytic flux and decreased lactate levels.
Tyrosine PTMs can also be studied using ncAA analogues. 4-carboxymethyl-Phe (4-CmPhe) was used as a mimic of phosphotyrosine to study how the PTM regulates function in the SAM-dependent enzyme protein arginine methyltransferase 1 (PRMT1). (275) Tyr291(4-CmPhe) substitution did not affect the kinetic parameters for methylation of histone H4, but caused a large increase in the KM for two peptides that mimic either the histone H4 tail region or the site of methylation. These data suggest that the phosphorylation of Tyr291 plays a role in the modulation of the substrate specificity of PRMT1 in vivo.
3.4.2. Noncanonical Amino Acids to Mimic Post-Translational Cross-Links
Thioether-bonded tyrosine–cysteine cross-links (Tyr-Cys cross-links) are a PTM common to a range of metalloenzymes, including galactose oxidase, (300) cytochrome c nitrite reductase, (301) and cysteine dioxygenase. (302) To explore the role of this PTM, a model of T. nitratireducens cytochrome c nitrite reductase (TvNiR) was developed in sperm whale Mb with either Tyr or 2-amino-3-(4-hydroxy-3-(methylthio)phenyl)propanoic acid (3-MeS-Tyr) at position 33, to mimic the Tyr-Cys cross-link of the WT nitrite reductase. (303) Hydroxylamine reduction activity increased 4-fold with 3-MeSTyr in place of Tyr33. These activity improvements can be attributed to the 3-methylthio modification lowering the pKa and reduction potential of the active site Tyr, leading to an increased rate of proton coupled electron transfer to the substrate.
NcAA incorporation can also be used to study Tyr-Cys cross-link formation, which is often challenging due to the rapidity of the oxidative cross-linking reaction. The nonheme iron enzyme cysteine dioxygenase (CDO) features a Tyr157-Cys93 cross-link adjacent to the iron center which increases catalytic efficiency by around 10-fold. (304) Site-specific substitution of Tyr157 for 3,5-F2-Tyr and subsequent crystal growth under anaerobic conditions enabled acquisition of a noncross-linked structure while retaining Cys93 (Figure 11). (305) Interestingly, exposure of CDO 3,5-F2-Tyr193 crystals to oxygen resulted in cross-link formation and cleavage of one of the C–F bonds of the ncAA. (306) The positioning of the precross-linked residues in the CDO 3,5-F2-Tyr193 structure suggests that the first target for oxidation during the cross-linking reaction is Cys93, given its proximity to the dioxygen binding site of the metal ion.
Figure 11
Figure 11. Anaerobic X-ray crystal structures of the active sites of Human Cysteine Dioxygenase (CDO, PDB: 6N43 (306)) and CDO Tyr157F2-Tyr (PDB: 6BPR (306)) in complex with the substrate cysteine and NO. CDO and CDO Tyr157F2-Tyr are shown as cartoons in blue and gray, respectively, with key active site residues and the substrate cysteine shown as atom-colored sticks with blue and gray carbon atoms. The noncanonical F2-Tyr157 is shown with orange carbon atoms.
OvoA is a nonheme iron enzyme that catalyzes the oxidative coupling of histidine and cysteine substrates, but also possesses ∼ 10% competing cysteine dioxygenase activity despite lacking a Tyr-Cys cross-link. Interestingly, replacement of active site Tyr417 with 3-MeSTyr alters the product distribution of the enzyme, with 30% of the Cys substrate oxidized to the corresponding sulfinic acid. (307)
The functional importance of post-translational Tyr-His cross-links can also be interrogated using ncAAs. Heme copper oxidases (HCOs) selectively catalyze the four electron reduction of molecular oxygen to water without releasing reactive oxygen species (ROS). (308) The copper center is coordinated by three His residues, one of which forms a cross-link from the Nε atom to the C6 of a neighboring Tyr residue. To better understand the mechanistic significance of this conserved cross-link, (309) Liu et al. incorporated (S)-2-amino-3-(4-hydroxy-3-(1H-imi-dazol-1-yl)phenyl)propanoic acid (ImiTyr) at position 33 in a previously reported functional HCO model based on Mb (vide supra). (310) The resulting enzyme performed over 1000 cycles of oxygen reduction with less than 6% ROS produced, demonstrating 8-fold higher H2O/ROS selectivity and 3 times as many turnovers as the previous HCO model that lacks a Tyr-imidazole cross-link. (311) A related study investigated the effect of Tyr33 pKa in the HCO model by incorporating a series of Tyr analogues by GCE. (312) The rate of O2 reduction was found to be inversely proportional to the pKa of the phenolic proton, consistent with a mechanism in which Tyr33 acts as a proton donor to facilitate O–O cleavage. Interestingly, introduction of 3-MeTyr, which has a similar pKa to Tyr but a lower reduction potential, also gave an increase in rate compared to the Tyr33 variant. (313)
4. Augmenting Function
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Augmenting the functions and properties of existing enzymes is a cornerstone of modern biocatalysis. The ability to improve and control enzyme activity, selectivity, and tolerance to process conditions is vital in enabling the full industrial potential of biocatalysts to be realized. Though a large degree of enzyme optimization can be attained using cAAs, the expanded structural and chemical diversity offered by ncAAs can provide new avenues to access optimized biocatalysts. Many enzyme properties can be augmented using ncAAs, including stability and reusability, substrate and product selectivity, catalytic efficiency, and regulation of activity.
4.1. Stability and Immobilization
Enzymes deployed in industrial processes must often withstand elevated temperatures and organic solvents, and are more cost-effective when recovered and reused across multiple reaction cycles. (314,315) NcAAs can be an effective tool to improve enzyme stability and solvent tolerance, and can open up new biorthogonal chemistries to immobilize enzymes in defined orientations on solid supports. A variety of approaches can be used to achieve stabilization and immobilization, reflecting the diversity of ncAAs available and the different attributes of SPI and SCS methodologies. This section reviews examples of enzyme stabilization achieved through ncAA-mediated noncovalent interactions, covalent cross-links within and between macromolecules, and site-specific immobilization on a range of solid supports.
4.1.1. Stabilization via Noncovalent Interactions
NcAAs can be used to stabilize enzymes by strengthening favorable noncovalent interactions without disrupting tertiary structure. Incorporation of halogenated ncAAs has often been shown to increase enzyme thermostability, (316−318) an effect attributed to dipolar interactions or the formation of halogen bonds between the positive σ-hole of the halogen atom and a neighboring lone pair or negatively charged group. (319) Halogenated ncAAs that are isostructural to their canonical counterparts are often well-accommodated in enzymes, with the similarity between C–F and C–H bond lengths being particularly advantageous in this regard. (25,320) Enzyme stabilization using less conservative ncAA modifications has also been investigated, most commonly employing aliphatic side chain substitutions designed to increase hydrophobic interactions. (321,322) The examples discussed below illustrate how ncAA incorporation can increase thermostability and solvent tolerance across a range of enzyme classes, and in some cases that these improvements can also correlate with enhanced catalytic performance.
Thermostable and solvent-tolerant lipases are widely used in many chemical industries. (323,324) Budisa et al. globally substituted 5-FTrp, 3-FTyr or 4-fluorophenylalanine (4-FPhe) for their respective cAAs in Candida antarctica lipase B (CalB) via SPI. (325) Despite some moderate changes in secondary structure (as observed by circular dichroism), all three variants retained lipase activity, and exhibited an increased shelf life. In particular, the 4-FPhe variant was 50% more active than the WT enzyme following storage for several months at 4 °C. A similar study globally incorporated 6-FTrp, 4-FPhe and 4-fluoroproline (4-FPro) simultaneously into Thermoanaerobacter thermohydrosulfuricus lipase (TTL), substituting almost 10% of the residues with fluorinated analogues. (326) Surprisingly, this extensive fluorination had minimal effects on secondary structure or enzyme activity, with the modified enzyme retaining around 60% of the WT activity. A subsequent study investigated the effect of separate global substitution of ten different ncAAs into TLL, including fluorinated Phe, Tyr and Pro, as well as norleucine and azidohomoalanine. (327) All variants evaluated retained at least some activity, with the 3-fluorophenylalanine (3-FPhe) variant displaying 25% greater efficiency than the WT. Interestingly, this variant also exhibited an expanded substrate scope, with a broader range of triacylglycerol chain lengths (C2-C18) accepted compared to the WT (C6-C10). A further study incorporated 13 different ncAA analogues into TTL and investigated the solvent and surfactant tolerance of the resulting variants. (321) In many cases, these substituted variants displayed an increased tolerance to organic solvents compared to the WT, with some exhibiting higher activity in solvent compared to in aqueous buffer. For example, SPI of 4-R-fluoroproline (4-R-FPro) displayed a 4.5-fold increase in activity in tert-butanol and 3.9-fold in acetonitrile. Many of the variants were also more resistant to denaturation, with 4-aminotryptophan (4-NH2Trp) and 3-FPhe substitutions enabling 70% of activity to be retained after incubation in 0.5 M guanidinium chloride, conditions which led to complete inactivation of the WT enzyme.
Transaminases are important biocatalysts due to their ability to produce enantiopure amines, a motif found in a large proportion of pharmaceutical intermediates and chemical commodities. (328,329) SPI of 3-FTyr into ω-transaminase from Vibrio fluvialis JS17 resulted in a soluble enzyme with 20% higher transaminase activity compared to the WT. (317) Thermostability was also enhanced, with a two hour 70 °C incubation reducing the activity of the fluorinated variant to 36%, compared to only 3.3% for the WT. The fluorinated enzyme also demonstrated increased solvent tolerance, with 90% of activity retained in 50% v/v DMSO cosolvent, whereas the WT exhibited a comparable loss in activity in less than 10% DMSO. Importantly, substrate specificity and enantioselectivity were unaffected by 3-FTyr incorporation, enabling the modified transaminase to catalyze the production of enantiopure (S)-1-phenylethylamine on a preparative scale in 89% isolated yield. A later study demonstrated in vivo biosynthesis of 2-fluorotyrosine (2-FTyr) via transgenic tyrosine phenol lyase and subsequent incorporation into two ω-transaminases from Sphaerobacter thermophilus. (330) The resulting enzymes exhibited high levels of 2-FTyr incorporation and increased thermostability compared to their parent enzymes. Again, substrate selectivity was preserved, and in a high temperature kinetic resolution of racemic α-methylbenzylamine the fluorinated variants yielded the R enantiomer of the amine to > 99% ee, compared to under 50% for the WT enzymes.
In some cases, global replacement of residues with halogenated analogues can simultaneously introduce both beneficial and deleterious mutations into an enzyme. Votchitseva et al. globally incorporated 3-FTyr into a phosphotriesterase (PTE), (331) an enzyme class which has garnered interest for their ability to degrade organophosphate-based pesticides and chemical warfare agents for the purposes of bioremediation. (332,333) The modified PTE exhibited an extended pH tolerance down to pH 5.5, plausibly due to the lower pKa of 3-FTyr compared to Tyr, as well as increased thermostability, with preincubation at 60 °C having minimal effect on paraoxon hydrolysis activity compared to ∼ 90% inactivation of the WT. However, under optimum conditions the catalytic efficiency of the variant was ∼ 400-fold lower than WT, plausibly due to Tyr309 substitution in the leaving group binding pocket, highlighting how ncAA incorporation at residue-level specificity can simultaneously introduce desirable and undesirable effects at different sites. A similar study globally replaced all 32 Pro residues in KlenTaq DNA polymerase with 4-R-FPro. (334) Despite the large number of substitutions made and the highly dynamic nature of polymerases during catalysis, the fluorinated variant retained DNA extension activity, exhibiting 94% of the WT dNTP consumption rate and a similar fidelity. However, thermostability was somewhat reduced, with residual activity only half that of the WT after a one hour incubation at 95 °C. Crystallization of the fluorinated polymerase (335) revealed that 4-R-FPro has a tendency to adopt an exo puckered conformation, forcing some positions to adopt this conformation rather than their WT endo pucker (Figure 12A), which may have contributed to the reduction in stability. Interestingly, the authors noted that the fluorinated variant exhibited enhanced crystallizability compared to the WT, with crystals formed under a broader range of conditions. Several surface 4-R-FPro positions were identified where the fluorine atoms were in close proximity to neighboring symmetry chains, potentially forming new crystallization contacts.
Figure 12
Figure 12. NcAA-mediated noncovalent interactions influence enzyme stability. (A) SPI of 4-R-FPro in KlenTaq DNA polymerase switches many Pro puckers from endo to exo, as illustrated by the substitution of Pro555 (left, gray carbons) to 4-R-FPro555 (right, orange carbons) (PDB: 4DLG, 4DLE (335)). (B) Evolutionary trajectory of TFLeu-incorporating CAT (orange bars) starting from WT CAT (gray bar) against the half-life of enzyme inactivation at 60 °C. (C) Structures of T4 lysozyme with canonical Tyr18 (left, gray carbons) and noncanonical 3-ClTyr18 (right, orange carbons). Glu11 and Gly28 backbone atoms shown (white carbons). Halogen bond between Gly28 backbone oxygen and 3-ClTyr18 chlorine atom indicated with a dashed line (PDB: 1L63, (340) 5V7E (339)).
Enzyme engineering can be used to overcome deleterious effects arising from global ncAA incorporation. Panchenko et al. investigated the effects of global leucine replacement with 5′,5′,5′-trifluoroleucine (TFLeu) in CAT. (336) The fluorinated enzyme retained 76% of WT activity but was much less thermostable, exhibiting a 27-fold reduction in half-life at 60 °C. TFLeu-containing CAT was subsequently evolved by screening acetyltransferase activity after preincubation of lysates at 60 °C for one hour. (90) After two rounds, a triple mutant was identified which exhibited comparable thermostability and activity to the nonfluorinated WT CAT (Figure 12B). Introduction of the same mutations into the nonfluorinated enzyme had a much smaller effect on half-life, demonstrating the specificity of the mutations for adapting the enzyme to TFLeu incorporation. Another study globally incorporated 4-FPhe into dimeric S5 PTE, leading to improved activity and stability. (316) The fluorinated enzyme displayed improved catalytic efficiency toward paraoxon and 2-napthylacetate hydrolysis (2.9-fold and 4.7-fold respectively). Preincubation at 65 °C led to complete inactivation of the WT, while the 4-FPhe variant retained up to 41% activity. Differential scanning calorimetry revealed that 4-FPhe incorporation increased the Tm of the two endothermic transitions by 1–2 °C. More notably, for samples subjected to a second heating scan, the WT showed no transitions whereas the two transitions were retained for the fluorinated variant, suggesting that the modified enzyme could refold after thermal denaturation. However, the authors noted the fluorinated variant gave substantially poorer yield of soluble protein compared with the WT enzyme, suggesting that not all 4-FPhe substitutions were beneficial. In a follow-up study, all phenylalanine positions in the PTE were interrogated in silico with Rosetta modeling software. (337) The positions were all replaced with 4-FPhe, then singly mutated to each of the 19 other cAA identities, to discover mutations which gave a better predicted energy score than 4-FPhe. Through this investigation, 4-FPhe104Ala was identified as potentially beneficial to enzyme stability, as it relieved a steric clash arising from 4-FPhe substitution at the dimer interface. Expression of this 4-FPhe-F104A mutant doubled soluble protein yield and increased the thermal unfolding transition temperatures. Paraoxon hydrolase activity was retained at similar levels to WT PTE, and the mutant was more stable under storage conditions, with 66% residual activity recorded after seven days compared to < 50% for WT PTE both with and without 4-FPhe incorporation. Interestingly, introducing the F104A substitution into otherwise unmodified PTE dramatically decreased stability and activity, demonstrating successful prediction of a 4-FPhe context-specific stabilizing mutation by the Rosetta protocol.
SCS offers a more targeted approach to ncAA-mediated enzyme stabilization, by enabling incorporation only at positions where the ncAA confers a benefit. Ohtake et al. systematically replaced each of the 15 Tyr residues in microbial transglutaminase (MTG) with 3-ClTyr via suppression of the UAG stop codon. (318) The single mutants at positions 20 and 62 exhibited increased residual activity relative to the WT following preincubation at 60 °C, while the other mutations tested were either neutral or deleterious. Testing combinations of the beneficial and neutral mutations revealed a triple mutant with a 5.1-fold longer half-life than the WT as well as a 1.4-fold increase in activity. The researchers then demonstrated simultaneous incorporation of an α-hydroxy acid analogue of Nε-allyloxycarbonyllysine (ALOLysOH) immediately after an N-terminal inhibitory peptide domain via sense codon reassignment. This generated a thermostabilized MTG variant which undergoes automaturation in basic conditions due to hydrolysis of the backbone ester bond.
Another study utilized 3-bromotyrosine (3-BrTyr) for the stabilization of azoreductase (AzoR). (338) Each Tyr residue in AzoR was individually replaced with 3-BrTyr, revealing three positions where bromination was beneficial to thermostability. Simultaneous substitution at these three sites yielded a variant with a 13-fold longer half-life than the WT enzyme at 78 °C. The authors also subjected GST to a similar engineering strategy, substituting Tyr residues with 3-ClTyr. Systematic identification and combination of beneficial substitutions generated a variant with four 3-ClTyr incorporation sites that exhibited 79% residual activity after a 10 min preincubation at 60 °C, compared to < 5% for WT GST.
SCS can also be used to install single substitutions explicitly designed to introduce new stabilizing interactions. By supplementing an in vitro translation reaction with chemically aminoacylated suppressor tRNAs, Mendel and co-workers substituted Leu133 in the hydrophobic core of T4 lysozyme with a variety of ncAA analogues chosen to sample a range of sizes and shapes for the aliphatic side chain without disrupting neighboring residues. (322) They correctly predicted that enzyme thermostability would correlate with ncAA side chain size, with norvaline and ethylglycine reducing Tm, and 2-amino-4-methylhexanoic acid and 2-amino-3-cyclopentylpropanoic acid increasing it by up to 4.3 °C, due to their larger side chains establishing more extensive hydrophobic interactions. In a different study, Scholfield et al. engineered a halogen bond into T4 lysozyme by substituting Tyr18 with 4-iodophenylalanine (4-IPhe). (320) Crystal structures of the modified and WT enzyme evidenced the formation of a halogen bond between the iodine atom and the backbone carbonyl of Glu11, coupled with displacement of the aromatic Tyr ring from its WT position. Replacement of the iodine with a methyl group did not result in the same displacement. However, 4-IPhe incorporation slightly decreased overall enzyme stability due to disruption of favorable hydrogen bonds made by the 4-hydroxyl of the WT tyrosine. A subsequent study incorporated 3-ClTyr at the same site. (339) This modification preserved hydrogen bond interactions made by the hydroxyl group and introduced a new halogen bond between the chlorine and the carbonyl oxygen of G28 (Figure 12C), leading to a 1.0 °C increase in Tm and a 15% increase in enzymatic activity at 40 °C compared to the WT.
4.1.2. Stabilization via Covalent Cross-Linking
Disulfide bridge formation through oxidative linking of Cys side chain thiols has long been known to enhance enzyme stability, (341,342) and engineering new disulfide bridges into enzymes has proven an effective method of stabilization. (343,344) However, disulfide links between canonical Cys residues are subject to geometric and chemical constraints that limit the range of enzymes in which these linkages can be successfully installed. (345) The use of noncanonical analogues can overcome some of these constraints, enabling more facile and diverse deployment of covalent cross-linking for enzyme stabilization (Figure 13A). Liu et al. developed noncanonical Tyr analogues derivatized with aliphatic thiols through the para hydroxyl motif, enabling formation of longer disulfide cross-links. (346) SCS was used to incorporate the thiol-containing ncAAs in a library of N-truncated TEM-1 β-lactamase where one random codon per gene was replaced with TAG. The N-terminal truncation was previously shown to destabilize the β-lactamase such that it could no longer confer antibiotic resistance to the host cell above 37 °C. (347) Growth of library-transformed cells in the presence of the lactam antibiotic ampicillin at 40 °C was used to select for active, stabilized enzyme mutants. A single hit was identified containing a disulfide bridge formed between a Cys residue at position 65 and the thiol ncAA O-(4-mercaptobutyl)tyrosine (SbuTyr) at position 184, which increased the Tm by 9 °C compared to the parent enzyme. Analysis of WT crystal structures revealed the cross-link was formed across the hinge region of two half-domains, which had previously been identified as a hotspot for stabilizing mutations. (348) Interestingly, the distance between the two β carbons of residues 65 and 184 was 11.1 Å, far exceeding the maximum distance of ∼ 5.5 Å for a canonical Cys disulfide. (345)
Figure 13
Figure 13. Covalent cross-links mediated by ncAAs. (A) Cross-links generated between cAAs (black) and ncAAs (orange). Cross-linking bonds shown in gray. Top left: canonical Cys-Cys cross-link. Top right: Cys-SbuTyr cross-link. Middle left: Cys-BpAla cross-link. Middle right: amino group-4-NCSPhe cross-link. Bottom left: Cys-O-2-BeTyr cross-link. Bottom right: Cys-4-CaaPhe cross-link. (B) Structures of Cys-O-2-BeTyr cross-link (left) and Cys-4-CaaPhe cross-link (right) in Mb(H64V,V68A), with Tm increases given by one and two cross-links indicated. ncAAs shown with orange carbons and Cys with white carbons (PDB: 7SPE, 7SPH (351)).
The use of ncAAs can also improve the chemical stability of covalent cross-links. Moore and co-workers developed redox-stable thioether “staples” formed between Cys thiols and O-2-bromoethyltyrosine (O-2-BeTyr) residues (349) (Figure 13B). The RosettaMatch algorithm was used to identify positions in a cyclopropanation-competent Mb variant which could accommodate Cys-O-2-BeTyr cross-links in a strain-free configuration, as well as compensatory mutations of surrounding residues to maximize favorable interactions around the staples. Out of nine designs experimentally evaluated, five successfully formed thioether cross-links, all of which exhibited Tm increases ranging from 3.9 to 10.0 °C over the parent enzyme. Substitution of O-2-BeTyr with isosteric but noncross-linking O-propargyltyrosine (O-PaTyr) lowered the Tm values to below WT, confirming the thioether cross-links were essential for the observed stability increases. Combination of the two most stabilized variants resulted in a doubly stapled design with a 16.8 °C higher Tm than the parent enzyme. Impressively, this variant also retained high levels of activity and stereoselectivity for styrene cyclopropanation, as well as exhibiting enhanced organic solvent tolerance. A different study conducted a similar RosettaMatch protocol on a multimodular pullulanase. (350) Installation of Cys-O-2-BeTyr cross-links in the N/C-terminal domains increased Tm by up to 7.0 °C, though hydrolase activity was adversely affected in the majority of designs. Compensatory mutations designed to improve packing around the cross-links yielded a stabilized variant with 44% higher activity than the WT. A later study investigated the use of alternative cross-linking ncAAs. (351) Substitution of O-2-BeTyr in the aformentioned Mb variants with phenylalanine derivatives containing a chloroacetamido (4-CaaPhe), acrylamido (4-AaPhe) or vinylsulfonamido (4-VsaPhe) group led to successful cross-link formation and increased Tm values relative to the WT enzyme. Notably, 4-CaaPhe incorporation led to even larger stability improvements than O-2-BeTyr, with up to a 16.4 °C increase in Tm given by a single Cys-4-CaaPhe cross-link, and a 26.2 °C increase for two cross-links. The 4-CaaPhe-stabilized mutants were shown to retain the same levels of cyclopropanation activity and stereoselectivity as the parent enzyme, with experimental crystal structures showing good agreement with the design models (Figure 13B). Intriguingly, 4-CaaPhe incorporation was also able to rescue some designs which had previously failed to form cross-links using O-2-BeTyr, suggesting a higher stapling efficiency and greater tolerance of 4-CaaPhe to different local environments.
NcAA-mediated cross-links can also be used to stabilize interactions between protein monomers. Homodimeric homoserine O-succinyltransferase (MetA) catalyzes an essential step in methionine biosynthesis in E. coli, but possesses only mesophilic thermostability which leads to inhibited cell growth above 44 °C. (352) Li et al. exploited this property by screening a BpAla scanning library of MetA variants for increased growth of cells lacking native MetA at 44 °C. (353) A consensus Phe21BpAla mutation emerged which conferred substantially faster growth on the host cells. Isolation of the mutant enzyme revealed a remarkable 21 °C increase in Tm over the WT. Position 21 is located near to the dimer interface, suggesting the possibility of a cross-link spanning the two subunits. Mutation of nucleophilic residues in the region facing Phe21 across the interface revealed that Cys90 was essential to the increase in stability, with a Cys90Ser substitution reverting Tm back to that of the WT. Further evidence of a Cys-BpAla cross-link was provided via trapping of the adduct with β-mercaptoethanol and 13C NMR studies. An alternative enzyme-stabilizing covalent linkage strategy was developed by Xuan et al.. (354) 4-Isothiocyanate phenylalanine (4-NCSPhe) was shown to react with Lys side chains, forming covalent thiourea adducts. The salt bridge formed between Lys17 and Asp123 in Mb was replaced with a thiourea cross-link via incorporation of 4-NCSPhe at position 123, which increased the Tm by 4.8 °C compared to the WT. A subsequent study utilized 4-NCSPhe to create stabilizing cross-links in MetA. (355) Using the same elevated temperature cell growth assay described above, a Phe264(4-NCSPhe) mutant was identified which gave a dramatic 24 °C increase in Tm over the WT. Though initially only the monomeric species was observed after SDS-PAGE analysis, it was found that incubation of the mutant at 37 °C led to the appearance of a dimeric species, suggesting that cross-link formation was temperature dependent. The increased thermostability corresponded with increased transferase activity at higher temperatures, with over 60% residual activity recorded at 60 °C, compared to negligible activity of the WT above 50 °C. Proteolysis and mass spectrometry (MS) fragment analysis revealed a temperature-dependent thiourea cross-link formed between 4-NCSPhe264 and N-terminal Pro2. Inspection of a WT crystal structure revealed that these two positions are over 30 Å apart across the dimer interface and are partially buried by neighboring loops, suggesting that thermally induced conformational changes are necessary to bring the two positions into closer contact, rationalizing why cross-linking only occurs at higher temperatures.
Enzyme stability and solvent tolerance can also be enhanced via ncAA-mediated cross-linking to solubilizing polymers. Deiters et al. reported the cross-linking of alkyne-derivatized polyethylene glycol (PEG) to 4-azidophenylalanine (4-AzPhe) incorporated at position 33 in superoxide dismutase. (356) The copper-catalyzed coupling reaction yielded singly PEGylated enzyme with 70–85% conversion, and with no significant loss in activity compared to the WT. A similar approach was used to generate singly PEGylated CalB. (357) Global substitution of the five methionine residues in the enzyme for azidohomoalanine (AzAla) yielded a variant with a single solvent-exposed azido group, as only one of the methionine residues was located on the enzyme surface. Covalent cross-linking to PEG via a CuAAC reaction gave a monofunctionalised species which displayed similar activity to that of the nonfunctionalized enzyme. Another study generated alkyne-decorated polymersomes, which were then cross-linked to AzAla-containing CalB. (358) The enzyme-decorated polymersomes displayed lipase activity, and could also be recovered from the aqueous phase via filtration, providing a facile method of enzyme recycling. Teeuwen et al. utilized the CuAAC reaction to cross-link the same AzAla CalB variant to the alkyne group of homopropargylglycine-containing elastin-like polypeptides (ELPs), which can be used to control enzyme solubility and aggregation. (359) The cross-linked species was purified from unreacted enzyme by taking advantage of the temperature-dependent phase transition of the ELPs, which reversibly form aggregates above a certain temperature. The residual lipase activity of the enzyme-elastin conjugate was found to be ca. 50% of the nonconjugated AzAla CalB variant. Enzymes containing ncAAs can also be cross-linked to solubilizing polymers using the copper-free SPAAC reaction. Debets et al. prepared dibenzocyclooctyne-functionalized PEG, which was then incubated with AzAla CalB for three hours. (360) Full conversion to the PEGylated enzyme conjugate was achieved, with the majority of the enzyme found to di-PEGylated, suggesting that the higher reactivity associated with the strain-promoted reaction enabled cross-linking to buried as well as surface AzAla residues. In a more recent study, Wilding et al. developed a CFE screening system to assess the effects of SPAAC-mediated PEGylation at six different positions in T4 lysozyme via site-specific incorporation of 4-AzPhe. (361) Interestingly, PEGylation efficiency did not correlate well with the solvent accessibility or hydrophobicity of each site, as previously assumed. In most positions, PEGylation led to slight increases in Tm with minimal impacts on activity. Interestingly however, PEGylation at position 91, which is located in an unstructured loop, was detrimental to both thermal stability and enzyme activity, despite this position previously being identified as optimal for lysozyme immobilization. (362)
4.1.3. Immobilization
A well-established strategy for enhancing enzyme stability is via immobilization on a solid support. (363) Immobilisation can increase enzyme thermostability, solvent tolerance and catalyst lifetime, as well as enabling facile biocatalyst recovery and reuse. (315) However, nonspecific immobilization methods, such as covalent cross-linking of surface amino groups with glutaraldehyde, can substantially decrease effective activity by immobilizing the enzymes in unproductive orientations where the active site is occluded, preventing substrate access (25,364) (Figure 14A). Site-specific ncAA incorporation enables precise immobilization of enzyme molecules in productive orientations (Figure 14B), with a range of biorthogonal cross-linking chemistries now available (Figure 14C). In some cases, ncAA-mediated immobilization can also be used to enhance enzyme activity by facilitating rapid electron exchange with functionalized electrodes. (365)
Figure 14
Figure 14. NcAA-mediated enzyme immobilization. (A) Schematic representation of nonspecific enzyme immobilization, mediated by cross-linking at multiple reactive surface residues (gray circles), resulting in multiple enzyme orientations relative to the solid support, as well as enzyme–enzyme cross-linking leading to multilayer immobilization. (B) Schematic representation of site-specific enzyme immobilization, mediated by a ncAA (orange circles) incorporated site specifically, resulting in a monolayer with a single defined enzyme orientation. (C) Immobilization chemistries utilizing ncAAs (orange). From top to bottom: CuAAC, SPAAC, DOPhe–amine coupling, tetrazine-sTCO Diels–Alder cycloaddition, 3-NH2Tyr-acryloyl Diels–Alder cycloaddition, Glaser–Hay alkynyl coupling, and 4-SHPhe-BODIPY coupling.
Azide-alkyne cycloadditions are an efficient method to generate biorthogonal cross-links for enzyme immobilization. Lim et al. site-specifically incorporated 4-ethynylphenylalanine (4-EthPhe) at position 43 of murine dihydrofolate reductase (mDHFR). (366) Optimisation of the CuAAC reaction conditions enabled efficient cross-linking while maximizing residual enzyme activity. Conjugation of 4-EthPhe to a biotin-PEG3-azide linker enabled immobilization of the enzyme to a streptavidin-coated plate which displayed DHFR activity after washing, while a control immobilization where the biotin linker was omitted resulted in an inactive plate. Another study investigated the effects of different immobilization sites on T4 lysozyme activity. (362) Site-specific incorporation of 4-propargyloxyphenylalanine (4-PaPhe) enabled enzyme immobilization onto azide-decorated magnetic beads via CuAAC. 4-PaPhe incorporation at the mouth of the active site led to a substantial 43% reduction in activity upon immobilization, whereas incorporation at more distal sites only resulted in 19–24% reductions. Additionally, one of these variants, Leu91 4-PaPhe, exhibited higher activity than the WT enzyme immobilized via nonspecific epoxy cross-linking. Enzyme lifetime was also improved, with the immobilized Leu91 4-PaPhe variant retaining 79% activity after three freeze–thaw cycles, compared to 42% residual activity for the epoxy-immobilized WT, and < 27% for nonimmobilized controls. A comparable trend was observed after incubation in 2 M urea. In a similar study, Wang et al. individually substituted five Tyr residues for 4-AzPhe in Geobacillus sp. lipase and immobilized the mutants on a cyclooctyne-decorated support via SPAAC. (367) Triolein hydrolysis activity of the mutants increased by 5–37% upon immobilization, apart from a variant cross-linked at a position adjacent to the active site, which exhibited a slight decrease in activity. All of the immobilized mutants compared favorably with the glutaraldehyde-immobilized WT, displaying up to 6.8-fold higher activity. Additionally, the SPAAC-linked mutants were modestly more thermostable than the randomly cross-linked WT, with up to 1.2-fold higher residual activity recorded after a 29 h incubation at 50 °C. Li et al. also utilized 4-AzPhe, immobilizing aldehyde ketone reductase (AKR) variants to a bicyclononyne-functionalized resin. (368) Variants containing 4-AzPhe at one of five positions were tested, all of which displayed 5–16% increased activity upon immobilization and were also more thermostable than the free unmodified enzyme. Impressively, combining all five 4-AzPhe substitutions into one variant enhanced thermostability further, with over 70% residual activity recorded after 24 h incubation at 60 °C, compared to 5% for the free enzyme.
Other biorthogonal chemistries can also be used to immobilize ncAA-substituted enzymes. DOPhe can be oxidized with sodium periodate to form an orthoquinone intermediate, which can then react with an amino-functionalized coupling partner. (369) Deepankumar and co-workers site-specifically incorporated DOPhe into an ω-transaminase and demonstrated successful immobilization on chitosan and polystyrene beads. (370) They combined this approach with global incorporation of 4-R-FPro, which substantially increased thermostability, doubling the half-life of the enzyme at 70 °C, as well as modestly improving tolerance of a range of organic solvents. Immobilisation of the stabilized variant reduced activity by less than 10% and enabled efficient biocatalyst recovery, with almost complete preservation of enzyme activity following 10 cycles of a 12 h kinetic resolution process. Another study utilized the inverse-electron-demand Diels–Alder (IEDDA) reaction between 1,2,4,5-tetrazines and strained trans-cyclooctenes (sTCOs) for immobilization of thermostable carbonic anhydrase II (tsCA). (371) This conjugation strategy was chosen for its high rate constant (∼72,000 M–1 s–1) which allowed a reduction in immobilization time, thereby minimizing nonspecific adsorption to the solid support. A previously developed tetrazine-containing ncAA, Tet2.0, (372) was incorporated into tsCA separately at three surface positions using SCS, chosen to orient the active site toward bulk solvent, toward the solid support, or parallel to the support. The cross-linking reaction between these variants and sTCO-decorated magnetic beads was found to be efficient enough to allow substoichiometric quantities of enzyme to be used, enabling precise control of the amount of enzyme immobilized on the beads. The orientation of the immobilized enzyme was found to control hydrolysis activity, with the solvent-facing variant retaining 90% activity compared to preimmobilization, the parallel-facing variant 75%, and the support-facing variant 60%. The researchers also demonstrated successful enzyme-limited immobilization onto an sTCO-functionalized flat surface, demonstrating the versatility of the technique. Switzer et al. used Glaser-Hay alkynyl coupling to immobilize a 4-PaPhe-substituted carboxylesterase to an alkyne-decorated Sepharose resin. (373) Four surface Tyr residues were identified and individually substituted with 4-PaPhe, with immobilization of all four variants yielding catalytically active resins. Solvent tolerance was greatly improved by immobilization, with the most active variant displaying increased activity in neat THF compared to neat aqueous buffer, whereas activity of the free WT enzyme was reduced by over 60% in a 1:1 mixture of buffer and THF. The immobilized carboxylesterase also displayed impressive recyclability and lifetime, with almost no activity lost after three years of storage and 18 reaction cycles, compared to the free WT which only retained 20% activity after 6 months of storage.
NcAA-mediated immobilization can also enhance direct electron transfer (DET) between redox enzymes and electrodes, enabling more efficient electrocatalysis, current generation, and reaction monitoring. Ray et al. site-specifically incorporated 3-aminotyrosine (3-NH2Tyr) at a surface position in Mb, which was used to immobilize the protein on an acryloyl-derivatized gold electrode via oxidation of the ncAA to the 2-iminoquinone and subsequent Diels–Alder cycloaddition. (374) As a control, WT Mb was randomly immobilized using a carbodiimide reagent. Atomic force microscopy (AFM) characterization revealed that the ncAA-immobilized protein formed a monolayer with an average feature height of 5 nm, whereas the WT was immobilized in an uneven, multilayer arrangement with an average feature height of 175 nm. Cyclic voltammetry measurements indicated DET could occur between the electrode and both forms of immobilized Mb. Electrocatalytic oxidation of thioanisole was then investigated, with randomly immobilized WT Mb exhibiting 87% conversion and the 3-NH2Tyr-immobilized Mb a comparable 81%, despite the far fewer number of enzyme molecules immobilized in the monolayer arrangement. A different study utilized the SPAAC reaction to immobilize 4-AzPhe-containing mutants of a small laccase to cyclooctyne-derivatized carbon nanotubes. (365) During electro-biocatalytic oxidation of 2,6-dimethoxyphenol, an electron transfer efficiency of 28.7% was recorded when 4-AzPhe was incorporated at position 47, compared to 1.9–7.5% at three other surface positions, and 1.3–3.4% when the enzyme was randomly immobilized by cross-linking surface amino groups to succinimidyl ester-decorated nanotubes. The success of this immobilization position was attributed to its proximity to a water channel connecting the catalytic copper cluster in the active site with the enzyme surface, which may have facilitated electron transfer to the electrode. The 4-AzPhe-immobilized enzyme also exhibited a longer lifetime, with catalytic current decreasing by only 13.6% after 8 days of storage at room temperature, compared to 47.0% for the randomly immobilized control. In another study, Xia and co-workers developed an orthogonal aaRS to enable site-selective incorporation of 4-thiolphenylalanine (4-SHPhe) into tryptophan oxidase (TrpOx). (375) This allowed the enzyme to be derivatized with a boron-dipyrromethene (BODIPY) moiety via a nucleophilic substitution reaction with the ncAA thiol. BODIPY molecules can bind to carbon nanotubes by forming strong π-π interactions, enabling immobilization of BODIPY-conjugated enzymes. AFM characterization of nanotubes incubated with 4-SHPhe-modified enzyme revealed 51.5% coverage when BODIPY was present and only 12.3% when it was absent, with the residual coverage attributed to nonspecific adsorption. On addition of the substrate tryptophan, an oxidative current was detected only from the electrode incubated with both BODIPY and enzyme, demonstrating the immobilized enzyme was functional and that site-specific immobilization was required for efficient electron transfer.
4.2. Improving Enzyme Selectivity
The introduction of ncAAs into enzymes can be used to reshape substrate binding pockets, leading to altered substrate profiles, product distributions and stereoselectivities.
4.2.1. Altering Substrate Profiles
The introduction of ncAAs has been used to alter the substrate preference of PikC, a P450 enzyme that catalyzes a C(sp3)–H oxidation step in the biosynthesis of macrolide antibiotics. (376) PikC substrates require an amino-sugar motif appended during the previous biosynthetic step by the glycosyltransferase DesVII, but it was demonstrated that installation of an ncAA could alleviate this requirement (Figure 15). Surveying a range of aromatic ncAAs at several positions around the substrate binding pocket revealed the mutation His238(4-AcPhe) enabled the site-selective hydroxylation of two different aglycone substrates (10-deoxymethonolide and narbonolide), differentiated from their respective final antibiotic by only the amino-sugar moiety (Figure 15, right). Oxidation site-selectivity for 10-deoxymethonolide could be improved further to 19:1 (methynolide:neomethynolide) by transplanting two known mutations (Glu85Gln and Glu94Gln) into the His238(4-AcPhe) construct. By contrast, the WT PikC oxidizes the glycosylated analogue of 10-deoxymethonolide in a ∼ 1:1 ratio. Structural analyses and docking studies suggested the changes in substrate specificity induced by 4-AcPhe incorporation were due to the acetyl motif supplying hydrogen-bonding and hydrophobic interactions that are normally provided by the desosamine motif in the native substrates. Additional hydrogen-bonding interactions were observed between the C5-hydroxyl group in narbonolide and the acetyl group. Interestingly, the His238(4-AcPhe) mutation also led to improvements in activity and site-selectivity toward the native substrate. The ability to reprogram biosynthetic sequences by altering substrate specificities could have applications in structural diversification for analogue discovery and enabling combinatorial biosynthesis.
Figure 15
Figure 15. Introduction of 4-AcPhe into PikC, a CYP450 enzyme, enabled biosynthetic reprogramming through allowing C(sp3)–H oxidation to occur in the absence of an amino-sugar moiety (brown).
4.2.2. Altering Product Distributions
Fasan and co-workers have investigated the effect of installing ncAAs into a P450 on oxidation regioselectivity. (377) Significant changes in oxidation product distribution were achieved when 4-AcPhe or O-BnTyr were incorporated at one of eleven positions located around the active site or proximal to the prosthetic heme ligand in P450BM3 (Figure 16). For (S)-ibuprofen methyl ester, the Ala78(4-AcPhe) mutation shifted the oxidation product ratio further toward the C1′ oxidation product (1a), whereas Ala181O-BnTyr led to a reversal in regioselectivity, furnishing C2′ oxidation product (1b) as the major species (15:85 1a:1b). For (+)-nootkatone, a new oxidation product, 2c, that was not formed by the WT enzyme was observed; the Ala78(4-AcPhe) mutant formed this as the major product (32:68 2a:2c). The Ala82(4-AcPhe) mutant reduced the native epoxidation activity and concomitantly increased C(sp3)–H oxidation activity; 2a and 2b were formed in a 38:62 ratio respectively. In these cases, canonical substitutions to Tyr at positions 78 and 181 did not give rise to altered product distributions, demonstrating how substrate binding pockets can be more extensively remodelled using ncAAs compared with using cAAs alone on the regioselectivity of P450 oxidations. (377)
Figure 16
Figure 16. Incorporation of ncAAs at various positions within P450BM3 alters the oxidation product distributions for (S)-ibuprofen-OMe and (+)-nootkatone substrates.
4.2.3. Improving Stereoselectivity
NcAA incorporation has been used to improve the stereoselectivity of Acinetobacter baylyi DKR. (378) The WT DKR used in this study catalyzes the reduction of 2-chloro-2-phenylethanone with modest selectivity (9% ee) in favor of the (R)-enantiomer. Mutating Trp222 to smaller cAAs led to a switch in enantio-preference to the (S)-enantiomer, suggesting that steric bulk at position 222 could be a determinant of facial selectivity for hydride delivery. A number of ncAAs with bulky aromatic side chains were incorporated at this position, with the largest improvement in stereoselectivity achieved with O-tBuTyr (up to 34% ee), the bulkiest ncAA tested.
An expanded genetic code has also been used to improve the diastereoselectivity of Pseudomonas alcaligenes lipase during menthol propionate hydrolysis through enhancing substrate specificity for L-menthol propionate over seven other stereoisomers present in the mixture. (379) Guided by MD simulations, a number of substituted aromatic ncAAs were installed at different positions around the active site. The most improved mutant identified was Ala253(2-BrPhe) which selectively catalyzed L-menthol propionate hydrolysis with 94% conversion and > 95% diastereomer selectivity. In another study, Kourist and co-workers investigated the effect of five different ncAAs at ten positions around the substrate binding pocket of the hydrolase Pseudomonas fluorescens esterase. (380) A split-GFP assay was employed to rapidly assess folding of the fifty possible variants, leading to the identification of several sites that are intolerant of ncAA incorporation. The activity and selectivity of the folded, soluble variants were assessed in the kinetic resolution of ethyl 3-phenylbutyrate. While conversions were lower than those achieved with the WT enzyme, enantioselectivity was improved from 27 to 68% ee with a Phe162(2-NapAla) variant, while an Ile224(4-AzPhe) substitution led to a switch in enantio-preference.
4.3. Improving Kinetic Parameters
In addition to improving enzyme stability and selectivity, the introduction of one or multiple ncAAs has been shown to improve enzyme kinetic parameters in effects not replicable with cAAs. Substitutions using both SPI and GCE have been shown to increase kcat, increase total turnover number (TTN), and improve substrate binding.
4.3.1. Noncanonical Ligands in Artificial Metalloenzymes
Heme peroxidases employ a conserved His residue as their axial iron ligand, which forms a strong hydrogen bond from the γ N–H to a neighboring aspartate, an interaction known to increase the electron donating capability of His (see section 3.3.4). Introduction of 3-methylhistidine (MeHis) by GCE into the axial position of an engineered ascorbate peroxidase (APX2) disrupts this hydrogen bond and surprisingly leads to a modest increase in the catalytic efficiency of guaiacol (2-methoxyphenol) oxidation (Figure 17A). (381) Remarkably, this structurally conservative substitution also greatly increases the TTN achieved prior to enzyme deactivation (31,300 and 6,200 for APX2 MeHis and APX2, respectively, Figure 17B).
Figure 17
Figure 17. Peroxidases with MeHis proximal ligands. (A) An overlay of the crystal structures of APX2 (PDB: 1OAG (382)) and APX2 MeHis163 (PDB: 5L86 (381)). Key active site residues and the heme are shown as atom-colored sticks with gray and blue carbons, respectively. MeHis is shown with brown carbons. (B) TTN achieved by APX2 and APX2 MeHis. (C) The catalytic efficiency toward guaiacol (2-methoxyphenol) oxidation for Mb variants and horseradish peroxidase (HRP). (D) An overlay of the crystal structures of Mb (PDB: 1A6K (383)) and Mb MeHis93 (PDB: 5OJ9 (384)). The protein backbones are shown as cartoons, and key active site residues and the heme are shown as atom-colored sticks with gray and blue carbons, respectively. MeHis93 is shown with brown carbon atoms.
The oxygen binding protein Mb lacks the proximal pocket aspartate, which is thought to be one contributing factor to the weak peroxidase activity displayed by this protein. (385) Inspired by the improved catalytic activity of APX2, the proximal ligand (His93) in sperm whale Mb was mutated to MeHis, causing a rotation of the imidazole plane (Figure 17D) and a ∼ 4-fold increase in the kcat of guaiacol oxidation (1.4 vs 6.6 s–1). (384) This increased activity was further improved by both rational mutation and directed evolution, to give a final variant with a catalytic efficiency for guaiacol oxidation that is 1,140-fold higher than WT Mb and in the order of natural peroxidases (Figure 17C).
Axial ligand substitutions in Mb have also been shown to improve nonbiological carbene transfer chemistry. Mutation of the axial iron binding His93 to MeHis in an engineered Mb (Mb*) obviates the requirement for a priming reductant and allows for efficient carbene transfer under aerobic conditions, which contrasts with the oxygen sensitivity of the His93-containing Mb*. (386) These properties were attributed to increased electrophilicity of the Fe(III) center, promoting direct attack of the substrate ethyl diazoacetate (EDA) to produce the carbenoid adduct. This intermediate was captured and characterized through X-ray crystallography and found to form an “unusual” bridging Fe(III)–C–N(pyrrole) geometry (Figure 18A), that the authors suggest is a reaction intermediate in Mb* MeHis93 and in the His-containing enzyme. (386)
Figure 18
Figure 18. Biocatalytic cyclopropanations by Mb* MeHis93. (A) The bridged ion carbenoid intermediate observed by X-ray crystallography (PDB: 6F17 (386)). A 2FO–FC map contoured at 1.5 σ is shown around the bridged carbenoid intermediate and the iron atom. (B) The cyclopropanation reaction catalyzed by engineered Mbs. (C) The non-native cofactor and MeHis ligand used to expand the scope of biocatalytic cyclopropanations. (388)
Pott et al. described the introduction of a wider range of noncanonical ligands into Mb, namely 5-thiazoylalanine (5-ThzAla), 4-thiazoylalanine (4-ThzAla) and 3-(3-thienyl)alanine (3-ThiAla), and found both Mb*(MeHis) and Mb*(5-ThzAla) led to improved oxygen tolerance cf. Mb*. (387) This augmented activity extended to abiological N–H insertion reactions, with MeHis93 and 5-ThzAla93. Introduction of 4-ThzAla as a ligand in Mb*, was found to give the most effective catalyst for S-H insertion reaction of EDA and thiophenol, plausibly due to the higher redox potential of the heme increasing the rate of radical recombination of the intermediate thiol radical with the iron-carbenoid.
Noncanonical ligand substitutions have also been used to expand the scope of biocatalytic cyclopropanations. Installing a non-native iron porphyrin cofactor and a noncanonical axial MeHis ligand into the Mb* variant of Mb afforded a selective artificial metalloenzyme that could catalyze cyclopropanation reactions with electron deficient alkenes (Figure 18B,C). (388) Mechanistic studies indicate that this expanded scope is enabled by radical-type carbene transfer reactivity from the combined effect of the non-native cofactor and axial ligand. In a subsequent study, a wider range of noncanonical ligands were explored at position 93, including 4-NH2Phe and 3-(3′-pyridyl)-Ala (3-PyrAla), albeit with reductions in TTN and/or selectivity compared to the MeHis-containing variant. (389)
Replacement of one key His ligand (His264) in the zinc metalloenzyme mannose-6-phosphate isomerase (ManA) by MeHis gave a functional enzyme that permitted growth of an E. coli strain lacking endogenous ManA activity, albeit at a lower growth rate. (390) Directed evolution of ManA His264MeHis led to the generation of an organism whose growth was strictly dependent on the presence of ncAA, providing a possible strategy for the biocontainment of recombinant organisms.
NcAAs have also been introduced to the distal pocket of heme proteins to tune the properties of the heme cofactor and augment catalytic function. Mutation of the distal pocket His64 of Mb to DOPhe led to a 54- and 10-fold increase in catalytic rate toward benzaldehyde oxidation and thioanisole sulfoxidation, respectively. (391) These improvements were proposed to arise from increased stabilization of the ferryl intermediate compound I from additional hydrogen bonding interactions in the His64DOPhe mutant, with the ncAA performing the hydrogen bonding role of the distal pocket His/Arg residues that are conserved in natural peroxidases.
4.3.2. Noncanonical Amino Acids to Tune Enzyme–Substrate Interactions
A range of cAAs and ncAAs were introduced into the Phe124 position of the prodrug activator nitroreductase, and the activity toward the prodrug CB1954 was found to increase 30-fold with 4-NO2Phe. (392) This residue forms π-stacking interactions with the aryl ring of the prodrug substrate, and previous studies interrogating this position had identified its importance to activity. (393) Interestingly, no correlation between ring electron density and catalytic rate was observed. Instead, it was hypothesized that a polarized aromatic ring at position 124 aids in π-stacking with the polarized aromatic substrate.
Mutation of the active site residue Tyr309 in a bacterial PTE to Hco increased the rate of hydrolysis of the insecticide paraoxon (∼8-fold improvement in kcat). (394) This improvement was suggested to arise from electrostatic repulsion between the negatively charged 4-nitrophenolate product and the anionic ncAA side chain to facilitate the rate-limiting product release step.
4.3.3. Noncanonical Amino Acids to Introduce Conformational Changes
Conformational changes resulting from ncAA incorporation have been reported to augment biocatalyst properties by stabilizing structural elements, improving substrate packing or increasing the rate of product release. Pagar et al. described the systematic incorporation of three ncAAs, 4-MePhe, 4-CF3Phe, and BpAla, into each of the positions 33, 86, and 88 in an (R)-selective transaminase, (R)-ATA, (395) which was developed for industrial sitagliptin production. (396) Phe88(4-MePhe), Phe88(4-CF3Phe), and Phe88BpAla variants all showed increased catalytic activity, the most active variant contained BpAla and gave a 15-fold improvement in activity with 1-phenylpropan-1-amine. Introduction of a Phe86Ala mutation into the BpAla containing variant afforded a Phe86Ala/Phe88BpAla double mutant with 30% higher residual activity after 55 °C incubation, which was used to perform kinetic resolution of 1-phenylpropan-1-amine with benzaldehyde as an amine acceptor.
In another study, the kcat of a transketolase engineered to accept aromatic aldehydes was improved by 2-fold with a Tyr385(4-CNPhe) substitution, with reduced substrate inhibition. (397) An alternative substitution, Tyr385(4-NH2Phe), increased the Tm by 5 °C. Structural modeling suggested this was due to the stabilization of an active site helix through an intersubunit hydrogen bond with Gly262.
SPI of 5-FTrp in place of four canonical Trp residues within the M1–1 isoenzyme of rat GST led to a 4-fold increase in kcat for the arylation of glutathione using 1-chloro-2,4-dinitrobenzene as a substrate, a reaction that is thought to be limited by the rate of product release. (398) Introduction of the noncanonical residues were shown by X-ray crystallography to introduce subtle conformational disorder into the active site, plausibly increasing the rate of product release. Interestingly, the kcat was unchanged using phenanthrene 9,10-oxide and 4-phenyl-3-buten-2-one as substrates, where the chemical step is rate limiting.
SPI was also used to replace the four native Phe residues in the restriction endonucleause PvuII with 2-FPhe, 3-FPhe, or 4-FPhe. (399) Of the three fluorinated Phe analogues, only 3-FPhe increased activity, by approximately 2-fold compared with the WT enzyme. The mutated residues were located distal to the catalytic center and DNA binding sites, with the observed activity increases attributed to conformational changes upon ncAA incorporation.
The specific activity of the lipase TTL toward 4-nitrophenyl palmitate hydrolysis increased upon substitution of 11 methionine residues with norleucine (Nle) using SPI. Activity increases were also observed upon substitution of Asp221 with BpAla by GCE. (400) Notably, simultaneous incorporation of both Nle and BpAla into TTL also gave an engineered variant with higher activity than the WT, albeit with slightly reduced activity compared with TTL containing only the Met to Nle substitutions. Similarly, SPI of Nle into 13 Met residue positions in the heme domain of cytohrome P450BM3 increased the rate of peroxygenase catalysis ∼ 2-fold. (401)
In a modified directed evolution experiment, ten ncAAs Phe analogues were introduced at random positions within TEM-1 β-lactamase using SCS. (402) The resulting library were evaluated using a cell viability assay, leading to identification of a Val216(4-acrylamido-phenylalanine) (4-AcrPhe) variant that increased kcat 14-fold with only a slightly increase in KM. Structural analysis of both apo enzymes and cephalexin acyl-enzyme intermediates by X-ray crystallography revealed conformational changes to key active site residues that likely lower the barrier to transacylation in the ncAA containing mutant.
4.3.4. Building Artificial Dimers Through Noncanonical Amino Acid Tethering
Genetic incorporation of the ncAA 4-AzPhe (403) introduces a ‘clickable’ functional group into proteins, which has been used to form artificial heterodimers using SPAAC reactions with bifunctional linkers. (404) Lim et al. used this approach to improve the cofactor shuttling and increase the efficiency of a two-enzyme cascade for D-mannitol production. (405) Introduction of 4-AzPhe into selected sites of formate dehydrogenase and mannitol dehydrogenase (MNDH) created bioorthogonal handles for SPAAC conjugation to either a heterobifunctional linker harboring a tetrazine handle, or an alternative linker with a cyclooctene handle. The tetrazine and cyclooctene moieties could then undergo an IEDDA reaction to give the artificial heterodimer, with the spatial relationship between the two proteins defined by the site of ncAA incorporation and the length of the linkers (Figure 19). Subsequent work described how the relative orientation of the two enzymes’ active sites affected catalysis. (406) With the active sites in closer proximity, D-mannitol production was 60% higher in comparison to the artificial heterodimer with active sites orientated further from one another.
Figure 19
Figure 19. Introduction of 4-AzPhe into selected sites of formate dehydrogenase (FDH) and mannitol dehydrogenase (MNDH) created bioorthogonal handles for SPAAC conjugation to either a heterobifunctional linker harboring a tetrazine handle or an alternative linker with a cyclooctene handle (PDB: 3WR5, (407) 1LJ8 (408)). FDH and MNDH are shown as gray and blue cartoons, respectively. The sites of 4-AzPhe incorporation are shown as red spheres.
4.4. Regulation of Enzyme Activity
NcAAs have been used to install artificial regulatory elements into biocatalysts to enable spatiotemporal control over enzyme activity in vivo or in vitro.
4.4.1. Chemical and Photochemical Decaging
Genetic code expansion has been used to install protected analogues of functional amino acids into enzymes. These caged amino acids offer an effective strategy to inhibit enzyme activity, either through the steric hindrance of ligand binding sites or by masking key catalytic groups, with facile restoration of activity achievable through cleavage of the obstructing group using chemical (409−411) or photochemical (412−424) methods.
Light offers a minimally invasive stimulus that can be used to regulate protein function both in vitro and in vivo. The most frequently employed photocaging moieties are based upon O-nitrobenzyl (O-NB) groups, which can be applied to cap hydroxy, carboxy, thiol or amino groups and are easily cleaved upon irradiation with long-wave UV light. (425,426) Chemical methods including both SPPS and the PTM of amino acid functional groups have proven effective in the photocaging of proteins. However, these techniques are usually restricted to smaller peptides or preferentially target reactive surface residues. One such example involved the photocaging of lysozyme through the reaction of solvent exposed Lys side chains with a photocleavable PEG reagent. (412) PEGylation of the enzyme resulted in a surrounding polymer layer that prevented interaction with substrate and inhibited enzyme activity, which could only be restored upon irradiation-induced cleavage of the bound PEG polymers.
Alternatively, GCE provides a more targeted approach to install new regulatory elements, and has been used to incorporate photocaged analogues of Tyr, Lys, Ser and Cys into enzyme active sites. (413−424) An early example from the Schultz lab reported the replacement of an active site Tyr503 with O-NBTyr in β-galactosidase to afford an inhibited variant that could be activated upon irradiation with 365 nm light to recover 67% of WT activity. (414) This approach was extended to the development of orthogonal translation components for incorporating photocaged tyrosine analogues in eukaryotic hosts, (427) which has enabled photochemical control of enzymes such Cre recombinases, (428) proteases, (422) or nucleases. (417) Zinc-finger nucleases (ZFNs) have been developed for the sequence-specific scission of double-stranded DNA to enable facile gene editing. (429−432) O-NBTyr was installed into ZFN to develop a photochemically activatable restriction enzyme. (417) Rational replacement of active site Tyr471 by O-NBTyr occludes the active site, preventing binding of the DNA substrate, meaning that phosphodiester cleavage was only observed after brief irradiation with 365 nm light. Crucially, this photocaging strategy is entirely compatible with further engineering of the ZFN, allowing for additional modifications to the dimer interface and introduction of additional mutations to further augment nuclease activity. (433) Photocaged analogues of Tyr have also enabled the regulation of polymerase activity. In the widely used Thermus aquaticus (Taq) polymerase, Tyr671 plays an important role in positioning the DNA template and the incoming dNTP. (434,435) Replacement of Tyr671 with O-NBTyr led to an inactivated variant of Taq polymerase, where the bulky O-NB group is believed to occupy dNTP binding cavity (Figure 20). Upon release of the O-NB caging group via short irradiation with 365 nm light, polymerase activity was restored to 71% of that of the WT. (415) This photocaged polymerase enabled development of a UV-inducible hot-start PCR method, a strategy which can reduce nonspecific DNA amplification and increase sensitivity and specificity. (436) Since various monomeric DNA and RNA polymerases also rely on an analogous active site Tyr, (437) recombinant replacement of this residue with O-NBTyr could provide a general approach to photochemical regulation of polymerase activity in these enzymes. To illustrate this broader applicability, Chou et al. engineered a photocaged variant of a bacteriophage T7 RNA polymerase (T7RNAP) with Tyr639 substituted with O-NBTyr. (416) As T7RNAP is orthogonal to all endogenous prokaryotic and eukaryotic RNA polymerases, expression of genes of interest under T7 promoters in bacterial and mammalian cells could be precisely controlled via light activation. Spatiotemporal activation of the polymerase was demonstrated through the engineering of E. coli to produce the photocaged T7RNAP alongside either GFP or luciferase reporter genes. In both cases, light irradiation successfully initiated RNA polymerization leading to downstream gene expression, where fluorescence intensity correlated with the duration of UV exposure. A more recent study targeted Lys631 for genetic replacement with methyl-2-nitropiperonyllysine (MNPLys), producing an additional light activatable variant of T7RNAP. (420)
Figure 20
Figure 20. Introduction of a photocaged ncAA into a DNA polymerase through GCE occludes the active site, preventing the diffusion of nucleotides for extension. Brief irradiation with UV light cleaves the O-NB moiety to reveal the catalytic Tyr and restore polymerase activity. Created with BioRender.com.
CRISPR/Cas9 is a powerful technology for genome editing that is widely used in medicine and biotechnology. The Deiters lab sought to add an optically controllable element to the Cas9 enzyme using GCE. (421) A combination of Ala and ncAA scanning experiments revealed the catalytic importance of Lys886 for Cas9 activity, so this residue was targeted for substitution with a photocaged analogue using SCS. The presence of MNPLys in the protein active site abolished DNA cleavage and nicking activity. Enzyme activity could be fully restored upon photodecaging of Lys886 using 365 nm light. This light-activated Cas9 system was successfully used for endogenous gene silencing, leading to reduced expression of the CD71 gene and 50% reduction of the CD71 transmembrane transferrin receptor on the cell surface.
Photocaged ncAAs have also found application for studying and modulating kinase activity. MNPLys was incorporated in place of the near-universally conserved Lys97 in the ATP binding domain of the MAP kinase MEK1, inhibiting its activity by blocking ATP binding and preventing phosphorylation. (419) Following photodecaging of Lys97, receptor-independent light activation of a designed subnetwork of the Raf/MEK/ERK signaling pathway was achieved in live mammalian cells, enabling study of the kinetics of individual steps in the transduction cascade.
The chemiluminescence activity of Renilla luciferase (RLuc) can also be controlled directly through the genetic replacement of a catalytic Cys residue with the photocaged analogue PCys using a previously engineered MbPylCKRS. (423,127) Following successful incorporation of the caged Cys, the activity of RLuc could be initiated with brief UV irradiation resulting in a > 150-fold increase in chemiluminescence activity over the caged enzyme. Protease activity can also be regulated via photocaging of catalytic Cys nucleophiles. An early study achieved genetic incorporation of O-NBCys in place of the active site Cys163 of capase-3 in yeast. (413) In lysate assays of the mutant enzyme, protease activity was only detectable after photodecaging upon irradiation with UV light. Interestingly, while expression of WT capase is toxic to yeast cells, expression of the photocaged variant was not detrimental to cell growth. This methodology was extended to protease expression in E. coli through the engineering of a PylRS/tRNACUA pair to encode photocaged Cys variants, allowing the development of light-activatable TEV protease variant. (422)
4.4.2. Azobenzene Photoswitches
Azobenzene derivatives have found diverse application in the reversible photocontrol of biomolecules, owing to their efficient light-induced E/Z isomerization which result in structurally disparate isomers (Figure 21). (438−447) Notably, a variety of azobenzene-based photoswitches can now be selectively incorporated into proteins using GCE. (448−456) In one study, incorporation of an azobenzene switch enabled photochemical control over an allosteric activation mechanism in imidazole glycerol phosphate synthase (ImGPS). (453) The HisH glutaminase subunit of this bienzyme complex is allosterically stimulated by binding of a regulatory ribonucleotide to the HisF cyclase subunit. Light-mediated isomerization (356 nm to induce E to Z isomerization and 420 nm for Z to E) of 4-azobenzylphenylalanine (AzoPhe) introduced at position 55 of HisF resulted in reversible 10-fold regulation of HisH activity. In another study, AzoPhe, 4-azo-(2’,6’-difluorobenzyl)phenylalanine (F2AzoPhe), and 4-azo-(2’,6’-difluorobenzyl)-3,5-difluorophenylalanine (F4AzoPhe) were incorporated into firefly luciferase (FLuc) in both prokaryotic and eukaryotic cells. (454) Unsubstituted AzoPhe demonstrates UV light-induced E to Z photoswitching but spontaneously reverts to the more stable E-isomer, posing challenges for long-term control of protein function. In contrast, F4AzoPhe possesses enhanced thermal stability in the Z state. Furthermore, the fluorine substituents lead to a red-shifted absorption spectrum enabling efficient E to Z and Z to E isomerizations to be initiated by visible light, which facilitates longer-term control of enzyme activity in vivo than would be possible with UV irradiation. Computational modeling, guided by local repacking analysis, identified potential allosteric sites in FLuc to aid rational amino acid substitution. Of the seven sites identified, it was found that reversible on/off switching of luciferase activity could be achieved through azobenzene incorporation at Trp417, enabling up to five cycles of photoswitching.
Figure 21
Figure 21. Photoresponsive ncAAs used in the allosteric light regulation of ImGPS. AzoPhe undergoes light induced reversible E/Z isomerizations enabling on–off switching of HisH activity.
4.4.3. Metal Responsive Regulation
In a recent study, Zubi et al. developed a metal-responsive system for protein (in)activation involving the genetic incorporation of two spatially separated bidentate (2,2’-bipyridin-5-yl)alanine (BpyAla) residues to impart conformational control over proteins that do not otherwise exhibit allostery (Figure 22). (457) The serine protease Pyrococcus furiosus (Pfu) prolyl oligopeptidase (POP) was selected as a model enzyme due to the dynamic domain opening/closing it undergoes to allow substrate entry and positioning of the catalytic triad, as revealed by MD simulations. To minimize screening efforts, pairs of positions for BpyAla incorporation were identified through parametrized MD simulations, which highlighted residues that were appropriately positioned to chelate metal ions in the closed conformational state to occlude the active site, but not in the open state. When incubated in excess divalent metal salts such as those of Ni(II), Cu(II), Co(II), and Zn(II), protease activity of selected POP variants was almost entirely inhibited as intended, with near quantitative recovery of enzyme activity achieved upon addition of EDTA for up to 24 on/off cycles. Spectroscopic, and computational investigations collectively suggested that the Bpy pairs engage in reversible metal binding to generate the targeted M(II)(BpyAla)2 complex, resulting in a conformational change which inhibits catalytic turnover. To illustrate the generality of this approach, the study was extended to develop a metal dependent Photinus pyralis luciferase (Pluc), with the best variant showing a 20-fold decreased Vmax in the presence of Ni(II).
Figure 22
Figure 22. Introduction of a pair of BpyAlas into Pfu POP (PDB: 5T88 (458)) enabled inhibition of protease activity when incubated in divalent metal salts. Metal binding of the noncanonical ligands holds POP in a closed inactive conformation, which can be released through chelation of metal ions with EDTA addition, thereby allowing reversible allosteric control of biocatalyst activity. Created with BioRender.com.
5. Designing New Catalytic Mechanisms and Functions
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A major objective of modern biocatalysis is to broaden scope of chemical transformations beyond those known in nature, by developing enzymes that operate through new catalytic manifolds. To maximize the breadth of chemistry accessible with designed biocatalysts, it is important to be able to introduce a wide variety of functional groups within enzyme active sites. One way to achieve this is to embed new catalytic elements as ncAA side chains. A major advantage of this approach for enzyme design is its versatility. Having established suitable aaRS/tRNA pairs to encode a functional amino acid of interest, this residue can be positioned at various sites in diverse protein scaffolds. (14,27) Importantly, it has been shown that directed evolution workflows can be adapted to optimize designed enzymes made from an expanded set of amino acid residues. (28,459,460) As a result of rapid advances in the field, a variety of enzymes have now been developed that use ncAAs as key catalytic motifs.
5.1. Metalloenzymes
2,2′-Bipyridine is a widely used bidentate ligand for divalent cations. (461) Xie et al. expressed T4 lysozyme mutants with BpyAla site-specifically incorporated at several positions on the protein surface. (462) Incubation of the modified lysozyme variants with CuCl2 and subsequent MS and spectroscopic analyses confirmed Cu(II) binding, which was dependent on the BpyAla ligand. Subsequently, BpyAla was used to confer DNA cleavage activity on E. coli catabolite activator protein (CAP). (463) Guided by a crystal structure of dimeric CAP bound to DNA, the surface residue Lys26 was selected for substitution to BpyAla due to its proximity to the DNA–protein interface. When incubated with redox active metal ions such as Fe(II) or Cu(II), a reducing agent and the allosteric activator cAMP, CAP Lys26BpyAla promoted site-selective oxidative cleavage of a bound DNA fragment. Cleavage occurred at either side of a specific nucleobase, suggesting that a freely diffusible oxidizing agent generated at the metal center is responsible for DNA cleavage.
The transcription factor Lactoccocal multidrug resistance Regulator (LmrR) has proven to be a versatile scaffold (464) for developing metalloenzymes using BpyAla. Drienovská et al. generated copper metalloproteins for enantioselective vinylogous Friedel–Crafts alkylations by incorporating BpyAla into LmrR at position 89 (Figure 23A). (465) Copper-loaded LmrR Met89BpyAla promoted alkylations of electron-rich indoles with 1-(1-methyl-1H-imidazol-2-yl)but-2-en-1-one (Figure 23B,C) with modest levels of stereocontrol. Reaction selectivity could be further improved through targeted mutagenesis of residues in proximity to the BpyAla ligand. In a later study, a combination of quantum mechanics, docking and MD calculations facilitated the design an LmrR-based metallohydratase. (466) The designs utilized BpyAla-complexed Cu(II) to catalyze enantioselective hydration of α,β-unsaturated 2-acyl pyridines (Figure 23C), achieving up to 64% ee. The same group also demonstrated the binding and stabilization of 2-semiquinone radicals by artificial metalloproteins containing a variety of first row transition metal ions in the LmrR Met89BpyAla template. (467) This ability to stabilize reactive radical species in the binding pocket of a protein could facilitate the development of artificial enzymes for controlling transformations involving radical intermediates.
Figure 23
Figure 23. Catalytic metal-coordinating ncAAs. (A) Crystal structure of dimeric LmrR, with the positions Val15, Met89, and Trp96 in the binding pocket shown with blue carbons (PDB: 3F8B (474)). (B) BpyAla-coordinated Cu(II) complex which activates 1-(1-methyl-1H-imidazol-2-yl)but-2-en-1-one toward nucleophilic attack. (C) Schemes of vinylogous Friedel–Crafts alkylations (top) and α,β-unsaturated 2-acyl pyridine hydrations (bottom) catalyzed by BpyAla-Cu(II) or 3-HqAla-Cu(II) metalloenzymes.
In addition to LmrR, several other protein scaffolds have been elaborated into artificial copper metalloenzymes by incorporating BpyAla. For example, a Tyr123BpyAla variant of QacR proved to be an effective biocatalyst for stereocontrolled vinylogous Friedel–Crafts alkylations of indoles with 1-(1-methyl-1H-imidazol-2-yl)but-2-en-1-one (Figure 23C). (468) Interestingly, this metalloprotein gave the opposite product enantiomers to those formed with LmrR Met89BpyAla. Another study installed BpyAla in a homohexameric acetyltransferase to create a dinuclear copper oxidase. (469) The innate symmetry in the scaffold allowed the distance between the Cu(II) sites to be varied, inducing electronically coupled dinuclear behavior in variants where the sites were proximal and mononuclear behavior in variants where they were distal. Interestingly, the dinuclear copper variants were found to oxidize ascorbate more rapidly than their mononuclear counterparts.
Beyond BpyAla, other metal-binding ncAAs have been used to generate artificial metalloenzymes. The development of an MjTyrRS pair to incorporate 3-HqAla (150) enabled production of LmrR variants with 3-HqAla ligands at Val15 or Met89 (470) (Figure 23A) which can be used to chelate Cu(II), Zn(II) and Rh(II) ions. Slow amide bond hydrolysis activity was observed for Zn(II)-containing variants, which did not occur in the presence of the Zn(NO3)2 salt alone, suggesting that catalysis occurs within the LmrR binding pocket. Cu(II)-loaded variants were found to be competent catalysts for vinylogous Friedel–Crafts alkylations and hydration of α,β-unsaturated carbonyls, albeit with modest selectivities (Figure 23C). More recently, Stein et al. utilized a regioisomer of 3-HqAla, (8-hydroxyquinolin-5-yl)alanine (5-HqAla) to generate a Ru-based metalloenzyme for allylic deamination. (471) Halotag, a self-labeling protein derived from Rhodococcus dehalogenase, (472) was expressed with 5-HqAla incorporated at positions Phe144, Ala145 or Met175 in the hydrophobic binding cleft. After incubation with [Cp*Ru(MeCN)3]PF6, all three variants displayed increased deamination activity toward an O-allyl carbamate-protected coumarin compared to the WT, with the 5-HqAla175 variant performing up to 9 turnovers. In another study, 3-pyridylalanine (3-PyrAla) was successfully used to transform dimeric bovine pancreatic polypeptide (bPP) into an enantioselective biocatalyst for Diels–Alder cycloadditions and Michael reactions. (473) A Tyr7 to 3-PyrAla mutation was proposed to create a bidentate metal binding site at the dimer interface. 3-PyrAla-modified bPP was produced by SPPS and incubated with Cu(H2O)6(NO3)2. The resulting metalloprotein was found to promote Diels–Alder cycloadditons of α,β-unsaturated carbonyls and cyclopentadiene, and Michael additions of dimethylmalonate and α,β-unsaturated 2-acyl imidazoles, with appreciable levels of stereocontrol (up to 83% and 86% ee, respectively).
Artificial metalloenzymes can also be created by using ncAAs to covalently tether metal complexes to proteins. Biorthogonal SPAACs to 4-AzPhe are well-suited for this purpose. (475) Yang et al. reported the incorporation of 4-AzPhe into tHisF, a thermostable α,β-barrel protein, and subsequent SPAAC conjugation of metal complexes derivatized with bicyclo[6.1.0]nonyne (BCN). (476) Tethering of tetracarboxylate dirhodium complexes and Cu- or Mn-terpyridines was demonstrated, with 50–90% conversion to the conjugated species observed. The artificial metalloenzymes were then evaluated for intermolecular cyclopropanation and Si–H insertion activities. Unfortunately, the catalysts gave reduced activity compared with the free metal complex and only minimal selectivity, suggesting the location of the metal complexes near the mouth of the α,β-barrel did not provide a conducive environment for selective catalysis. Greater success was found using POP as a scaffold, chosen for its thermostability and large internal volume suitable for hosting metal complexes. (477) Initial efforts to conjugate POP variants modified with 4-AzPhe at various positions in the active site with a BCN-derivatized dirhodium paddlewheel complex (Figure 24A) failed, likely due to the enzyme adopting a closed conformation which shields the active site from solvent. Mutation of four residues lining the pore of the β-barrel domain to alanine overcame these issues (Figure 24B), enabling rapid conjugation to form the metalated enzyme. Catalysis of styrene cyclopropanation with a diazo ester (Figure 24C) gave a single diastereomer with 19% conversion and 11% ee, which could be further improved to 38% ee upon optimization of reaction conditions. Additional improvements were achieved with the introduction of an active site His to coordinate the proximal rhodium center and phenylalanine mutations around the distal rhodium, with the best variant achieving 74% conversion and 92% ee. In a subsequent study, a directed evolution platform was established to allow more extensive engineering of artificial metalloenzymes generated using SPAAC. (458) Starting from the aforementioned quadruple alanine POP mutant, three rounds of mutagenesis and screening generated the improved variant 3-VRVH, which contained 12 mutations and achieved 92% ee for 4-methyoxystyrene cyclopropanation, and a significantly higher reaction rate than the optimal variant from the previous study. Directed evolution was also used to optimize the performance of artificial POP metalloenzymes containing dirhodium paddlewheel complexes for efficient cross-coupling of diazoesters. (478) The most highly evolved metalloenzyme, 5-G, was able to perform an impressive 40,000 turnovers and delivered the cross-coupled products with high levels of stereocontrol (14.9:1 E/Z selectivity). 5-G was subsequently integrated into a biocatalytic cascade with an alkene reductase and glucose dehydrogenase for NADPH recycling (Figure 24C), leading to the production of derivatized succinate products with up to 61% conversion and > 99% ee.
Figure 24
Figure 24. 4-AzPhe-anchored metalloenzymes. (A) BCN-Derivatised dirhodium complex. OAc– = acetate anion. (B) Crystal structure of POP, with positions of 4-AzPhe incorporation (orange spheres) and pore-opening alanine mutations (blue spheres) shown (PDB: 5T88 (479)). (C) Schemes of styrene cyclopropanations (top) and the diazo cross-coupling cascade (bottom) catalyzed by POP variants containing 4-AzPhe-tethered dirhodium complexes.
5.2. Nucleophilic Catalysis
Nucleophilic catalysis is a versatile strategy used in chemistry and biology to accelerate diverse transformations. For example, natural hydrolases or acyltransferases often feature an activated Ser or Cys nucleophile embedded within catalytic diads or triads. (480−482) Efforts to recapitulate these mechanisms in designed enzymes commonly results in proteins where catalysis stalls due to the formation of stable acyl-enzyme intermediates that are resistant to hydrolysis. (483−485) To overcome these limitations, our lab used GCE to reengineer a computationally designed protein BH32 into an efficient hydrolase. (486) BH32 was originally designed to catalyze the Morita-Baylis-Hillman (MBH) reaction using a His23 nucleophile, (487) but was also found to possess promiscuous ester hydrolase activity. (470) Similar to previously designed hydrolases, BH32 displayed a biphasic reaction profile consistent with rapid acylation of His23 generating a stable acyl-imidazole intermediate that is resistant to hydrolysis. To resolve this bottleneck in catalysis, His23 was replaced by a noncanonical MeHis, which lead to the formation of more reactive acyl-imidazolium intermediates (Figure 25A). Hydrolase activity was subsequently enhanced by directed evolution, affording the enzyme OE1.3 with six mutations (Figure 25B) that is ca. 9,000-fold more active than free MeHis in solution, along with an enantioselective hydrolase OE1.4 that contains an additional three mutations (cf. OE1.3). MeHis has also been shown to be a valuable catalytic nucleophile for more complex chemical conversions. Installation of MeHis into an engineered BH32 template followed by extensive evolutionary optimization afforded a highly efficient and enantioselective enzyme (BHMeHis1.8) for Morita-Baylis-Hillman reactions, which are valuable C–C bond forming processes for which there are no natural enzymes known. (488) BHMeHis1.8 is more than an order of magnitude more active than our earlier BH32.14 enzyme which contains a His23 nucleophile, (489) and can also promote challenging MBH conversions of electron-rich aromatic aldehydes that were inaccessible with BH32.14. Interestingly, introduction of MeHis led to a dramatically altered mechanistic outcome following evolution, where a key catalytic Arg124 found in BH32.14 was abandoned in favor of a Glu26 residue that mediates a rate-limiting proton transfer step (Figure 25C).
Figure 25
Figure 25. Nucleophilic catalysis utilizing MeHis. (A) Scheme of ester hydrolysis, showing the reactive covalent intermediate formed between the substrate and MeHis23 (orange). (B) Structure of OE1.3, with MeHis23 (orange carbons) and sites of mutations installed during evolution (blue spheres) shown (PDB: 6Q7Q (486)). (C) Scheme highlighting the proton transfer role of Glu26 (gray) in the evolved MBHase BHMeHis1.8. Intermediates 2 (left) and 3 (right) are shown, covalently bound to MeHis23 (orange).
Aniline nucleophiles are known to catalyze hydrazone and oxime formations. (490,491) LmrR was engineered to efficiently catalyze hydrazone formations by embedding an aniline nucleophile. (492) A two-step protocol involving initial installation of 4-AzPhe by GCE followed by chemical reduction of the aromatic azide was used. The resulting enzyme, LmrR_Val15(4-NH2Phe), achieved 72% conversion for a hydrazone formation between 4-methoxybenzaldehyde and a benzoxadiazole, and was also found to catalyze the analogous oxime formation (Figure 26A), with a kcat/KM 37-fold higher than WT LmrR. Hydrazone formation activity was subsequently optimized through directed evolution, (460) affording triple and quadruple mutants that displayed 57- and 74-fold improved catalytic efficiency respectively compared to the parent enzyme. Mutation of the 4-NH2Phe nucleophile to an isosteric Tyr led to dramatic activity reductions in both variants, highlighting the critical role played by the ncAA in accelerating hydrazone formation. The evolved hydrazone-forming enzymes have subsequently been used in combination with alcohol oxidases and carboxylic acid reductases for in vivo biocatalytic cascades. (493) The reaction scope of 4-NH2Phe-containing LmrR variants has been extended to include enantioselective Friedel–Crafts alkylations of indoles (Figure 26B) (459,494) and enantioselective Michael additions of enals and 2-acylimidazoles. (495,496) In the latter case, catalysis is dependent upon the synergistic action of the 4-NH2Phe nucleophile and a Cu(II)-phenanthroline complex that is sandwiched between two tryptophan residues within the hydrophobic binding pocket, with up to 99% ee observed.
Figure 26
Figure 26. Nucleophilic catalysis utilizing 4-NH2Phe. (A) Scheme of hydrazone (X = N) and oxime (X = O) formations catalyzed by 4-NH2Phe (orange) incorporated into LmrR, with the covalent adduct formed by the carbonyl substrate and 4-NH2Phe15 shown. (B) Scheme of vinylogous Friedel–Crafts alkylations catalyzed by LmrR_V15_4-NH2Phe_RGN, with the activated imine intermediate formed between 4-NH2Phe15 (orange) and the aldehyde substrate shown. At the end of the reaction time NaBH4 is added to reduce the enzymatic product to the corresponding alcohol (right).
More recent studies have disclosed other catalytic ncAAs. Gran-Scheuch et al. reported the synthesis and incorporation of several ncAAs featuring secondary amine motifs using the MbPylRS system. (497) LmrR containing a pyrrolidine-inspired ncAA at position 15 was found to catalyze Michael addition of nitromethane to cinnamaldehyde with moderate conversions and up to 38% ee. Incubation of the enzyme with cinnamaldehyde and NaBH3CN gave rise to an MS peak shift consistent with formation of a reduced Schiff-base adduct, implicating ncAA-mediated iminium ion activation of the aldehyde. Another study reported the use of 4-boronophenylalanine (4-BoPhe) installed in engineered LmrR variants to catalyze condensation of α-hydroxyketones with hydroxylamine to form enantioenriched oximes in a kinetic resolution process. (498) MS and 11B NMR studies provided evidence for the catalytic role of 4-BoPhe, which was proposed to form transient boronate adducts with vicinal diol reaction intermediates in a stereoselective manner.
5.3. Photocatalysis
Photons provide a convenient and tuneable source of energy to selectively access reactive excited state intermediates under mild reaction conditions. This use of light energy opens up new modes of reactivity that are challenging to access in the ground state, resulting in the development of diverse synthetic methods for constructing carbon-carbon and carbon-heteroatom bonds. (499,500) Light-driven reactions can be broadly divided into photoinduced electron transfer (photoredox) processes and triplet energy transfer processes. There are a handful of natural photoenzymes (501−503) and several engineered photoenzymes (504−506) that operate via photoredox mechanisms. In contrast, there are no natural enzymes known to mediate stereocontrolled transformations through energy transfer mechanisms. To address this limitation, Trimble et al. and Sun et al. independently developed enantioselective enzymes for thermally forbidden [2 + 2] cycloadditions. In the former study, a BpAla triplet sensitizer was incorporated into the active site of a previously designed Diels–Alderase, DA_20_00, (507) giving rise to a first generation photocatalyst EnT1.0 that could mediate intramolecular [2 + 2] cycloadditions of quinolone substrates (Figure 27A) with modest regio- and enantioselectivity upon irradiation with 365 nm light. (28) Introduction of five additional mutations via directed evolution afforded an optimized photoenzyme EnT1.3 with substantially improved activity, regioselectivity and enantioselectivity (>99% ee). This energy transfer photoenzyme can operate efficiently at ambient temperatures and in the presence of oxygen, and can also mediate bimolecular [2 + 2] cycloadditions with high levels of stereocontrol. A product-bound crystal structure of EnT1.3 reveals that the ligand is sandwiched between the BpAla side chain and an active site His (Figure 27B), an arrangement conducive to efficient energy transfer between the photosensitizer and substrate. Sun et al. used a similar approach to develop enantioselective photoenzymes for intramolecular cycloadditions of N-substituted indoles using LmrR as a protein scaffold. (508) In this case, a fluorinated analogue of BpAla (3′-FBpAla) led to improved conversions and selectivities for a number of substrates compared with the parent sensitizer.
Figure 27
Figure 27. [2 + 2] Photocycloadditions catalyzed by BpAla. (A) Schemes of intramolecular [2 + 2] photocycloadditions of derivatized quinolones (top) and indoles (bottom). X = O or C, n = 1 or 2. (B) Crystal structure of EnT1.3 with product (green carbons) bound between BpAla (orange carbons), Trp244, and His287 (blue carbons) (PDB: 7ZP7 (28)).
A large number of chemical transformations can be driven by photoinduced electron transfer (PET) processes. (509,510) Incorporation of ncAAs into fluorescent proteins has delivered photoredox catalysts by tuning the absorption profiles and redox properties of the chromophore. Liu et al. generated a miniature photocatalytic CO2-reducing enzyme by modifying superfolder yellow fluorescent protein (sfYFP), (506) which features a chromophore generated by autocatalytic cyclization and oxidation of residues Ser65, Tyr66 and Gly67 (511) (Figure 28A, top). Substitution of Tyr66 with BpAla (Figure 28A, bottom) in a His148Glu Phe203Asp mutant of sfYFP generated the photosensitizer protein PSP2. (506) Photochemical reduction of PSP2 with sacrificial reductants produced super-reducing radicals (PSP2•) which were sufficiently potent to drive CO2 reduction by a nickel-terpyridine complex ligated to PSP2 through a Cys residue introduced at position 95 (Figure 28B). Introduction of proton-donating Tyr residues around the nickel complex further improved activity, with the resulting enzyme PSP2T2 exhibiting a CO2/CO conversion quantum efficiency of 2.6%. PSP2T2 was also found to catalyze light-driven dehalogenations of simple aryl halides to phenolic products with up to 98% conversion (Figure 28C). (512)
Figure 28
Figure 28. Metal-dependent ncAA-incorporating photoenzymes. (A) Chromophore autocatalytically generated in sfYFP and in PSP2, which incorporates BpAla (orange side chain) at position 66. (B) Structure of PSP2, with a chromophore shown (backbone indicated with gray carbons, BpAla side chain with orange carbons). The Cys95 site of nickel–terpyridine complex ligation is shown in dark gray (PDB: 5YR3 (506)). (C) Scheme of dehalogenation reactions catalyzed by BpAla-incorporating PSP2T2 or by BpyAla-incorporating Mb. X = Cl, Br, or I. (D) Structure of Mb incorporating BpyAla (orange carbons) and with an iridium photocatalyst (green carbons) ligated to Cys45 (gray carbons) (PDB: 7YLK (516)).
Photoenzymes can also be generated via biorthogonal conjugation of organic or metal photocatalysts to 4-AzPhe incorporated into proteins. Gu et al. anchored a BCN-derivatized 9-mesityl-10-methylacridinium cofactor into the active site of POP. (513) Upon irradiation with 450 nm light, the modified enzyme catalyzed the conversion of thioanisoles to the corresponding sulfoxides, albeit with somewhat lower conversions than the free cofactor. More success was found by anchoring a BCN-derivatized Ru(Bpy)32+ complex into POP. (514) The conjugated enzyme catalyzed photoreductive cyclization of a dienone substrate with 72% conversion, compared to 36% with the free ruthenium complex. Similarly, [2 + 2] photocycloaddition of 4-methoxystyrene and cinnamoyl imidazole proceeded with 81% conversion using the conjugated enzyme but only 25% with the free complex. The same reaction could also be catalyzed using polypyridyl iridium complexes anchored within POP. (515) Artificial photoenzymes have also been developed by incorporating multiple metal complexes within a protein scaffold. Lee and Song developed an artificial dehalogenase in Mb by introducing a genetically encoded BpyAla to chelate a nickel cofactor and an iridium photocatalyst ligated to a Cys at position 45 (Figure 28D). (516) The resulting enzyme catalyzed a mixture of light-driven hydrolytic and reductive dehalogenation of 4′-iodoacetophenone (Figure 28C), achieving 86% conversion to the phenolic product and ca. 10:1 selectivity over the reduced side product. Reaction selectivity could be further improved through judicious placement of the BpyAla ligated nickel cofactor. In contrast, using the free metal complexes in solution gave substantially lower conversion and chemoselectivity.
6. Conclusions and Outlook
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The ability to introduce new functional elements into proteins as ncAA side chains has opened a wealth of opportunities in the field of biocatalysis. In this review, we have highlighted how an expanded alphabet of amino acids can be used to study enzyme mechanisms, (255,271,392) augment biocatalyst properties, (351,362,370,373,339,517) and create enzymes with new catalytic functions. (450,470) Moving forward, there are opportunities for new innovations to maximize the impact of ncAAs in biocatalysis research.
First, continued expansion of the genetic code to include a greater repertoire of functional amino acids will be key to unlocking new modes of catalysis within protein active sites. For example, the recent encoding of phosphine-containing ncAAs holds great promise for metalloenzyme design and engineering. (518) Similarly, a recent article highlights the potential of genetically encoded boronic acids as catalytic motifs. (498) The introduction of a wider range of ncAAs harboring photoresponsive elements should also allow the development of selective biocatalysts for diverse photochemical reactions. Such photoresponsive ncAAs can also be interfaced with modern structural biology techniques, such as X-ray free electron laser crystallography, to provide new insights into conformational changes that take place during enzyme catalysis. (159) At present, the application of genetic code reprograming in biocatalysis has largely focused on the introduction of one or multiple copies of a single ncAA. However, with advances in synthetic genomics and the discovery of mutually orthogonal aaRS-tRNA pairs it is now possible to selectively introduce multiple ncAAs into proteins. (116,117,519−523) In principle, these advances will greatly expand the range of active site arrangements accessible in proteins, which should allow the development of increasingly sophisticated catalytic mechanisms.
Second, to fully capitalize on our ability to embed new functional motifs into proteins, we require accelerated enzyme engineering pipelines to deliver ncAA-containing biocatalysts with the properties required for target applications. For example, the design and evolution of new enzymes that use ncAAs as key functional elements typically takes several years with existing workflows. One approach to speed up biocatalyst development is to integrate ncAA mutagenesis with ultrahigh throughput screening methods (e.g., fluorescence-activated droplet sorting (524)), which allow greater exploration of protein sequence space to accelerate directed evolution campaigns. To complement experimental engineering techniques, the latest deep-learning methods for protein design (6,7,525−527) and structure prediction (528) also hold enormous promise. These methods have allowed the accurate design of proteins that bind metal ions, small molecules and peptides, and have recently been used to design new and improved enzymes. (8) A crucial next step is to integrate ncAAs into these deep-learning frameworks, which should enable the rapid design of ncAA-containing enzymes with a high degree of accuracy.
Finally, in the coming years it is essential to transition ncAA-containing enzymes from academic laboratories into large-scale biocatalytic applications. Current barriers to translation include the limited efficiency of orthogonal translation components and suboptimal performance of common production strains for genetic code reprogramming applications. These limitations lead to reduced titers of ncAA-containing proteins and the requirement for large excesses of ncAAs supplemented to the culture media, which results in prohibitively high production costs. To overcome these challenges, more efficient orthogonal translation components are required that can operate effectively at low ncAA concentrations. The development of such systems should be achievable through multiple rounds of laboratory evolution to progressively improve performance, or through advanced techniques such as multiplex automated genome engineering, (122) phage-assisted continuous evolution, (120) tRNA display (119) and computational approaches. (529,530) A recent study has also shown that exploration of a wider range of PylRS homologues can dramatically improve the efficiency of encoding catalytically important ncAAs. (531) To further improve protein titers, engineered or synthetic production strains can be used that have been specifically tailored for efficient ncAA incorporation. (532−536) Another attractive option to reduce the costs of producing ncAA-containing proteins is developing engineered hosts that contain the necessary biosynthetic machinery to produce target ncAAs in vivo, (330,537−539) thus avoiding the need to supply expensive ncAAs to the culture media.
For the reasons outlined above, we are optimistic that genetic code reprogramming methodologies will become increasingly important tools in enzymology and biocatalysis in the coming years. By overcoming the constraints of the genetic code, enzyme designers and engineers can now begin to tackle a vast array of chemical transformations that were previously thought inaccessible to biocatalysis.
Author Information
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- Anthony P. Green - Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester M1 7DN, U.K.;
https://orcid.org/0000-0003-0454-1798;
- Zachary Birch-Price - Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester M1 7DN, U.K.;https://orcid.org/0000-0002-2024-3005
- Florence J. Hardy - Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester M1 7DN, U.K.;https://orcid.org/0000-0003-0671-0209
Z.B.-P., F.J.H., T.M.L., and A.R.K.: writing─original draft, reviewing, and editing. A.P.G.: writing─reviewing and editing, supervision. CRediT: Zachary Birch-Price writing-original draft, writing-review & editing; Florence J. Hardy writing-original draft, writing-review & editing; Thomas M. Lister writing-original draft, writing-review & editing; Anna R. Kohn writing-original draft, writing-review & editing; Anthony P. Green supervision, writing-original draft, writing-review & editing.
Z.B.-P. and F.J.H. contributed equally. CRediT: Zachary Birch-Price writing-original draft, writing-review & editing; Florence J. Hardy writing-original draft, writing-review & editing; Thomas M. Lister writing-original draft, writing-review & editing; Anna R. Kohn writing-original draft, writing-review & editing; Anthony P. Green supervision, writing-original draft, writing-review & editing.
T.M.L. and A.R.K. contributed equally. CRediT: Zachary Birch-Price writing-original draft, writing-review & editing; Florence J. Hardy writing-original draft, writing-review & editing; Thomas M. Lister writing-original draft, writing-review & editing; Anna R. Kohn writing-original draft, writing-review & editing; Anthony P. Green supervision, writing-original draft, writing-review & editing.
Biographies
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Zachary Birch-Price
Zachary Birch-Price received his MSci degree in Natural Sciences (Biochemistry) from the University of Cambridge in 2020, working under the supervision of Prof. Ben Luisi. He was then awarded a BBSRC DTP studentship and moved to the University of Manchester, where he is currently studying for his doctorate under the supervision of Prof. Anthony Green, focusing on enzyme engineering and ncAAs.
Florence J. Hardy
Dr. Florence J. Hardy received her MChem degree in 2016 under the supervision of Prof. Chris Schofield at the University of Oxford. She moved to the University of Manchester to work with Prof. Anthony Green where she completed a Ph.D. in 2022, on engineering metalloenzymes with ncAAs. Currently, Florence is a postdoctoral researcher at the University of Manchester working on computational enzyme design.
Thomas M. Lister
Thomas M. Lister received his MChem degree from the University of Bath in 2020. During this time, he completed a year-long industrial placement at GSK working in medicinal chemistry and a final year project under the supervision of Dr. Alexander J. Cresswell. He is currently a Ph.D. student on the iCAT CDT at the University of Manchester working under the supervision of Profs. Igor Larrosa and Anthony P. Green.
Anna R. Kohn
Anna R. Kohn received her MChem degree from the University of Manchester in 2022, completing her final year project under the supervision of Prof. Anthony Green. She has since continued in the Green group as a Ph.D. student, where she is currently engineering photoenzymes containing ncAAs.
Anthony P. Green
Following his Ph.D. in synthetic organic chemistry under the supervision of E. J. Thomas, Anthony carried out postdoctoral research with N. J. Turner and S. L. Flitsch based in the Manchester Institute of Biotechnology and subsequently with D. Hilvert at ETH Zurich. Anthony started his independent research career in 2016 based in the Manchester Institute of Biotechnology at the University of Manchester, where he is a professor of organic and biological chemistry. His research interests lie in the design, evolution, and characterization of enzymes with a new function.
Acknowledgments
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We acknowledge the European Research Council (ERC Starting Grant no. 757991 to A.P.G.), the Biotechnology and Biological Sciences Research Council (David Phillips Fellowship BB/M027023/1 to A.P.G. and grants BB/W014483/1 and BB/X000974/1), and the Human Frontier Science Program research grant (RGP0004/2022). Z.B.-P. was supported by a BBSRC Doctoral Training Partnership (BB/T008725/1). F.J.H. was supported by the EPSRC Doctoral Prize Fellowship (EP/W524347/1). T.M.L. was supported by an integrated catalysis Doctoral Training Program (EP/023755/1). A.R.K. was supported by the Future Biomanufacturing Hub (EP/S01778X/1) on an EPSRC Industrial CASE PhD studentship with GSK.
| aaRS | Aminoacyl tRNA synthetase |
| AFM | Atomic force microscopy |
| AKR | Aldehyde ketone reductase |
| APX2 | Ascorbate peroxidase, engineered |
| AsSec | Aeromonas salmonicida selenocysteine synthase |
| AzoR | Azoreductase |
| BCN | Bicyclo[6.1.0]nonyne |
| BODIPY | Boron-dipyrromethene |
| bPP | Bovine pancreatic polypeptide |
| cAA | Canonical amino acid |
| CalB | Candida antartica lipase B |
| CAP | Catabolite activator protein |
| CAT | Chloramphenicol acetyltransferase |
| CcP | Cytochrome c peroxidase |
| CDO | Cysteine dioxygenase |
| CFE | Cell-free expression |
| ChPylRS | Chimeric pyrrolysyl tRNA synthetase |
| CuAAC | Copper-catalyzed azide–alkyne coupling |
| CYP | Cytochrome P450 |
| DADP | Diacetyl deuteroporphyrin |
| ddNTP | 2′,3′-dideoxynucleotide triphosphate |
| DEER | Double electron–electron resonance |
| DET | Direct electron transfer |
| DKR | Diketoreductase |
| DQF-COSY | Double quantum filtered correlation spectroscopy |
| dr | Diastereomeric ratio |
| EcLeuRS | Escherichia coli leucyl tRNA synthetase |
| EDA | Ethyl diazoacetate |
| EDTA | Ethylenediaminetetraacetic acid |
| ee | Enantiomeric excess |
| ELP | Elastin-like polypeptide |
| EPL | Expressed protein ligation |
| EPR | Electron paramagnetic resonance |
| FDH | Formate dehydrogenase |
| FLuc | Firefly luciferase |
| GCE | Genetic code expansion |
| GFP | Green fluorescent protein |
| GST | Glutathione S-transferase |
| HAT | Hydrogen atom transfer |
| HCO | Heme copper oxidase |
| HRP | Horseradish peroxidase |
| HSQC | Heteronuclear single quantum coherence |
| IEDDA | Inverse-electron-demand Diels–Alder |
| ImGPS | Imidazole glycerol phosphate synthase |
| IR | Infrared spectroscopy |
| KlenTaq | DNA polymerase I from Thermus aquaticus |
| KSI | Ketosteroid isomerase |
| LaL | Lysozyme from bacteriophage λ |
| LmrR | Lactoccocal multidrug resistance regulator |
| ManA | Mannose-6-phosphate isomerase |
| Mb | Myoglobin |
| MBH | Morita–Baylis–Hillman |
| MbPylCKRS | Methanosarcina barkeri pyrrolysyl photocaged lysine tRNA synthetase |
| MbPylRS | Methanosarcina barkeri pyrrolysyl tRNA synthetase |
| MD | Molecular dynamics |
| MLDH | Malate dehydrogenase |
| MNDH | Mannitol dehydrogenase |
| mDHFR | Murine dihydrofolate reductase |
| MjTyrRS | Methanocaldococcus jannaschii tyrosyl tRNA synthetase |
| MmPylRS | Methanosarcina mazeii pyrrolysyl tRNA synthetase |
| MmSepRS | Methanococcus maripaludis phosphoseryl-tRNA synthetase |
| MS | Mass spectrometry |
| MTG | Microbial transglutaminase |
| mtRNAP | Mitochondrial RNA polymerase |
| ncAA | Noncanonical amino acid |
| NCL | Native chemical ligation |
| NiSOD | Nickel-dependent superoxide dismutase |
| NMR | Nuclear magnetic resonance |
| PCR | Polymerase chain reaction |
| pdCpA | 5′-phospho-2′-deoxyribocytidylriboadenosine |
| PEG | Polyethylene glycol |
| PET | Photoinduced electron transfer |
| PLA | Phospholipase A2 |
| PLuc | Photinus pyralis luciferase |
| POP | Prolyl oligopeptidase |
| POR | Protochlorophyllide oxidoreductase |
| PRMT1 | Protein arginine methyltransferase 1 |
| PTE | Phosphotriesterase |
| PTM | Post-translational modification |
| RLuc | Renilla luciferase |
| RNase | Ribonuclease |
| RNR | Ribonucleotide reductase |
| ROS | Reactive oxygen species |
| SAD | Single-wavelength anomalous diffraction |
| SAM | S-adenosylmethionine |
| SCS | Stop codon suppression |
| sfYFP | Superfolder yellow fluorescent protein |
| SHC | Squalene-hopene cyclase |
| SPAAC | Strain-promoted azide–alkyne coupling |
| SPI | Selective pressure incorporation |
| SPPS | Solid-phase peptide synthesis |
| sTCO | Strained trans-cyclooctene |
| T7RNAP | T7 RNA polymerase |
| tHisF | Thermotoga maritima synthase subunit of ImGPS |
| TMS | Trimethylsilyl |
| TOCSY | Total correlation spectroscopy |
| TR | Thioredoxin |
| TrpOx | Tryptophan oxidase |
| tsCA | Thermostable carbonic anhydrase II |
| TTL | Thermoanaerobacter thermohydrosulfuricus lipase |
| TTN | Total turnover number |
| TvNiR | Thioalkalivibrio nitratireducens cytochrome c nitrite reductase |
| VHR | Vaccinia H1-related |
| VSE | Vibrational Stark effect |
| WT | Wild type |
| XANES | X-ray absorption near edge structure |
| XFEL | X-ray free-electron laser |
| ZFN | Zinc finger nuclease |
References
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- 1Punekar, N. S. ENZYMES: Catalysis, Kinetics and Mechanisms; Springer Nature Singapore, 2018. DOI: 10.1007/978-981-13-0785-0Google ScholarThere is no corresponding record for this reference.
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- 3Wagner, C. R.; Benkovic, S. J. Site Directed Mutagenesis: a Tool for Enzyme Mechanism Dissection. Trends Biotechnol. 1990, 8, 263– 270, DOI: 10.1016/0167-7799(90)90189-5Google ScholarThere is no corresponding record for this reference.
- 4Arnold, F. H. Directed Evolution: Bringing New Chemistry to Life. Angew. Chem. Int. Ed. 2018, 57 (16), 4143– 4148, DOI: 10.1002/anie.201708408Google Scholar4Directed evolution: Bringing new chemistry to lifeArnold, Frances H.Angewandte Chemie, International Edition (2018), 57 (16), 4143-4148CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Directed evolution mimics evolution by artificial selection, and is accelerated in the lab. setting by focusing on individual genes expressed in fast-growing microorganisms. We start with existing proteins (sourced from Nature or engineered), introduce mutations, and then screen for the progeny proteins with enhanced activity (or other desirable traits). We use the improved enzymes as parents for the next round of mutation and screening, recombining beneficial mutations as needed, and continuing until we reach the target level of performance. Thus, the evolution of Nature's enzymes can lead to the discovery of new reactivity, transformations not known in biol., and reactivity inaccessible by small-mol. catalysis.
- 5Turner, N. J. Directed Evolution Drives the Next Generation of Biocatalysts. Nat. Chem. Biol. 2009, 5, 567– 573, DOI: 10.1038/nchembio.203Google Scholar5Directed evolution drives the next generation of biocatalystsTurner, Nicholas J.Nature Chemical Biology (2009), 5 (8), 567-573CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)A review. Enzymes are increasingly being used as biocatalysts in the generation of products that have until now been derived using traditional chem. processes. Such products range from pharmaceutical and agrochem. building blocks to fine and bulk chems. and, more recently, components of biofuels. For a biocatalyst to be effective in an industrial process, it must be subjected to improvement and optimization, and in this respect the directed evolution of enzymes has emerged as a powerful enabling technol. Directed evolution involves repeated rounds of (1) random gene library generation, (2) expression of genes in a suitable host, and (3) screening of libraries of variant enzymes for the property of interest. Both in vitro screening-based methods and in vivo selection-based methods have been applied to the evolution of enzyme function and properties. Significant developments have occurred recently, particularly with respect to library design, screening methodol., applications in synthetic transformations, and strategies for the generation of new enzyme function.
- 6Dauparas, J.; Anishchenko, I.; Bennett, N.; Bai, H.; Ragotte, R. J.; Milles, L. F.; Wicky, B. I. M.; Courbet, A.; de Haas, R. J.; Bethel, N. Robust Deep Learning-Based Protein Sequence Design Using ProteinMPNN. Science 2022, 378 (6615), 49– 56, DOI: 10.1126/science.add2187Google Scholar6Robust deep learning-based protein sequence design using ProteinMPNNDauparas, J.; Anishchenko, I.; Bennett, N.; Bai, H.; Ragotte, R. J.; Milles, L. F.; Wicky, B. I. M.; Courbet, A.; de Haas, R. J.; Bethel, N.; Leung, P. J. Y.; Huddy, T. F.; Pellock, S.; Tischer, D.; Chan, F.; Koepnick, B.; Nguyen, H.; Kang, A.; Sankaran, B.; Bera, A. K.; King, N. P.; Baker, D.Science (Washington, DC, United States) (2022), 378 (6615), 49-56CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Although deep learning has revolutionized protein structure prediction, almost all exptl. characterized de novo protein designs have been generated using phys. based approaches such as Rosetta. Here, we describe a deep learning-based protein sequence design method, ProteinMPNN, that has outstanding performance in both in silico and exptl. tests. On native protein backbones, ProteinMPNN has a sequence recovery of 52.4% compared with 32.9% for Rosetta. The amino acid sequence at different positions can be coupled between single or multiple chains, enabling application to a wide range of current protein design challenges. We demonstrate the broad utility and high accuracy of ProteinMPNN using x-ray crystallog., cryo-electron microscopy, and functional studies by rescuing previously failed designs, which were made using Rosetta or AlphaFold, of protein monomers, cyclic homo-oligomers, tetrahedral nanoparticles, and target-binding proteins.
- 7Watson, J. L.; Juergens, D.; Bennett, N. R.; Trippe, B. L.; Yim, J.; Eisenach, H. E.; Ahern, W.; Borst, A. J.; Ragotte, R. J.; Milles, L. F. De Novo Design of Protein Structure and Function with RFdiffusion. Nature 2023, 620, 1089– 1100, DOI: 10.1038/s41586-023-06415-8Google Scholar7De novo design of protein structure and function with RFdiffusionWatson, Joseph L.; Juergens, David; Bennett, Nathaniel R.; Trippe, Brian L.; Yim, Jason; Eisenach, Helen E.; Ahern, Woody; Borst, Andrew J.; Ragotte, Robert J.; Milles, Lukas F.; Wicky, Basile I. M.; Hanikel, Nikita; Pellock, Samuel J.; Courbet, Alexis; Sheffler, William; Wang, Jue; Venkatesh, Preetham; Sappington, Isaac; Torres, Susana Vazquez; Lauko, Anna; De Bortoli, Valentin; Mathieu, Emile; Ovchinnikov, Sergey; Barzilay, Regina; Jaakkola, Tommi S.; DiMaio, Frank; Baek, Minkyung; Baker, DavidNature (London, United Kingdom) (2023), 620 (7976), 1089-1100CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)There has been considerable recent progress in designing new proteins using deep-learning methods1-9. Despite this progress, a general deep-learning framework for protein design that enables soln. of a wide range of design challenges, including de novo binder design and design of higher-order sym. architectures, has yet to be described. Diffusion models10,11 have had considerable success in image and language generative modeling but limited success when applied to protein modeling, probably due to the complexity of protein backbone geometry and sequence-structure relationships. Here we show that by fine-tuning the RoseTTAFold structure prediction network on protein structure denoising tasks, we obtain a generative model of protein backbones that achieves outstanding performance on unconditional and topol.-constrained protein monomer design, protein binder design, sym. oligomer design, enzyme active site scaffolding and sym. motif scaffolding for therapeutic and metal-binding protein design. We demonstrate the power and generality of the method, called RoseTTAFold diffusion (RFdiffusion), by exptl. characterizing the structures and functions of hundreds of designed sym. assemblies, metal-binding proteins and protein binders. The accuracy of RFdiffusion is confirmed by the cryogenic electron microscopy structure of a designed binder in complex with influenza haemagglutinin that is nearly identical to the design model. In a manner analogous to networks that produce images from user-specified inputs, RFdiffusion enables the design of diverse functional proteins from simple mol. specifications.
- 8Sumida, K. H.; Núñez-Franco, R.; Kalvet, I.; Pellock, S. J.; Wicky, B. I. M.; Milles, L. F.; Dauparas, J.; Wang, J.; Kipnis, Y.; Jameson, N. Improving Protein Expression, Stability, and Function with ProteinMPNN. J. Am. Chem. Soc. 2024, 146 (3), 2054– 2061, DOI: 10.1021/jacs.3c10941Google ScholarThere is no corresponding record for this reference.
- 9Gargiulo, S.; Soumillion, P. Directed Evolution for Enzyme Development in Biocatalysis. Curr. Opin. Chem. Biol. 2021, 61, 107– 113, DOI: 10.1016/j.cbpa.2020.11.006Google Scholar9Directed evolution for enzyme development in biocatalysisGargiulo, Serena; Soumillion, PatriceCurrent Opinion in Chemical Biology (2021), 61 (), 107-113CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. As an important sector of the chem. industry, biocatalysis requires the continuous development of enzymes with tailor-made activity, selectivity, stability, or tolerance to unnatural environments. This is now routinely achieved by directed evolution based on iterative cycles of genetic diversification and activity screening. Here, we highlight its recent developments. First, the design of "smarter" libraries by focused mutagenesis may be a crucial start-up for a fast and successful outcome. Then library assembly and expression are also key steps that benefits from modern mol. biol. progresses. Finally, various strategies may be considered for library screening depending on the final objective: while low-throughput direct assays have been very successful in generating enzymes for important biocatalytic processes, even in bringing completely new chemistries to the enzyme world, ultrahigh-throughput screening methods are emerging as powerful approaches for engineering the next generation of industrial enzymes.
- 10Planas-Iglesias, J.; Marques, S. M.; Pinto, G. P.; Musil, M.; Stourac, J.; Damborsky, J.; Bednar, D. Computational Design of Enzymes for Biotechnological Applications. Biotechnol. Adv. 2021, 47, 107696, DOI: 10.1016/j.biotechadv.2021.107696Google Scholar10Computational design of enzymes for biotechnological applicationsPlanas-Iglesias, Joan; Marques, Sergio M.; Pinto, Gaspar P.; Musil, Milos; Stourac, Jan; Damborsky, Jiri; Bednar, DavidBiotechnology Advances (2021), 47 (), 107696CODEN: BIADDD; ISSN:0734-9750. (Elsevier Inc.)A review. Enzymes are the natural catalysts that execute biochem. reactions upholding life. Their natural effectiveness has been fine-tuned as a result of millions of years of natural evolution. Such catalytic effectiveness has prompted the use of biocatalysts from multiple sources on different applications, including the industrial prodn. of goods (food and beverages, detergents, textile, and pharmaceutics), environmental protection, and biomedical applications. Natural enzymes often need to be improved by protein engineering to optimize their function in non-native environments. Recent technol. advances have greatly facilitated this process by providing the exptl. approaches of directed evolution or by enabling computer-assisted applications. Directed evolution mimics the natural selection process in a highly accelerated fashion at the expense of arduous lab. work and economic resources. Theor. methods provide predictions and represent an attractive complement to such expts. by waiving their inherent costs. Computational techniques can be used to engineer enzymic reactivity, substrate specificity and ligand binding, access pathways and ligand transport, and global properties like protein stability, soly., and flexibility. Theor. approaches can also identify hotspots on the protein sequence for mutagenesis and predict suitable alternatives for selected positions with expected outcomes. This review covers the latest advances in computational methods for enzyme engineering and presents many successful case studies.
- 11Świderek, K.; Tuñón, I.; Moliner, V.; Bertran, J. Computational Strategies for the Design of New Enzymatic Functions. Arch. Biochem. Biophys. 2015, 582, 68– 79, DOI: 10.1016/j.abb.2015.03.013Google Scholar11Computational strategies for the design of new enzymatic functionsSwiderek, K.; Tunon, I.; Moliner, V.; Bertran, J.Archives of Biochemistry and Biophysics (2015), 582 (), 68-79CODEN: ABBIA4; ISSN:0003-9861. (Elsevier B.V.)A review. In this contribution, recent developments in the design of biocatalysts are reviewed with particular emphasis in the de novo strategy. Studies based on three different reactions, Kemp elimination, Diels-Alder and Retro-Aldolase, are used to illustrate different success achieved during the last years. Finally, a section is devoted to the particular case of designed metalloenzymes. As a general conclusion, the interplay between new and more sophisticated engineering protocols and computational methods, based on mol. dynamics simulations with Quantum Mechanics/Mol. Mechanics potentials and fully flexible models, seems to constitute the bed rock for present and future successful design strategies.
- 12Renata, H.; Wang, Z. J.; Arnold, F. H. Expanding the Enzyme Universe: Accessing Non-Natural Reactions by Mechanism-Guided Directed Evolution. Angew. Chem. Int. Ed. 2015, 54 (11), 3351– 3367, DOI: 10.1002/anie.201409470Google Scholar12Expanding the Enzyme Universe: Accessing Non-Natural Reactions by Mechanism-Guided Directed EvolutionRenata, Hans; Wang, Z. Jane; Arnold, Frances H.Angewandte Chemie, International Edition (2015), 54 (11), 3351-3367CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. High selectivity and exquisite control over the outcome of reactions entice chemists to use biocatalysts in org. synthesis. However, many useful reactions are not accessible because they are not in nature's known repertoire. In this Review, we outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progression has been recapitulated in the lab. starting from extant enzymes. We then examine non-native enzyme activities that have been exploited for chem. synthesis, with an emphasis on reactions that do not have natural counterparts. Non-natural activities can be improved by directed evolution, thus mimicking the process used by nature to create new catalysts. Finally, we describe the discovery of non-native catalytic functions that may provide future opportunities for the expansion of the enzyme universe.
- 13Pagar, A. D.; Patil, M. D.; Flood, D. T.; Yoo, T. H.; Dawson, P. E.; Yun, H. Recent Advances in Biocatalysis with Chemical Modification and Expanded Amino Acid Alphabet. Chem. Rev. 2021, 121 (10), 6173– 6245, DOI: 10.1021/acs.chemrev.0c01201Google Scholar13Recent Advances in Biocatalysis with Chemical Modification and Expanded Amino Acid AlphabetPagar, Amol D.; Patil, Mahesh D.; Flood, Dillon T.; Yoo, Tae Hyeon; Dawson, Philip E.; Yun, HyungdonChemical Reviews (Washington, DC, United States) (2021), 121 (10), 6173-6245CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. The two main strategies for enzyme engineering, directed evolution and rational design, have found widespread applications in improving the intrinsic activities of proteins. Although numerous advances have been achieved using these ground-breaking methods, the limited chem. diversity of the biopolymers, restricted to the 20 canonical amino acids, hampers creation of novel enzymes that Nature has never made thus far. To address this, much research has been devoted to expanding the protein sequence space via chem. modifications and/or incorporation of noncanonical amino acids (ncAAs). This review provides a balanced discussion and crit. evaluation of the applications, recent advances, and tech. breakthroughs in biocatalysis for three approaches: (i) chem. modification of cAAs, (ii) incorporation of ncAAs, and (iii) chem. modification of incorporated ncAAs. Furthermore, the applications of these approaches and the result on the functional properties and mechanistic study of the enzymes are extensively reviewed. We also discuss the design of artificial enzymes and directed evolution strategies for enzymes with ncAAs incorporated. Finally, we discuss the current challenges and future perspectives for biocatalysis using the expanded amino acid alphabet.
- 14Zhao, J.; Burke, A. J.; Green, A. P. Enzymes with Noncanonical Amino Acids. Curr. Opin. Chem. Biol. 2020, 55, 136– 144, DOI: 10.1016/j.cbpa.2020.01.006Google Scholar14Enzymes with noncanonical amino acidsZhao, Jingming; Burke, Ashleigh J.; Green, Anthony P.Current Opinion in Chemical Biology (2020), 55 (), 136-144CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. Enzyme design and engineering strategies rely almost exclusively on nature's alphabet of twenty canonical amino acids. Recent years have seen the emergence of powerful genetic code expansion methods that allow hundreds of structurally diverse amino acids to be installed into proteins in a site-selective manner. Here, we will highlight how the availability of an expanded alphabet of amino acids has opened new avenues in enzyme engineering research. Genetically encoded noncanonical amino acids have provided new tools to probe complex enzyme mechanisms, improve biocatalyst activity and stability, and most ambitiously to design enzymes with new catalytic mechanisms that would be difficult to access within the constraints of the genetic code. We anticipate that the studies highlighted in this article, coupled with the continuing advancements in genetic code expansion technol., will promote the widespread use of noncanonical amino acids in biocatalysis research in the coming years.
- 15Huang, Y.; Liu, T. Therapeutic Applications of Genetic Code Expansion. Synth. Syst. Biotechnol. 2018, 3 (3), 150– 158, DOI: 10.1016/j.synbio.2018.09.003Google Scholar15Therapeutic applications of genetic code expansionHuang Yujia; Liu TaoSynthetic and systems biotechnology (2018), 3 (3), 150-158 ISSN:.In nature, a limited, conservative set of amino acids are utilized to synthesize proteins. Genetic code expansion technique reassigns codons and incorporates noncanonical amino acids (ncAAs) through orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs. The past decade has witnessed the rapid growth in diversity and scope for therapeutic applications of this technology. Here, we provided an update on the recent progress using genetic code expansion in the following areas: antibody-drug conjugates (ADCs), bispecific antibodies (BsAb), immunotherapies, long-lasting protein therapeutics, biosynthesized peptides, engineered viruses and cells, as well as other therapeutic related applications, where the technique was used to elucidate the mechanisms of biotherapeutics and drug targets.
- 16Icking, L.-S.; Riedlberger, A. M.; Krause, F.; Widder, J.; Frederiksen; Anne, S.; Stockert, F.; Spädt, M.; Edel, N.; Armbruster, D.; Forlani, G. iNClusive: a Database Collecting Useful Information on Non-Canonical Amino Acids and Their Incorporation into Proteins for Easier Genetic Code Expansion Implementation. Nucleic Acids Res. 2024, 52, D476, DOI: 10.1093/nar/gkad1090Google ScholarThere is no corresponding record for this reference.
- 17Chatterjee, A.; Guo, J.; Lee, H. S.; Schultz, P. G. A Genetically Encoded Fluorescent Probe in Mammalian Cells. J. Am. Chem. Soc. 2013, 135 (34), 12540– 12543, DOI: 10.1021/ja4059553Google Scholar17A Genetically Encoded Fluorescent Probe in Mammalian CellsChatterjee, Abhishek; Guo, Jiantao; Lee, Hyun Soo; Schultz, Peter G.Journal of the American Chemical Society (2013), 135 (34), 12540-12543CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Fluorescent reporters are useful in vitro and in vivo probes of protein structure, function, and localization. Here we report that the fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), can be site-specifically incorporated into proteins in mammalian cells in response to the TAG codon with high efficiency using an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair. We further demonstrate that Anap can be used to image the subcellular localization of proteins in live mammalian cells. The small size of Anap, its environment-sensitive fluorescence, and the ability to introduce Anap at specific sites in the proteome by simple mutagenesis make it a unique and valuable tool in eukaryotic cell biol.
- 18Evans, H. T.; Benetatos, J.; van Roijen, M.; Bodea, L. G.; Götz, J. Decreased Synthesis of Ribosomal Proteins in Tauopathy Revealed by Non-Canonical Amino Acid Labelling. EMBO J. 2019, 38 (13), e101174 DOI: 10.15252/embj.2018101174Google ScholarThere is no corresponding record for this reference.
- 19Schultz, K. C.; Supekova, L.; Ryu, Y.; Xie, J.; Perera, R.; Schultz, P. G. A Genetically Encoded Infrared Probe. J. Am. Chem. Soc. 2006, 128 (43), 13984– 13985, DOI: 10.1021/ja0636690Google Scholar19A Genetically Encoded Infrared ProbeSchultz, Kathryn C.; Supekova, Lubica; Ryu, Youngha; Xie, Jianming; Perera, Roshan; Schultz, Peter G.Journal of the American Chemical Society (2006), 128 (43), 13984-13985CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)An orthogonal tRNA/aminoacyl-tRNA synthetase pair has been evolved that makes it possible to selectively and efficiently incorporate p-cyanophenylalanine (pCNPhe) into proteins in E. coli at sites specified by the amber nonsense codon, TAG. Substitution of pCNPhe for histidine-64 in myoglobin (Mb) affords a sensitive vibrational probe of ligand binding. This methodol. provides a useful IR reporter of protein structure, biomol. interactions, and conformational changes.
- 20Goettig, P.; Koch, N. G.; Budisa, N. Non-Canonical Amino Acids in Analyses of Protease Structure and Function. Int. J. Mol. Sci. 2023, 24 (18), 14035, DOI: 10.3390/ijms241814035Google ScholarThere is no corresponding record for this reference.
- 21Tinzl, M.; Hilvert, D. Trapping Transient Protein Species by Genetic Code Expansion. ChemBioChem 2021, 22 (1), 92– 99, DOI: 10.1002/cbic.202000523Google Scholar21Trapping Transient Protein Species by Genetic Code ExpansionTinzl, Matthias; Hilvert, DonaldChemBioChem (2021), 22 (1), 92-99CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Nature employs a limited no. of genetically encoded amino acids for the construction of functional proteins. By engineering components of the cellular translation machinery, however, it is now possible to genetically encode noncanonical building blocks with tailored electronic and structural properties. The ability to incorporate unique chem. functionality into proteins provides a powerful tool to probe mechanism and create novel function. In this minireview, we highlight several recent studies that illustrate how noncanonical amino acids have been used to capture and characterize reactive intermediates, fine-tune the catalytic properties of enzymes, and stabilize short-lived protein-protein complexes.
- 22Birch-Price, Z.; Taylor, C. J.; Ortmayer, M.; Green, A. P. Engineering Enzyme Activity Using an Expanded Amino Acid Alphabet. Protein Eng. Des. Sel. 2023, 36, gzac013, DOI: 10.1093/protein/gzac013Google ScholarThere is no corresponding record for this reference.
- 23Giger, S.; Buller, R. Advances in Noncanonical Amino Acid Incorporation for Enzyme Engineering Applications. CHIMIA 2023, 77 (6), 395– 402, DOI: 10.2533/chimia.2023.395Google ScholarThere is no corresponding record for this reference.
- 24Hayashi, T.; Hilvert, D.; Green, A. P. Engineered Metalloenzymes with Non-Canonical Coordination Environments. Chem. Eur. J. 2018, 24 (46), 11821– 11830, DOI: 10.1002/chem.201800975Google ScholarThere is no corresponding record for this reference.
- 25Lugtenburg, T.; Gran-Scheuch, A.; Drienovská, I. Non-Canonical Amino Acids as a Tool for the Thermal Stabilization of Enzymes. Protein Eng. Des. Sel. 2023, 36, gzad003, DOI: 10.1093/protein/gzad003Google ScholarThere is no corresponding record for this reference.
- 26Mirts, E. N.; Bhagi-Damodaran, A.; Lu, Y. Understanding and Modulating Metalloenzymes with Unnatural Amino Acids, Non-Native Metal Ions, and Non-Native Metallocofactors. Acc. Chem. Res. 2019, 52 (4), 935– 944, DOI: 10.1021/acs.accounts.9b00011Google Scholar26Understanding and Modulating Metalloenzymes with Unnatural Amino Acids, Non-Native Metal Ions, and Non-Native MetallocofactorsMirts, Evan N.; Bhagi-Damodaran, Ambika; Lu, YiAccounts of Chemical Research (2019), 52 (4), 935-944CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. Metalloproteins set the gold std. for performing important functions, including catalyzing demanding reactions under mild conditions. Designing artificial metalloenzymes (ArMs) to catalyze abiol. reactions has been a major endeavor for many years, but most ArMs' activities are far below those of native enzymes, making them unsuitable for most pratical applications. A crit. step to advance the field is to fundamentally understand what it takes to not only confer but also fine-tune ArM activities so they match native enzymes. Indeed, only once we can freely modulate ArM activity to rival (or surpass!) natural enzymes can the potential of ArMs be fully realized. A key to unlocking ArM potential is the observation that one metal primary coordination sphere (PCS) can display a range of functions and levels of activity, leading to the realization that secondary coordination sphere (SCS) interactions are critically important. However, SCS interactions are numerous, long-range, and weak, making them very difficult to reproduce in ArMs. Furthermore, natural enzymes are tied to a small set of biol. available functional moieties from canonical amino acids and the physiol. available metal ions and metallocofactors, severely limiting the chem. space available to probe and tune ArMs. In this Account, we summarize our group's use of unnatural amino acids (UAAs) and non-native metal ions and metallocofactors to probe and modulate ArM functions. We incorporated isostructural UAAs in a type 1 copper (T1Cu) protein azurin to provide conclusive evidence that the axial ligand hydrophobicity is a major determinant of T1Cu redunction potential (E'°). We also probed the role of protein backbone interactions that cannot be altered by std. mutagenesis by replacing the peptide bond with an ester linkage. We used insight gained from these studies to tune the E'° of azurin across the entire physiol. range, the broadest range ever achieved in a single metalloprotein. Introducing UAA analogs of Tyr into ArM models of heme-copper oxidase (HCO) revealed a linear relationship between pKa, E'°, and activity. We have also substituted non-native hemes and non-native metal ions for their native equiv. in these models to resolve several issues that were intractable in native HCOs and the closely related nitric oxide reductases (NOR), such as their roles in modulating substrate affinity, ET rate, and activity. We have incorporated abiol. cofactors such as ferrocene and Mn(salen) into azurin and myoglobin, resp., to stabilize these inorg. and organometallic compds. in water, confer abiol. functions, tune their E'° and activity through SCS interactions, and show that the approach to metallocofactor anchoring and orientation can tune enantioselectivity and alter function. Replacing Cu in azurin with non-native Fe or Ni can impart novel activities, such as superoxide redn. and C-C bond formation. While progress has been made, we have identified only a small fraction of the interactions that can be generally applied to ArMs to fine-tune their functions. Because SCS interactions are subtle and heavily interconnected, it has been difficult to characterize their effects quant. It is vital to develop spectroscopic and computational techniques to detect and quantify their effects in both resting states and catalytic intermediates.
- 27Drienovská, I.; Roelfes, G. Expanding the Enzyme Universe with Genetically Encoded Unnatural Amino Acids. Nat. Catal. 2020, 3 (3), 193– 202, DOI: 10.1038/s41929-019-0410-8Google Scholar27Expanding the enzyme universe with genetically encoded unnatural amino acidsDrienovska, Ivana; Roelfes, GerardNature Catalysis (2020), 3 (3), 193-202CODEN: NCAACP; ISSN:2520-1158. (Nature Research)A review. The emergence of robust methods to expand the genetic code allows incorporation of non-canonical amino acids into the polypeptide chain of proteins, thus making it possible to introduce unnatural chem. functionalities in enzymes. In this Perspective, we show how this powerful methodol. is used to create enzymes with improved and novel, even new-to-nature, catalytic activities. We provide an overview of the current state of the art, and discuss the potential benefits of developing and using enzymes with genetically encoded non-canonical amino acids compared with enzymes contg. only canonical amino acids.
- 28Trimble, J. S.; Crawshaw, R.; Hardy, F. J.; Levy, C. W.; Brown, M. J. B.; Fuerst, D. E.; Heyes, D. J.; Obexer, R.; Green, A. P. A Designed Photoenzyme for Enantioselective [2 + 2] Cycloadditions. Nature 2022, 611 (7937), 709– 714, DOI: 10.1038/s41586-022-05335-3Google Scholar28A designed photoenzyme for enantioselective [2+2] cycloadditionsTrimble, Jonathan S.; Crawshaw, Rebecca; Hardy, Florence J.; Levy, Colin W.; Brown, Murray J. B.; Fuerst, Douglas E.; Heyes, Derren J.; Obexer, Richard; Green, Anthony P.Nature (London, United Kingdom) (2022), 611 (7937), 709-714CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)The ability to program new modes of catalysis into proteins would allow the development of enzyme families with functions beyond those found in nature. To this end, genetic code expansion methodol. holds particular promise, as it allows the site-selective introduction of new functional elements into proteins as noncanonical amino acid side chains1-4. Here we exploit an expanded genetic code to develop a photoenzyme that operates by means of triplet energy transfer (EnT) catalysis, a versatile mode of reactivity in org. synthesis that is not accessible to biocatalysis at present5-12. Installation of a genetically encoded photosensitizer into the beta-propeller scaffold of DA_20_00 (ref. 13) converts a de novo Diels-Alderase into a photoenzyme for [2+2] cycloaddns. (EnT1.0). Subsequent development and implementation of a platform for photoenzyme evolution afforded an efficient and enantioselective enzyme (EnT1.3, up to 99% enantiomeric excess (e.e.)) that can promote intramol. and bimol. cycloaddns., including transformations that have proved challenging to achieve selectively with small-mol. catalysts. EnT1.3 performs >300 turnovers and, in contrast to small-mol. photocatalysts, can operate effectively under aerobic conditions and at ambient temps. An X-ray crystal structure of an EnT1.3-product complex shows how multiple functional components work in synergy to promote efficient and selective photocatalysis. This study opens up a wealth of new excited-state chem. in protein active sites and establishes the framework for developing a new generation of enantioselective photocatalysts.
- 29Boutureira, O.; Bernardes, G. J. L. Advances in Chemical Protein Modification. Chem. Rev. 2015, 115 (5), 2174– 2195, DOI: 10.1021/cr500399pGoogle Scholar29Advances in Chemical Protein ModificationBoutureira, Omar; Bernardes, Goncalo J. L.Chemical Reviews (Washington, DC, United States) (2015), 115 (5), 2174-2195CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Transition metal-free and -mediated approaches are covered.
- 30Pfleiderer, G. Chemical Modification of Proteins. In Review Articles, including those from an International Conference held in Bielefeld, FR of Germany, June 1–2, 1984 , 2021; Walter de Gruyter GmbH & Co KG, p 207. DOI: 10.1515/9783112393048-011Google ScholarThere is no corresponding record for this reference.
- 31Gunnoo, S. B.; Madder, A. Chemical Protein Modification through Cysteine. ChemBioChem 2016, 17 (7), 529– 553, DOI: 10.1002/cbic.201500667Google Scholar31Chemical Protein Modification through CysteineGunnoo, Smita B.; Madder, AnnemiekeChemBioChem (2016), 17 (7), 529-553CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)The modification of proteins with non-protein entities is important for a wealth of applications, and methods for chem. modifying proteins attract considerable attention. Generally, modification is desired at a single site to maintain homogeneity and to minimise loss of function. Though protein modification can be achieved by targeting some natural amino acid side chains, this often leads to ill-defined and randomly modified proteins. Amongst the natural amino acids, cysteine combines advantageous properties contributing to its suitability for site-selective modification, including a unique nucleophilicity, and a low natural abundance-both allowing chemo- and regioselectivity. Native cysteine residues can be targeted, or Cys can be introduced at a desired site in a protein by means of reliable genetic engineering techniques. This review on chem. protein modification through cysteine should appeal to those interested in modifying proteins for a range of applications.
- 32Bischak, C. G.; Longhi, S.; Snead, D. M.; Costanzo, S.; Terrer, E.; Londergan, C. H. Probing Structural Transitions in the Intrinsically Disordered C-Terminal Domain of the Measles Virus Nucleoprotein by Vibrational Spectroscopy of Cyanylated Cysteines. Biophys. J. 2010, 99 (5), 1676– 1683, DOI: 10.1016/j.bpj.2010.06.060Google Scholar32Probing Structural Transitions in the Intrinsically Disordered C-Terminal Domain of the Measles Virus Nucleoprotein by Vibrational Spectroscopy of Cyanylated CysteinesBischak, Connor G.; Longhi, Sonia; Snead, David M.; Costanzo, Stephanie; Terrer, Elodie; Londergan, Casey H.Biophysical Journal (2010), 99 (5), 1676-1683CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)Four single-cysteine variants of the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) were cyanylated at cysteine and their IR spectra in the C≡N stretching region were recorded both in the absence and in the presence of one of the physiol. partners of NTAIL, namely the C-terminal X domain (XD) of the viral phosphoprotein. Consistent with previous studies showing that XD triggers a disorder-to-order transition within NTAIL, the C≡N stretching bands of the IR probe were found to be significantly affected by XD, with this effect being position-dependent. When the cyanylated cysteine side chain is solvent-exposed throughout the structural transition, its changing linewidth reflects a local gain of structure. When the probe becomes partially buried due to binding, its frequency reports on the mean hydrophobicity of the microenvironment surrounding the labeled side chain of the bound form. The probe moiety is small compared to other common covalently attached spectroscopic probes, thereby minimizing possible steric hindrance/perturbation at the binding interface. These results show for the first time to our knowledge the suitability of site-specific cysteine mutagenesis followed by cyanylation and IR spectroscopy to document structural transitions occurring within intrinsically disordered regions, with regions involved in binding and folding being identifiable at the residue level.
- 33Fafarman, A. T.; Webb, L. J.; Chuang, J. I.; Boxer, S. G. Site-Specific Conversion of Cysteine Thiols into Thiocyanate Creates an IR Probe for Electric Fields in Proteins. J. Am. Chem. Soc. 2006, 128 (41), 13356– 13357, DOI: 10.1021/ja0650403Google Scholar33Site-Specific Conversion of Cysteine Thiols into Thiocyanate Creates an IR Probe for Electric Fields in ProteinsFafarman, Aaron T.; Webb, Lauren J.; Chuang, Jessica I.; Boxer, Steven G.Journal of the American Chemical Society (2006), 128 (41), 13356-13357CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The nitrile stretching mode of the thiocyanate moiety is a nearly ideal probe for measuring the local elec. field arising from the organized environment of the interior of a protein. Nitriles were introduced into three proteins: RNase S, human aldose reductase (hALR2), and the reaction center (RC) of Rhodobacter capsulatus, through a facile synthetic scheme for the transformation of cysteine residues into thiocyanatoalanine. Vibrational Stark effect spectroscopy and Fourier transform IR spectroscopy on the modified proteins demonstrated that thiocyanate residues are a highly general tool for probing electrostatic fields in proteins.
- 34Weeks, C. L.; Jo, H.; Kier, B.; DeGrado, W. F.; Spiro, T. G. Cysteine-Linked Aromatic Nitriles as UV Resonance Raman Probes of Protein Structure. J. Raman Spectrosc. 2012, 43 (9), 1244– 1249, DOI: 10.1002/jrs.3167Google ScholarThere is no corresponding record for this reference.
- 35Boutureira, O.; Bernardes, G. J.; D’Hooge, F.; Davis, B. G. Direct Radiolabelling of Proteins at Cysteine Using [18 F]-Fluorosugars. Chem. Commun. 2011, 47 (36), 10010– 10012, DOI: 10.1039/c1cc13524dGoogle ScholarThere is no corresponding record for this reference.
- 36Kaiser, E. T.; Lawrence, D. S. Chemical Mutation of Enzyme Active Sites. Science 1984, 226 (4674), 505– 511, DOI: 10.1126/science.6238407Google ScholarThere is no corresponding record for this reference.
- 37Levine, H. L.; Kaiser, E. Oxidation of Dihydronicotinamides by Flavopapain. J. Am. Chem. Soc. 1978, 100 (24), 7670– 7677, DOI: 10.1021/ja00492a040Google ScholarThere is no corresponding record for this reference.
- 38Levine, H. L.; Nakagawa, Y.; Kaiser, E. Flavopapain: Synthesis and Properties of Semi-Synthetic Enzymes. Biochem. Biophys. Res. Commun. 1977, 76 (1), 64– 70, DOI: 10.1016/0006-291X(77)91668-0Google ScholarThere is no corresponding record for this reference.
- 39Mayer, C.; Gillingham, D. G.; Ward, T. R.; Hilvert, D. An Artificial Metalloenzyme for Olefin Metathesis. Chem. Commun. 2011, 47 (44), 12068– 12070, DOI: 10.1039/c1cc15005gGoogle Scholar39An artificial metalloenzyme for olefin metathesisMayer, Clemens; Gillingham, Dennis G.; Ward, Thomas R.; Hilvert, DonaldChemical Communications (Cambridge, United Kingdom) (2011), 47 (44), 12068-12070CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)A Grubbs-Hoveyda type olefin metathesis catalyst, equipped with an electrophilic bromoacetamide group, was used to modify a cysteine-contg. variant of a small heat shock protein from Methanocaldococcus jannaschii. The resulting artificial metalloenzyme was found to be active under acidic conditions in a benchmark ring closing metathesis reaction.
- 40den Heeten, R.; Muñoz, B. K.; Popa, G.; Laan, W.; Kamer, P. C. J. Synthesis of Hybrid Transition-Metalloproteins via Thiol-Selective Covalent Anchoring of Rh-Phosphine and Ru-Phenanthroline Complexes. Dalton Trans. 2010, 39 (36), 8477– 8483, DOI: 10.1039/c0dt00239aGoogle ScholarThere is no corresponding record for this reference.
- 41Deuss, P. J.; Popa, G.; Botting, C. H.; Laan, W.; Kamer, P. C. J. Highly Efficient and Site-Selective Phosphane Modification of Proteins through Hydrazone Linkage: Development of Artificial Metalloenzymes. Angew. Chem. Int. Ed. 2010, 49 (31), 5315– 5317, DOI: 10.1002/anie.201002174Google ScholarThere is no corresponding record for this reference.
- 42Jarvis, A. G.; Obrecht, L.; Deuss, P. J.; Laan, W.; Gibson, E. K.; Wells, P. P.; Kamer, P. C. J. Enzyme Activity by Design: An Artificial Rhodium Hydroformylase for Linear Aldehydes. Angew. Chem. Int. Ed. 2017, 56 (44), 13596– 13600, DOI: 10.1002/anie.201705753Google ScholarThere is no corresponding record for this reference.
- 43Merrifield, R. B. Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J. Am. Chem. Soc. 1963, 85 (14), 2149– 2154, DOI: 10.1021/ja00897a025Google Scholar43Solid phase peptide synthesis. I. The synthesis of a tetrapeptideMerrifield, R. B.Journal of the American Chemical Society (1963), 85 (14), 2149-54CODEN: JACSAT; ISSN:0002-7863.A new approach to the chem. synthesis of polypeptides was investigated. It involved the stepwise addition of protected amino acids to a growing peptide chain which was bound by a covalent bond to a solid resin particle. This provided a procedure whereby reagents anti by-products were removed by filtration, and the recrystn. of intermediates was eliminated. The advantages of the new method were speed and simplicity of operation. The feasibility of the idea was demonstrated by the synthesis of the model tetrapeptide L-leucyl-L-alanylglycyl-L-valine. The peptide was identical with a sample prepd. by the standard p-nitrophenyl ester procedure.
- 44Eckert, D. M.; Malashkevich, V. N.; Hong, L. H.; Carr, P. A.; Kim, P. S. Inhibiting HIV-1 Entry: Discovery of D-Peptide Inhibitors that Target the gp41 Coiled-Coil Pocket. Cell 1999, 99 (1), 103– 115, DOI: 10.1016/S0092-8674(00)80066-5Google Scholar44Inhibiting HIV-1 entry: discovery of D-peptide inhibitors that target the gp41 coiled-coil pocketEckert, Debra M.; Malashkevich, Vladimir N.; Hong, Lily H.; Carr, Peter A.; Kim, Peter S.Cell (Cambridge, Massachusetts) (1999), 99 (1), 103-115CODEN: CELLB5; ISSN:0092-8674. (Cell Press)The HIV-1 gp41 protein promotes viral entry by mediating the fusion of viral and cellular membranes. A prominent pocket on the surface of a central trimeric coiled coil within gp41 was previously identified as a potential target for drugs that inhibit HIV-1 entry. The authors designed a peptide, IQN17, which properly presents this pocket. Utilizing IQN17 and mirror-image phage display, the authors identified cyclic, D-peptide inhibitors of HIV-1 infection that share a sequence motif. A 1.5 Å cocrystal structure of IQN17 in complex with a D-peptide, and NMR studies, show that conserved residues of these inhibitors make intimate contact with the gp41 pocket. The authors studies validate the pocket per se as a target for drug development. IQN17 and these D-peptide inhibitors are likely to be useful for development and identification of a new class of orally bioavailable anti-HIV drugs.
- 45Kent, S.; Sohma, Y.; Liu, S.; Bang, D.; Pentelute, B.; Mandal, K. Through The Looking Glass - a New World of Proteins Enabled by Chemical Synthesis. J. Pept. Sci. 2012, 18 (7), 428– 436, DOI: 10.1002/psc.2421Google ScholarThere is no corresponding record for this reference.
- 46Schumacher, T. N. M.; Mayr, L. M.; Minor, D. L.; Milhollen, M. A.; Burgess, M. W.; Kim, P. S. Identification of D-Peptide Ligands Through Mirror-Image Phage Display. Science 1996, 271 (5257), 1854– 1857, DOI: 10.1126/science.271.5257.1854Google Scholar46Identification of D-peptide ligands through mirror-image phage displaySchumacher, Ton N. M.; Mayr, Lorenz M.; Minor, Daniel L., Jr.; Milhollen, Michael A.; Burgess, Michael W.; Kim, Peter S.Science (Washington, D. C.) (1996), 271 (5257), 1854-7CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Genetically encoded libraries of peptides and oligonucleotides are well suited for the identification of ligands for many macromols. A major drawback of these techniques is that the resultant ligands are subject to degrdn. by naturally occurring enzymes. Here, a method is described that uses a biol. encoded library for the identification of D-peptide ligands, which should be resistant to proteolytic degrdn. In this approach, a protein is synthesized in the D-amino acid configuration and used to select peptides from a phage display library expressing random L-amino acid peptides. For reasons of sym., the mirror images of these phage-displayed peptides interact with the target protein of the natural handedness. The value of this approach was demonstrated by the identification of a cyclic D-peptide partially overlaps the site for the physiol. ligands of this domain.
- 47Pech, A.; Achenbach, J.; Jahnz, M.; Schülzchen, S.; Jarosch, F.; Bordusa, F.; Klussmann, S. A Thermostable D-Polymerase for Mirror-Image PCR. Nucleic Acids Res. 2017, 45 (7), 3997– 4005, DOI: 10.1093/nar/gkx079Google Scholar47A thermostable D-polymerase for mirror-image PCRPech, Andreas; Achenbach, John; Jahnz, Michael; Schulzchen, Simone; Jarosch, Florian; Bordusa, Frank; Klussmann, SvenNucleic Acids Research (2017), 45 (7), 3997-4005CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)Biol. evolution resulted in a homochiral world in which nucleic acids consist exclusively of Dnucleotides and proteins made by ribosomal translation of L-amino acids. From the perspective of synthetic biol., however, particularly anabolic enzymes that could build the mirror-image counterparts of biol. macromols. such as L-DNA or L-RNA are lacking. Based on a convergent synthesis strategy, we have chem. produced and characterized a thermostable mirror-image polymerase that efficiently replicates and amplifies mirror-image (L)-DNA. This artificial enzyme, dubbed D-Dpo4-3C, is a mutant of Sulfolobus solfataricus DNA polymerase IV consisting of 352 D-amino acids. D-Dpo4-3C was reliably deployed in classical polymerase chain reactions (PCR) and it was used to assemble a first mirror-image gene coding for the protein Sso7d. We believe that this D-polymerase provides a valuable tool to further investigate the mysteries of biol. (homo) chirali ty and to pave the way for potential novel life forms running on a mirror-image genome.
- 48Jackson, D. Y.; Burnier, J.; Quan, C.; Stanley, M.; Tom, J.; Wells, J. A. A Designed Peptide Ligase for Total Synthesis of Ribonuclease A with Unnatural Catalytic Residues. Science 1994, 266 (5183), 243– 247, DOI: 10.1126/science.7939659Google ScholarThere is no corresponding record for this reference.
- 49Kaiser, E. Synthetic Approaches to Biologically Active Peptides and Proteins Including Enzymes. Acc. Chem. Res. 1989, 22 (2), 47– 54, DOI: 10.1021/ar00158a001Google ScholarThere is no corresponding record for this reference.
- 50Schnölzer, M.; Kent, S. B. Constructing Proteins by Dovetailing Unprotected Synthetic Peptides: Backbone-Engineered HIV Protease. Science 1992, 256 (5054), 221– 225, DOI: 10.1126/science.1566069Google Scholar50Constructing proteins by dovetailing unprotected synthetic peptides: backbone-engineered HIV proteaseSchnolzer M; Kent S BScience (New York, N.Y.) (1992), 256 (5054), 221-5 ISSN:0036-8075.Backbone-engineered HIV-1 protease was prepared by a total chemical synthesis approach that combines the act of joining two peptides with the generation of an analog structure. Unprotected synthetic peptide segments corresponding to the two halves of the HIV-1 protease monomer polypeptide chain were joined cleanly and in high yield through unique mutually reactive functional groups, one on each segment. Ligation was performed in 6 molar guanidine hydrochloride, thus circumventing limited solubility of protected peptide segments, the principal problem of the classical approach to the chemical synthesis of proteins. The resulting fully active HIV-1 protease analog contained a thioester replacement for the natural peptide bond between Gly51-Gly52 in each of the two active site flaps, a region known to be highly sensitive to mutational changes of amino acid side chains.
- 51Bode, J. W. Chemical Protein Synthesis with the α-Ketoacid-Hydroxylamine Ligation. Acc. Chem. Res. 2017, 50 (9), 2104– 2115, DOI: 10.1021/acs.accounts.7b00277Google Scholar51Chemical Protein Synthesis with the α-Ketoacid-Hydroxylamine LigationBode, Jeffrey W.Accounts of Chemical Research (2017), 50 (9), 2104-2115CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. The coupling of an α-ketoacid and a hydroxylamine (KAHA ligation) affords amide bonds under aq., acidic conditions without the need for protecting groups or coupling agents. Translating this finding into a general approach to chem. protein synthesis required the identification of methods to incorporate the key functional groups into unprotected peptide segments-ideally using well-established Fmoc solid-phase peptide synthesis protocols. A decade of effort has now led to robust, convenient methods for prepg. peptides bearing free or masked C-terminal α-ketoacids and N-terminal hydroxylamines. The facile synthesis of the segments and the aq., acidic conditions of the KAHA ligation make it ideal for the construction of small proteins (up to 200 residues), including SUMO and related modifier proteins, betatrophin and other protein hormones, nitrophorin 4, S100A4, and the cyclic protein AS-48.Key to the successful development of this protein synthesis platform was the identification and gram-scale synthesis of (S)-5-oxaproline. This hydroxylamine monomer is completely stable toward std. methods and practices of solid-phase peptide synthesis while still performing very well in the KAHA ligation. This reaction partner-in contrast to all others examd.-affords esters rather than amides as the primary ligation product. The resulting depsipeptides often offer superior soly. and handling and have been key in the chem. synthesis of hydrophobic and amphiphilic proteins. Upon facile O-to-N acyl shift, peptides bearing a noncanonical homoserine residue at the ligation site are formed. With proper choice of the ligation site, the incorporation of this unnatural amino acid does not appear to affect the structure or biol. activity of the protein targets. The development of the chem. methods for prepg. and masking peptide α-ketoacids and hydroxyalmines, the prepn. of several protein targets by convergent ligation strategies, and the synthesis of new hydroxylamine monomers affording either natural or unnatural residues at the ligation site are discussed. By operation under acidic conditions and with a distinct preference for the ligation site, these efforts establish KAHA ligation as a complementary method to the venerable native chem. ligation (NCL) for chem. protein synthesis. This Account documents both the state of the KAHA ligation and the challenges in identifying, inventing, and optimizing new reactions and building blocks needed to interface KAHA ligation with Fmoc solid-phase peptide chem. With these challenges largely addressed, peptide segments ready for ligation are formed directly upon resin cleavage, facilitating rapid assembly of four to five segments into proteins. This work sets the stage for applications of the KAHA ligation to chem. biol. and protein therapeutics.
- 52Liu, H.; Li, X. Serine/Threonine Ligation: Origin, Mechanistic Aspects, and Applications. Acc. Chem. Res. 2018, 51 (7), 1643– 1655, DOI: 10.1021/acs.accounts.8b00151Google Scholar52Serine/Threonine Ligation: Origin, Mechanistic Aspects, and ApplicationsLiu, Han; Li, XuechenAccounts of Chemical Research (2018), 51 (7), 1643-1655CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)Review. Synthetic proteins are expected to go beyond the boundary of recombinant DNA expression systems, by being flexibly installed with the site-specific natural or unnatural modification structures along synthesis. To enable protein chem. synthesis, peptide ligations provide effective strategies to assemble short peptide fragments obtained from solid phase peptide synthesis (SPPS) into long peptides and proteins. In this regard, chemoselective peptide ligation represents a simple but powerful transformation realizing selective amide formation between the C-terminus and N-terminus of two side-chain unprotected peptide fragments. These reactions are highly chemo- and regioselective to tolerate the side-chain functionalities present on the unprotected peptides, highly reactive to work with mM or sub-mM concn. of the substrates, and operationally simple with mild conditions and accessible building blocks. This Account focuses on our work in the development of serine/threonine ligation (STL), which originates from a chemoselective reaction between an unprotected peptide with the C-terminal salicylaldehyde ester and another unprotected peptide with N-terminal serine or threonine residues. Mechanistically, STL involves imine capture, 5-endo-trig ring-chain tautomerization, O-to-N [1,5] acyl transfer to afford the N,O-benzylidene acetal linked peptide, followed by acidolysis to regenerate the Xaa-Ser/Thr linkage (Xaa is the amino acid) at the ligation site. The high abundance of serine and threonine residues (12.7%) in naturally occurring proteins and good compatibility of STL with variable C-terminal residues provide multiple choices for ligation sites. The requisite peptide C-terminal salicylaldehyde (SAL) esters can be prepd. from the peptide fragments obtained from both Fmoc-SPPS and Boc-SPPS through four available methods (safety catch strategy based on phenolysis, direct coupling, ozonolysis and n+1 strategy). In the synthesis of proteins (e.g., ACYP enzyme, MUC1 glycopeptide 40-mer to 80-mer, interleukin 25 and HMGA1a with variable post-translational modification patterns), both C-to-N and N-to-C sequential STL strategies have been developed, through selection of temporal N-terminal protecting groups and proper design of the switch-on/off C-terminal SAL ester surrogate, resp. In the synthesis of cyclic peptide natural products (e.g., daptomycin, teixobactin, cyclomontanin B, yunnanin C) and their analogs, the intramol. head-to-tail STL has been implemented on linear peptide SAL ester precursors contg. four to ten amino acid residues, with good efficiency and minimized oligomerization. As a thiol-independent chemoselective ligation complementary to native chem. ligation (NCL), STL provides an alternative tool to chem. synthesize homogeneous proteins with site specific and structure defined modifications and cyclic peptide natural products, which lays foundation for chem. biol. and medicinal study on those mols. with biol. importance and therapeutic potentials.
- 53Muir, T. W. Semisynthesis of Proteins by Expressed Protein Ligation. Annu. Rev. Biochem. 2003, 72 (1), 249– 289, DOI: 10.1146/annurev.biochem.72.121801.161900Google ScholarThere is no corresponding record for this reference.
- 54Antos, J. M.; Truttmann, M. C.; Ploegh, H. L. Recent Advances in Sortase-Catalyzed Ligation Methodology. Curr. Opin. Struct. Biol. 2016, 38, 111– 118, DOI: 10.1016/j.sbi.2016.05.021Google Scholar54Recent advances in sortase-catalyzed ligation methodologyAntos, John M.; Truttmann, Matthias C.; Ploegh, Hidde L.Current Opinion in Structural Biology (2016), 38 (), 111-118CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. The transpeptidation reaction catalyzed by bacterial sortases continues to see increasing use in the construction of novel protein derivs. In addn. to growth in the no. of applications that rely on sortase, this field has also seen methodol. improvements that enhance reaction performance and scope. In this opinion, we present an overview of key developments in the practise and implementation of sortase-based strategies, including applications relevant to structural biol. Topics include the use of engineered sortases to increase reaction rates, the use of redesigned acyl donors and acceptors to mitigate reaction reversibility, and strategies for expanding the range of substrates that are compatible with a sortase-based approach.
- 55Chen, S.-Y.; Cressman, S.; Mao, F.; Shao, H.; Low, D. W.; Beilan, H. S.; Cagle, E. N.; Carnevali, M.; Gueriguian, V.; Keogh, P. J. Synthetic Erythropoietic Proteins: Tuning Biological Performance by Site-Specific Polymer Attachment. Chem. Biol. 2005, 12 (3), 371– 383, DOI: 10.1016/j.chembiol.2005.01.017Google Scholar55Synthetic erythropoietic proteins: tuning biological performance by site-specific polymer attachmentChen, Shiah-Yun; Cressman, Sonya; Mao, Feng; Shao, Haiyan; Low, Donald W.; Beilan, Hal S.; Cagle, E. Neil; Carnevali, Maia; Gueriguian, Vincent; Keogh, Peter J.; Porter, Heather; Stratton, Stephen M.; Wiedeke, M. Con; Savatski, Laura; Adamson, John W.; Bozzini, Carlos E.; Kung, Ada; Kent, Stephen B. H.; Bradburne, James A.; Kochendoerfer, Gerd G.Chemistry & Biology (2005), 12 (3), 371-383CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)Chem. synthesis in combination with precision polymer modification allows the systematic exploration of the effect of protein properties, such as charge and hydrodynamic radius, on potency using defined, homogeneous conjugates. A series of polymer-modified synthetic erythropoiesis proteins were constructed that had a polypeptide chain similar to the amino acid sequence of human erythropoietin but differed significantly in the no. and type of attached polymers. The analogs differed in charge from +5 to -26 at neutral pH and varied in mol. wt. from 30 to 54 kDa. All were active in an in vitro cell proliferation assay. However, in vivo potency was found to be strongly dependent on overall charge and size. The trends obsd. in this study may serve as starting points for the construction of more potent synthetic EPO analogs in the future.
- 56Kochendoerfer, G. G.; Chen, S.-Y.; Mao, F.; Cressman, S.; Traviglia, S.; Shao, H.; Hunter, C. L.; Low, D. W.; Cagle, E. N.; Carnevali, M. Design and Chemical Synthesis of a Homogeneous Polymer-Modified Erythropoiesis Protein. Science 2003, 299 (5608), 884– 887, DOI: 10.1126/science.1079085Google Scholar56Design and Chemical Synthesis of a Homogeneous Polymer-Modified Erythropoiesis ProteinKochendoerfer, Gerd G.; Chen, Shiah-Yun; Mao, Feng; Cressman, Sonya; Traviglia, Stacey; Shao, Haiyan; Hunter, Christie L.; Low, Donald W.; Cagle, E. Neil; Carnevali, Maia; Gueriguian, Vincent; Keogh, Peter J.; Porter, Heather; Stratton, Stephen M.; Wiedeke, M. Con; Wilken, Jill; Tang, Jie; Levy, Jay J.; Miranda, Les P.; Crnogorac, Milan M.; Kalbag, Suresh; Botti, Paolo; Schindler-Horvat, Janice; Savatski, Laura; Adamson, John W.; Kung, Ada; Kent, Stephen B. H.; Bradburne, James A.Science (Washington, DC, United States) (2003), 299 (5608), 884-887CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The authors report the design and total chem. synthesis of "synthetic erythropoiesis protein" (SEP), a 51-kilodalton protein-polymer construct consisting of a 166-amino-acid polypeptide chain and two covalently attached, branched, and monodisperse polymer moieties that are neg. charged. The ability to control the chem. allowed the authors to synthesize a macromol. of precisely defined covalent structure. SEP was homogeneous as shown by high-resoln. anal. techniques, with a mass of 50,825 ±10 daltons by electrospray mass spectrometry, and with a pI of 5.0. In cell and animal assays for erythropoiesis, SEP displayed potent biol. activity and had significantly prolonged duration of action in vivo. These chem. methods are a powerful tool in the rational design of protein constructs with potential therapeutic applications.
- 57Kent, S. B. H. Bringing the Science of Proteins into the Realm of Organic Chemistry: Total Chemical Synthesis of SEP (Synthetic Erythropoiesis Protein). Angew. Chem. Int. Ed. 2013, 52 (46), 11988– 11996, DOI: 10.1002/anie.201304116Google ScholarThere is no corresponding record for this reference.
- 58Liu, S.; Pentelute, B. L.; Kent, S. B. H. Convergent Chemical Synthesis of [Lysine24, 38, 83] Human Erythropoietin. Angew. Chem. Int. Ed. 2012, 51 (4), 993– 999, DOI: 10.1002/anie.201106060Google ScholarThere is no corresponding record for this reference.
- 59Baca, M.; Kent, S. B. Catalytic Contribution of Flap-Substrate Hydrogen Bonds in ″HIV-1 Protease″ Explored by Chemical Synthesis. Proc. Natl. Acad. Sci. U.S.A. 1993, 90 (24), 11638– 11642, DOI: 10.1073/pnas.90.24.11638Google ScholarThere is no corresponding record for this reference.
- 60Beadle, J. D.; Knuhtsen, A.; Hoose, A.; Raubo, P.; Jamieson, A. G.; Shipman, M. Solid-Phase Synthesis of Oxetane Modified Peptides. Org. Lett. 2017, 19 (12), 3303– 3306, DOI: 10.1021/acs.orglett.7b01466Google ScholarThere is no corresponding record for this reference.
- 61Abdildinova, A.; Kurth, M. J.; Gong, Y.-D. Solid-Phase Synthesis of Peptidomimetics with Peptide Backbone Modifications. Asian J. Org. Chem. 2021, 10 (9), 2300– 2317, DOI: 10.1002/ajoc.202100264Google Scholar61Solid-phase Synthesis of Peptidomimetics with Peptide Backbone ModificationsAbdildinova, Aizhan; Kurth, Mark J.; Gong, Young-DaeAsian Journal of Organic Chemistry (2021), 10 (9), 2300-2317CODEN: AJOCC7; ISSN:2193-5807. (Wiley-VCH Verlag GmbH & Co. KGaA)Peptidomimetics are a class of compds. with promising pharmacol. properties. Peptidomimetics reduce limitations of peptides including low bioavailability, poor stability, and poor cell-permeability. Peptide backbone modifications are a frequently used manipulation to reach desirable properties of the peptide mols. The development of accessible synthetic methodologies plays an important role in peptidomimetic progress. Synthesis of peptidomimetics proceeds via soln. and solid-phase synthesis strategies. Solid-phase org. synthesis serves as a powerful tool for the prepn. of peptidomimetic mols., thus, numerous strategies have been developed over the years. In this review, we discuss solid-phase synthetic approaches for peptide backbone modifications that were presented in the last two decades.
- 62Flavell, R. R.; Muir, T. W. Expressed Protein Ligation (EPL) in the Study of Signal Transduction, Ion Conduction, And Chromatin Biology. Acc. Chem. Res. 2009, 42 (1), 107– 116, DOI: 10.1021/ar800129cGoogle Scholar62Expressed protein ligation (EPL) in the study of signal transduction, ion conduction, and chromatin biologyFlavell, Robert R.; Muir, Tom W.Accounts of Chemical Research (2009), 42 (1), 107-116CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. EPL is a semisynthetic technique in which a recombinant protein thioester, generated by thiolysis of an intein fusion protein, is reacted with a synthetic or recombinant peptide with an N-terminal cysteine to produce a native peptide bond. This method has been used extensively for the incorporation of biophys. probes, unnatural amino acids, and post-translational modifications in proteins. In the 10 years since this technique was developed, the applications of EPL to studying protein structure and function have grown ever more sophisticated. In this account, we review the use of EPL in selected systems in which substantial mechanistic insights have recently been gained through the use of the semisynthetic protein derivs. EPL has been used in many studies to unravel the complexity of signaling networks and subcellular trafficking. Herein, we highlight this application to 2 different systems. First, we describe how phosphorylated or otherwise modified proteins in the TGF-β signaling network were prepd. and how they were applied to understanding the complexities of this pathway, from receptor activation to nuclear import. Second, Rab-GTPases are multiply modified with lipid derivs., and EPL-based techniques were used to incorporate these modifications, allowing for the elucidation of the biophys. basis of membrane assocn. and dissocn. We also review the use of EPL to understand the biol. of 2 other systems, the potassium channel KcsA and histones. EPL was used to incorporate D-alanine and an amide-to-ester backbone modification in the selectivity filter of the KcsA potassium channel, providing insight into the mechanism of selectivity in ion conduction. In the case of histones, which are among the most heavily post-translationally modified proteins, the modifications play a key role in the regulation of gene transcription and chromatin structure. We describe how native chem. ligation and EPL were used to generate acetylated, phosphorylated, methylated, and ubiquitylated histones and how these modified histones were used to interrogate chromatin biol. Collectively, these studies demonstrate the utility of EPL in protein science. These techniques and concepts are applicable to many other systems, and ongoing advances promise to extend this semisynthetic technique to increasingly complex biol. problems.
- 63Valiyaveetil, F. I.; Leonetti, M.; Muir, T. W.; MacKinnon, R. Ion Selectivity in a Semisynthetic K+ Channel Locked in the Conductive Conformation. Science 2006, 314 (5801), 1004– 1007, DOI: 10.1126/science.1133415Google ScholarThere is no corresponding record for this reference.
- 64Vázquez, M. E.; Nitz, M.; Stehn, J.; Yaffe, M. B.; Imperiali, B. Fluorescent Caged Phosphoserine Peptides as Probes to Investigate Phosphorylation-Dependent Protein Associations. J. Am. Chem. Soc. 2003, 125 (34), 10150– 10151, DOI: 10.1021/ja0351847Google Scholar64Fluorescent caged phosphoserine peptides as probes to investigate phosphorylation-dependent protein associationsVazquez, M. Eugenio; Nitz, Mark; Stehn, Justine; Yaffe, Michael B.; Imperiali, BarbaraJournal of the American Chemical Society (2003), 125 (34), 10150-10151CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The development of chem. probes for the investigation of the complex phosphorylation signaling cascades that regulate biol. events is crucial to understanding these processes. We describe herein a bifunctional probe that enables spatial and temporal release of a biol. active ligand while allowing simultaneous monitoring of its binding to the protein of interest. Substitution of Tyr(-2) for the environmentally sensitive fluorescent amino acid DANA in the sequence RLYRpSLPA which is known to bind the 14-3-3 protein does not adversely affect binding affinity and allows monitoring of the binding process. The binding of the peptide to 14-3-3 places the fluorescent reporter unit into a hydrophobic pocket, which changes the fluorescent max. emission intensity and wavelength. At the same time, the newly developed photolabile 1-(2-nitrophenyl)ethyl-caged phosphoserine allows control of the release of the biol. active ligand through unmasking of the key phosphoserine functionality upon UV irradn.
- 65Müller, M. M.; Kries, H.; Csuhai, E.; Kast, P.; Hilvert, D. Design, Selection, and Characterization of a Split Chorismate Mutase. Protein Sci. 2010, 19 (5), 1000– 1010, DOI: 10.1002/pro.377Google ScholarThere is no corresponding record for this reference.
- 66Choi, Y.; Shin, S. H.; Jung, H.; Kwon, O.; Seo, J. K.; Kee, J.-M. Specific Fluorescent Probe for Protein Histidine Phosphatase Activity. ACS sensors 2019, 4 (4), 1055– 1062, DOI: 10.1021/acssensors.9b00242Google ScholarThere is no corresponding record for this reference.
- 67Sainlos, M.; Imperiali, B. Tools For Investigating Peptide-Protein Interactions: Peptide Incorporation of Environment-Sensitive Fluorophores Through SPPS-Based ’Building Block’ Approach. Nat. Protoc. 2007, 2 (12), 3210– 3218, DOI: 10.1038/nprot.2007.443Google ScholarThere is no corresponding record for this reference.
- 68Wu, Y.; Tam, W.-S.; Chau, H.-F.; Kaur, S.; Thor, W.; Aik, W. S.; Chan, W.-L.; Zweckstetter, M.; Wong, K.-L. Solid-Phase Fluorescent BODIPY-Peptide Synthesis via in situ Dipyrrin Construction. Chem. Sci. 2020, 11 (41), 11266– 11273, DOI: 10.1039/D0SC04849FGoogle ScholarThere is no corresponding record for this reference.
- 69Kienhöfer, A.; Kast, P.; Hilvert, D. Selective Stabilization of the Chorismate Mutase Transition State by a Positively Charged Hydrogen Bond Donor. J. Am. Chem. Soc. 2003, 125 (11), 3206– 3207, DOI: 10.1021/ja0341992Google Scholar69Selective stabilization of the chorismate mutase transition state by a positively charged hydrogen bond donorKienhofer Alexander; Kast Peter; Hilvert DonaldJournal of the American Chemical Society (2003), 125 (11), 3206-7 ISSN:0002-7863.Citrulline was incorporated via chemical semisynthesis at position 90 in the active site of the AroH chorismate mutase from Bacillus subtilis. The wild-type arginine at this position makes hydrogen-bonding interactions with the ether oxygen of chorismate. Replacement of the positively charged guanidinium group with the isosteric but neutral urea has a dramatic effect on the ability of the enzyme to convert chorismate into prephenate. The Arg90Cit variant exhibits a >104-fold decrease in the catalytic rate constant kcat with a 2.7-fold increase in the Michaelis constant Km. In contrast, its affinity for a conformationally constrained inhibitor molecule that effectively mimics the geometry but not the dissociative character of the transition state is only reduced by a factor of approximately 6. These results show that an active site merely complementary to the reactive conformation of chorismate is insufficient for catalysis of the mutase reaction. Instead, electrostatic stabilization of the polarized transition state by provision of a cationic hydrogen bond donor proximal to the oxygen in the breaking C-O bond is essential for high catalytic efficiency.
- 70Roy, R. S.; Imperiali, B. Pyridoxamine-Amino Acid Chimeras in Semisynthetic Aminotransferase Mimics. Protein eng. 1997, 10 (6), 691– 698, DOI: 10.1093/protein/10.6.691Google ScholarThere is no corresponding record for this reference.
- 71Lopez, G.; Anderson, J. C. Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a BL21 (DE3) Biosafety Strain. ACS Synth. Biol. 2015, 4 (12), 1279– 1286, DOI: 10.1021/acssynbio.5b00085Google ScholarThere is no corresponding record for this reference.
- 72Budisa, N. Amino Acids and Codons - Code Organization and Protein Structure. Engineering the Genetic Code 2005, 66– 89, DOI: 10.1002/3527607188.ch4Google ScholarThere is no corresponding record for this reference.
- 73Budisa, N.; Steipe, B.; Demange, P.; Eckerskorn, C.; Kellermann, J.; Huber, R. High-Level Biosynthetic Substitution of Methionine in Proteins by Its Analogs 2-Aminohexanoic Acid, Selenomethionine, Telluromethionine and Ethionine in Escherichia coli. Eur. J. Biochem. 1995, 230 (2), 788– 796, DOI: 10.1111/j.1432-1033.1995.0788h.xGoogle ScholarThere is no corresponding record for this reference.
- 74Cowie, D. B.; Cohen, G. N. Biosynthesis by Escherichia coli of Active Altered Proteins Containing Selenium Instead of Sulfur. Biochim. Biophys. Acta 1957, 26 (2), 252– 261, DOI: 10.1016/0006-3002(57)90003-3Google ScholarThere is no corresponding record for this reference.
- 75Wong, J. Membership Mutation of the Genetic Code: Loss of Fitness by Tryptophan. Proc. Natl. Acad. Sci. U.S.A. 1983, 80 (20), 6303– 6306, DOI: 10.1073/pnas.80.20.6303Google Scholar75Membership mutation of the genetic code: Loss of fitness by tryptophanWong, J. Tze FeiProceedings of the National Academy of Sciences of the United States of America (1983), 80 (20), 6303-6CODEN: PNASA6; ISSN:0027-8424.Bacillus subtilis Strain QB928, a tryptophan auxotroph, was serially mutated to yield strain HR15. For QB928, tryptophan functioned as a competent amino acid and 4-fluorotryptophan as merely an inferior analog. For HR15, these roles were reversed. The tryptophan/4-fluorotryptophan growth ratio decreased by a factor of 2 × 104 in the transition from QB928 to HR15.
- 76Dóring, V.; Mootz, H. D.; Nangle, L. A.; Hendrickson, T. L.; de Crécy-Lagard, V.; Schimmel, P.; Marliere, P. Enlarging the Amino Acid Set of Escherichia coli by Infiltration of the Valine Coding Pathway. Science 2001, 292 (5516), 501– 504, DOI: 10.1126/science.1057718Google ScholarThere is no corresponding record for this reference.
- 77Datta, D.; Wang, P.; Carrico, I. S.; Mayo, S. L.; Tirrell, D. A. A Designed Phenylalanyl-tRNA Synthetase Variant Allows Efficient in Vivo Incorporation of Aryl Ketone Functionality into Proteins. J. Am. Chem. Soc. 2002, 124 (20), 5652– 5653, DOI: 10.1021/ja0177096Google Scholar77A Designed Phenylalanyl-tRNA Synthetase Variant Allows Efficient in Vivo Incorporation of Aryl Ketone Functionality into ProteinsDatta, Deepshikha; Wang, Pin; Carrico, Isaac S.; Mayo, Stephen L.; Tirrell, David A.Journal of the American Chemical Society (2002), 124 (20), 5652-5653CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Incorporation of non-natural amino acids into proteins in vivo expands the scope of protein synthesis and design. P-Acetylphenylalanine was incorporated into recombinant dihydrofolate reductase (DHFR) in Escherichia coli via a computationally designed mutant form of the phenylalanyl-tRNA synthetase of the host. DHFR outfitted with ketone functionality can be chemoselectively ligated with hydrazide reagents under mild conditions.
- 78Sharma, N.; Furter, R.; Kast, P.; Tirrell, D. A. Efficient Introduction of Aryl Bromide Functionality into Proteins in vivo. FEBS Lett. 2000, 467 (1), 37– 40, DOI: 10.1016/S0014-5793(00)01120-0Google Scholar78Efficient introduction of aryl bromide functionality into proteins in vivoSharma, N.; Furter, R.; Kast, P.; Tirrell, D. A.FEBS Letters (2000), 467 (1), 37-40CODEN: FEBLAL; ISSN:0014-5793. (Elsevier Science B.V.)Artificial proteins can be engineered to exhibit interesting solid state, liq. crystal or interfacial properties and may ultimately serve as important alternatives to conventional polymeric materials. The utility of protein-based materials is limited, however, by the availability of just the 20 amino acids that are normally recognized and utilized by biol. systems; many desirable functional groups cannot be incorporated directly into proteins by biosynthetic means. In this study, we incorporate para-bromophenylalanine (p-Br-phe) into a model target protein, mouse dihydrofolate reductase (DHFR), by using a bacterial phenylalanyl-tRNA synthetase (PheRS) variant with relaxed substrate specificity. Coexpression of the mutant PheRS and DHFR in a phenylalanine auxotrophic Escherichia coli host strain grown in p-Br-phe-supplemented minimal medium resulted in 88% replacement of phenylalanine residues by p-Br-phe; variation in the relative amts. of phe and p-Br-phe in the medium allows control of the degree of substitution by the analog. Protein expression yields of 20-25 mg/l were obtained from cultures supplemented with p-Br-phe; this corresponds to about two-thirds of the expression levels characteristic of cultures supplemented with phe. The aryl bromide function is stable under the conditions used to purify DHFR and creates new opportunities for post-translational derivatization of brominated proteins via metal-catalyzed coupling reactions. In addn., bromination may be useful in X-ray studies of proteins via the multiwavelength anomalous diffraction (MAD) technique.
- 79Kiick, K. L.; van Hest, J. C.; Tirrell, D. A. Expanding the Scope of Protein Biosynthesis by Altering the Methionyl-tRNA Synthetase Activity of a Bacterial Expression Host. Angew. Chem. Int. Ed. 2000, 39 (12), 2148– 2152, DOI: 10.1002/1521-3773(20000616)39:12<2148::AID-ANIE2148>3.0.CO;2-7Google ScholarThere is no corresponding record for this reference.
- 80Hendrickson, W. A.; Horton, J. R.; LeMaster, D. M. Selenomethionyl Proteins Produced for Analysis by Multiwavelength Anomalous Diffraction (MAD): a Vehicle for Direct Determination of Three-Dimensional Structure. EMBO J. 1990, 9 (5), 1665– 1672, DOI: 10.1002/j.1460-2075.1990.tb08287.xGoogle Scholar80Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structureHendrickson, Wayne A.; Horton, John R.; LeMaster, David M.EMBO Journal (1990), 9 (5), 1665-72CODEN: EMJODG; ISSN:0261-4189.An expression system has been established for the incorporation of selenomethionine into recombinant proteins produced from plasmids in Escherichia coli. Replacement of methionine by selenomethionine is demonstrated at the level of 100% for both T4 and E. coli thioredoxins. The natural recombinant proteins and the selenomethionyl variants of both thioredoxins crystallize isomorphously. Anomalous scattering factors were deduced from synchrotron x-ray absorption measurements of crystals of the selenomethionyl proteins. Taken with ref. to experience in the structural anal. of selenobiotinyl streptavidin by the method of MAD, these data indicate that recombinant selenomethionyl proteins analyzed by MAD phasing offer a rather general means for the elucidation of at. structures.
- 81Boles, J. O.; Lewinski, K.; Kunkle, M.; Odom, J. D.; Dunlap, R. B.; Lebioda, L.; Hatada, M. Bio-Incorporation of Telluromethionine into Buried Residues of Dihydrofolate Reductase. Nat. Struct. Biol. 1994, 1 (5), 283– 284, DOI: 10.1038/nsb0594-283Google ScholarThere is no corresponding record for this reference.
- 82Strub, M. P.; Hoh, F.; Sanchez, J. F.; Strub, J. M.; Böck, A.; Aumelas, A.; Dumas, C. Selenomethionine and Selenocysteine Double Labeling Strategy for Crystallographic Phasing. Structure 2003, 11 (11), 1359– 1367, DOI: 10.1016/j.str.2003.09.014Google ScholarThere is no corresponding record for this reference.
- 83Budisa, N.; Karnbrock, W.; Steinbacher, S.; Humm, A.; Prade, L.; Neuefeind, T.; Moroder, L.; Huber, R. Bioincorporation of Telluromethionine into Proteins: A Promising New Approach for X-Ray Structure Analysis of Proteins. J. Mol. Biol. 1997, 270 (4), 616– 623, DOI: 10.1006/jmbi.1997.1132Google ScholarThere is no corresponding record for this reference.
- 84Bae, J. H.; Alefelder, S.; Kaiser, J. T.; Friedrich, R.; Moroder, L.; Huber, R.; Budisa, N. Incorporation of β-Selenolo[3,2-b]Pyrrolyl-Alanine into Proteins for Phase Determination in Protein X-Ray Crystallography. J. Mol. Biol. 2001, 309 (4), 925– 936, DOI: 10.1006/jmbi.2001.4699Google ScholarThere is no corresponding record for this reference.
- 85Minks, C.; Huber, R.; Moroder, L.; Budisa, N. Atomic Mutations at the Single Tryptophan Residue of Human Recombinant Annexin V: Effects on Structure, Stability, and Activity. Biochemistry 1999, 38 (33), 10649– 10659, DOI: 10.1021/bi990580gGoogle Scholar85Atomic mutations at the single tryptophan residue of human recombinant annexin V: Effects on structure, stability, and activityMinks, Caroline; Huber, Robert; Moroder, Luis; Budisa, NediljkoBiochemistry (1999), 38 (33), 10649-10659CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The single tryptophan residue (Trp187) of human recombinant annexin V, contg. 320 residues and 5328 atoms, was replaced with three different isosteric analogs where hydrogen atoms at positions 4, 5, and 6 in the indole ring were exchanged with fluorine. Such single atom exchanges of H → F represent at. mutations that result in slightly increased covalent bond lengths and inverted polarities in the residue side-chain structure. These minimal changes in the local geometry do not affect the secondary and tertiary structures of the mutants, which were identical to those of wild-type protein in the crystal form. But the mutants exhibit significant differences in stability, folding cooperativity, biol. activity, and fluorescence properties if compared to the wild-type protein. These rather large global effects, resulting from the minimal local changes, have to be attributed either to the relatively strong changes in polar interactions of the indole ring or to differences in the van der Waals radii or to a combination of both facts. The changes in local geometry that are below resoln. of protein X-ray crystallog. studies are probably of secondary importance in comparison to the strong electronegativity introduced by the fluorine atom. Correspondingly, these types of mutations provide an interesting approach to study cooperative functions of integrated residues and modulation of particular physicochem. properties, in the present case of electronegativity, in a uniquely structured and hierarchically organized protein mol.
- 86Renner, C.; Alefelder, S.; Bae, J. H.; Budisa, N.; Huber, R.; Moroder, L. Fluoroprolines as Tools for Protein Design and Engineering. Angew. Chem. Int. Ed. 2001, 40 (5), 923– 925, DOI: 10.1002/1521-3773(20010302)40:5<923::AID-ANIE923>3.0.CO;2-#Google Scholar86Fluoroprolines as tools for protein design and engineeringRenner, Christian; Alefelder, Stefan; Bae, Jae H.; Budisa, Nediljko; Huber, Robert; Moroder, LuisAngewandte Chemie, International Edition (2001), 40 (5), 923-925CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)The preference of the peptidyl-fluoroproline amide bond for the cis or trans conformation in the model compds. N-acetyl-4-fluoroproline Me esters fully correlates with the thermostability of the related mutants of the model protein barstar. Thus, the (4S)-L-FPro mutants show a higher and the(4R)-L-FPro mutants a lower thermal stability than barstar.
- 87Tang, Y.; Ghirlanda, G.; Petka, W. A.; Nakajima, T.; DeGrado, W. F.; Tirrell, D. A. Fluorinated Coiled-Coil Proteins Prepared in Vivo Display Enhanced Thermal and Chemical Stability. Angew. Chem. Int. Ed. 2001, 40 (8), 1494– 1496, DOI: 10.1002/1521-3773(20010417)40:8<1494::AID-ANIE1494>3.0.CO;2-XGoogle Scholar87Fluorinated coiled-coil proteins prepared in vivo display enhanced thermal and chemical stabilityTang, Yi; Ghirlanda, Giovanna; Petka, Wendy A.; Nakajima, Tadashi; DeGrado, William F.; Tirrell, David A.Angewandte Chemie, International Edition (2001), 40 (8), 1494-1496CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)In this article the authors present a general approach to the stabilization of leucine-zipper peptides and coiled-coil proteins by incorporation of the hyperhydrophobic leucine isostere trifluoroleucine.
- 88Tang, Y.; Tirrell, D. A. Biosynthesis of a Highly Stable Coiled-Coil Protein Containing Hexafluoroleucine in an Engineered Bacterial Host. J. Am. Chem. Soc. 2001, 123 (44), 11089– 11090, DOI: 10.1021/ja016652kGoogle Scholar88Biosynthesis of a highly stable coiled-coil protein containing hexafluoroleucine in an engineered bacterial hostTang, Yi; Tirrell, David A.Journal of the American Chemical Society (2001), 123 (44), 11089-11090CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Modification of leucyl-tRNA synthetase (LeuRS) of the host is reported to allow efficient incorporation of hexafluoroleucine (I) into recombinant proteins prepd. in Escherichia coli. The E. coli leuS gene and its endogenous promoter were amplified from genomic DNA and ligated into the expression vector pQEA1 to yield pA1EL; the LeuRS activity of the new strains was approx. 8-fold higher than that of pQEA1-carrying strains and LeuRS was overexpressed. Only the strain carrying pQEA1 supported protein synthesis with I. The compn. and phys. properties of the I-contg. recombinant protein were detd. following affinity chromatog. The yield was 8 mg/L with 74% replacement of leucine by I. The secondary structure was >90% α-helical, and the predominant structure was dimeric. The free energy of unfolding was elevated 3.7 kcal over the leucine-contg. protein and the protein showed remarkable resistance to urea denaturation.
- 89Wang, P.; Tang, Y.; Tirrell, D. A. Incorporation of Trifluoroisoleucine into Proteins in Vivo. J. Am. Chem. Soc. 2003, 125 (23), 6900– 6906, DOI: 10.1021/ja0298287Google Scholar89Incorporation of Trifluoroisoleucine into Proteins in VivoWang, Pin; Tang, Yi; Tirrell, David A.Journal of the American Chemical Society (2003), 125 (23), 6900-6906CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Two fluorinated derivs. of isoleucine: D,L-2-amino-3-trifluoromethyl pentanoic acid (3TFI, 2) and D,L-2-amino-5,5,5-trifluoro-3-Me pentanoic acid (5TFI, 3) were prepd. 5TFI was incorporated into a model target protein, murine dihydrofolate reductase (mDHFR), in an isoleucine auxotrophic Escherichia coli host strain suspended in 5TFI-supplemented minimal medium depleted of isoleucine. Incorporation of 5TFI was confirmed by tryptic peptide anal. and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) of the protein product. Amino acid anal. showed that more than 93% of the encoded isoleucine residues were replaced by 5TFI. Measurement of the rate of activation of 5TFI by the E. coli isoleucyl-tRNA synthetase (IleRS) yielded a specificity const. (kcat/Km) 134-fold lower than that for isoleucine. 5TFI was successfully introduced into the cytokine murine interleukin-2 (mIL-2) at the encoded isoleucine positions. The concn. of fluorinated protein that elicits 50% of the maximal proliferative response is 3.87 ng/mL, about 30% higher than that of wild-type mIL-2 (EC50 = 2.70 ng/mL). The maximal responses are equiv. for the fluorinated and wild-type cytokines, indicating that fluorinated proteins can fold into stable and functional structures. 3TFI yielded no evidence for in vivo incorporation into recombinant proteins, and no evidence for activation by IleRS in vitro.
- 90Montclare, J. K.; Tirrell, D. A. Evolving Proteins of Novel Composition. Angew. Chem. Int. Ed. 2006, 45 (27), 4518– 4521, DOI: 10.1002/anie.200600088Google Scholar90Evolving proteins of novel compositionMontclare, Jin Kim; Tirrell, David A.Angewandte Chemie, International Edition (2006), 45 (27), 4518-4521CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Changing its nature: Global incorporation of noncanonical amino acids can alter the behavior of proteins in useful ways. In some cases, however, replacement of natural amino acids by noncanonical analogs can cause loss of protein stability. After several generations, functional proteins of non-natural compn. were prepd. through residue-specific incorporation combined with directed evolution.
- 91Hyun Bae, J.; Rubini, M.; Jung, G.; Wiegand, G.; Seifert, M. H.J.; Azim, M.K.; Kim, J.-S.; Zumbusch, A.; Holak, T. A.; Moroder, L.; Huber, R.; Budisa, N. Expansion of the Genetic Code Enables Design of a Novel “Gold” Class of Green Fluorescent Proteins. J. Mol. Biol. 2003, 328 (5), 1071– 1081, DOI: 10.1016/S0022-2836(03)00364-4Google ScholarThere is no corresponding record for this reference.
- 92Budisa, N.; Rubini, M.; Bae, J. H.; Weyher, E.; Wenger, W.; Golbik, R.; Huber, R.; Moroder, L. Global Replacement of Tryptophan with Aminotryptophans Generates Non-Invasive Protein-Based Optical pH Sensors. Angew. Chem. Int. Ed. 2002, 41 (21), 4066– 4069, DOI: 10.1002/1521-3773(20021104)41:21<4066::AID-ANIE4066>3.0.CO;2-6Google ScholarThere is no corresponding record for this reference.
- 93Duewel, H. S.; Daub, E.; Robinson, V.; Honek, J. F. Elucidation of Solvent Exposure, Side-Chain Reactivity, and Steric Demands of the Trifluoromethionine Residue in a Recombinant Protein. Biochemistry 2001, 40 (44), 13167– 13176, DOI: 10.1021/bi011381bGoogle Scholar93Elucidation of solvent exposure, side-chain reactivity, and steric demands of the trifluoromethionine residue in a recombinant proteinDuewel, Henry S.; Daub, Elisabeth; Robinson, Valerie; Honek, John F.Biochemistry (2001), 40 (44), 13167-13176CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)When incorporated into proteins, fluorinated amino acids have been utilized as 19F NMR probes of protein structure and protein-ligand interactions, and as subtle structural replacements for their parent amino acids which is not possible using the std. 20-amino acid repertoire. Recent investigations have shown the ability of various fluorinated methionines, such as difluoromethionine (DFM) and trifluoromethionine (TFM), to be bioincorporated into recombinant proteins and to be extremely useful as 19F NMR biophys. probes. Interestingly, in the case of the bacteriophage lambda lysozyme (LaL) which contains only three Met residues (at positions 1, 14, and 107), four 19F NMR resonances are obsd. when TFM is incorporated into LaL. To elucidate the underlying structural reasons for this anomalous observation and to more fully explore the effect of TFM on protein structure, site-directed mutagenesis was used to assign each 19F NMR resonance. Incorporation of TFM into the M14L mutant resulted in the collapse of the two 19F resonances assocd. with TFM at position 107 into a single resonance, suggesting that when position 14 in wild-type protein contains TFM, a subtle but different environment exists for the methionine at position 107. In addn., 19F and [1H-13C]-HMQC NMR expts. have been utilized with paramagnetic line broadening and K2PtCl4 reactivity expts. to obtain information about the probable spatial position of each Met in the protein. These results are compared with the recently detd. crystal structure of LaL and allow for a more detailed structural explanation for the effect of fluorination on protein structure.
- 94Seifert, M. H. J.; Ksiazek, D.; Azim, M. K.; Smialowski, P.; Budisa, N.; Holak, T. A. Slow Exchange in the Chromophore of a Green Fluorescent Protein Variant. J. Am. Chem. Soc. 2002, 124 (27), 7932– 7942, DOI: 10.1021/ja0257725Google Scholar94Slow exchange in the chromophore of a green fluorescent protein variantSeifert, Markus H. J.; Ksiazek, Dorota; Azim, M. Kamran; Smialowski, Pawel; Budisa, Nediljko; Holak, Tad A.Journal of the American Chemical Society (2002), 124 (27), 7932-7942CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Green fluorescent protein and its mutants have become valuable tools in mol. biol. They also provide systems rich in photophys. and photochem. phenomena of which an understanding is important for the development of new and optimized variants of GFP. Surprisingly, not a single NMR study has been reported on GFPs until now, possibly because of their high tendency to aggregate. ABS Here, we report the 19F NMR studies on mutants of the green fluorescent protein (GFP) and cyan fluorescent protein (CFP) labeled with fluorinated tryptophans that enabled the detection of slow mol. motions in these proteins. The concerted use of dynamic NMR and 19F relaxation measurements, supported by temp., concn.- and folding-dependent expts. provides direct evidence for the existence of a slow exchange process between two different conformational states of CFP. 19F NMR relaxation and line shape anal. indicate that the time scale of exchange between these states is in the range of 1.2-1.4 ms. Thermodn. anal. revealed a difference in enthalpy ΔH0 = (18.2±3.8) kJ/mol and entropy TΔS0 = (19.6±1.2) kJ/mol at T = 303 K for the two states involved in the exchange process, indicating an entropy-enthalpy compensation. The free energy of activation was estd. to be approx. 60 kJ/mol. Exchange between two conformations, either of the chromophore itself or more likely of the closely related histidine 148, is suggested to be the structural process underlying the conformational mobility of GFPs. The possibility to generate a series of single-atom exchanges ("at. mutations") like H → F in this study offers a useful approach for characterizing and quantifying dynamic processes in proteins by NMR.
- 95Bann, J. G.; Pinkner, J.; Hultgren, S. J.; Frieden, C. Real-Time and Equilibrium 19F-NMR Studies Reveal the Role of Domain-Domain Interactions in the Folding of the Chaperone PapD. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (2), 709– 714, DOI: 10.1073/pnas.022649599Google ScholarThere is no corresponding record for this reference.
- 96Deming, T. J.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A. Biosynthetic Incorporation and Chemical Modification of Alkene Functionality in Genetically Engineered Polymers. Journal of Macromolecular Science, Part A 1997, 34 (10), 2143– 2150, DOI: 10.1080/10601329708010331Google ScholarThere is no corresponding record for this reference.
- 97Kiick, K. L.; Saxon, E.; Tirrell, D. A.; Bertozzi, C. R. Incorporation of Azides into Recombinant Proteins for Chemoselective Modification by the Staudinger Ligation. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (1), 19– 24, DOI: 10.1073/pnas.012583299Google Scholar97Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligationKiick, Kristi L.; Saxon, Eliana; Tirrell, David A.; Bertozzi, Carolyn R.Proceedings of the National Academy of Sciences of the United States of America (2002), 99 (1), 19-24CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The introduction of chem. unique groups into proteins by means of non-natural amino acids has numerous applications in protein engineering and functional studies. One method to achieve this involves the utilization of a non-natural amino acid by the cell's native translational app. Here we demonstrate that a methionine surrogate, azidohomoalanine, is activated by the methionyl-tRNA synthetase of Escherichia coli and replaces methionine in proteins expressed in methionine-depleted bacterial cultures. We further show that proteins contg. azidohomoalanine can be selectively modified in the presence of other cellular proteins by means of Staudinger ligation with triarylphosphine reagents. Incorporation of azide-functionalized amino acids into proteins in vivo provides opportunities for protein modification under native conditions and selective labeling of proteins in the intracellular environment.
- 98Kothakota, S.; Mason, T. L.; Tirrell, D. A.; Fournier, M. J. Biosynthesis of a Periodic Protein Containing 3-Thienylalanine: A Step Toward Genetically Engineered Conducting Polymers. J. Am. Chem. Soc. 1995, 117 (1), 536– 537, DOI: 10.1021/ja00106a064Google Scholar98Biosynthesis of a Periodic Protein Containing 3-Thienylalanine: A Step Toward Genetically Engineered Conducting PolymersKothakota, Srinivas; Mason, Thomas L.; Tirrell, David A.; Fournier, Maurille J.Journal of the American Chemical Society (1995), 117 (1), 536-7CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The authors present evidence for the use of 3-thienylalanine (3-TA), a phenylalanine analog, by the Escherichia coli biosynthetic machinery. The analog was incorporated into a periodic polymer of sequence [(GlyAla)3GlyPhe]13, in place of phenylalanine. The extent of substitution was at least 80%, as shown by UV spectroscopy, NMR spectroscopy and amino acid anal. No evidence for modification of the analog was found. The 3-alkylthiophene side chain of 3-TA should be susceptible to oxidative crosslinking or grafting of conventional 3-alkylthiophene monomers, opening a route to genetically engineered polymeric materials with useful properties.
- 99Link, A. J.; Tirrell, D. A. Cell Surface Labeling of Escherichia coli via Copper(I)-Catalyzed [3 + 2] Cycloaddition. J. Am. Chem. Soc. 2003, 125 (37), 11164– 11165, DOI: 10.1021/ja036765zGoogle Scholar99Cell surface labeling of Escherichia coli via copper(I)-catalyzed [3+2] cycloadditionLink, A. James; Tirrell, David A.Journal of the American Chemical Society (2003), 125 (37), 11164-11165CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Labeling of the cell surface of Escherichia coli was accomplished by expression of a recombinant outer membrane protein, OmpC, in the presence of the unnatural amino acid azidohomoalanine, which acts as a methionine surrogate. The surface-exposed azide moieties of whole cells were biotinylated via Cu(1)-catalyzed [3+2] azide-alkyne cycloaddn. The specificity of labeling of both wild-type OmpC and a mutant contg. addnl. methionine sites for azidohomoalanine incorporation was confirmed by Western blotting. Flow cytometry was performed to examine the specificity of the labeling. Cells that express the mutant form of OmpC in the presence of azidohomoalanine, which were biotinylated and stained with fluorescent avidin, exhibit a mean fluorescence 10-fold higher than the background. Incorporation of an unnatural amino acid can thus be detd. on a single-cell basis.
- 100van Hest, J. C. M.; Kiick, K. L.; Tirrell, D. A. Efficient Incorporation of Unsaturated Methionine Analogues into Proteins in Vivo. J. Am. Chem. Soc. 2000, 122 (7), 1282– 1288, DOI: 10.1021/ja992749jGoogle Scholar100Efficient incorporation of unsaturated methionine analogues into proteins in vivoVan Hest, Jan C. M.; Kiick, Kristi L.; Tirrell, David A.Journal of the American Chemical Society (2000), 122 (7), 1282-1288CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A set of eight methionine analogs was assayed for translational activity in Escherichia coli. Norvaline and norleucine, which are com. available, were assayed along with 2-amino-5-hexenoic acid (I), 2-amino-5-hexynoic acid (II), cis-2-amino-4-hexenoic acid, trans-2-amino-4-hexenoic acid, 6,6,6-trifluoro-2-aminohexanoic acid, and 2-aminoheptanoic acid, each of which was prepd. by alkylation of di-Et acetamidomalonate with the appropriate tosylate, followed by hydrolysis. The E. coli methionine auxotroph CAG18491, transformed with plasmids pREP4 and pQE15, was used as the expression host, and translational activity was assayed by detn. of the capacity of the analog to support synthesis of the test protein dihydrofolate reductase (DHFR) in the absence of added methionine. The importance of amino acid side chain length was illustrated by the fact that neither norvaline nor 2-aminoheptanoic acid showed translational activity, in contrast to norleucine, which does support protein synthesis under the assay conditions. The internal alkene functions of cis-2-amino-4-hexenoic acid trans-2-amino-4-hexenoic acid prevented incorporation of these analogs into test protein, and the fluorinated analog 6,6,6-trifluoro-2-aminohexanoic acid yielded no evidence of translational activity. The terminally unsatd. compds. I and II, however, proved to be excellent methionine surrogates: 1H NMR spectroscopy, amino acid anal., and N-terminal sequencing indicated ∼85% substitution of methionine by I, while II showed 90-100% replacement. Both analogs also function efficiently in the initiation step of protein synthesis, as shown by their near-quant. occupancy of the N-terminal amino acid site in DHFR. Enzyme kinetics assays were conducted to det. the rate of activation of each of the methionine analogs by methionyl tRNA synthetase (MetRS); results of the in vitro assays corroborate the in vivo incorporation results, suggesting that success or failure of analog incorporation in vivo is controlled by MetRS.
- 101van Hest, J. C. M.; Tirrell, D. A. Efficient introduction of alkene functionality into proteins in vivo. FEBS Lett. 1998, 428 (1–2), 68– 70, DOI: 10.1016/S0014-5793(98)00489-XGoogle ScholarThere is no corresponding record for this reference.
- 102Omari, K. E.; Ren, J.; Bird, L. E.; Bona, M. K.; Klarmann, G.; LeGrice, S. F. J.; Stammers, D. K. Molecular Architecture and Ligand Recognition Determinants for T4 RNA Ligase. J. Biol. Chem. 2006, 281 (3), 1573– 1579, DOI: 10.1074/jbc.M509658200Google ScholarThere is no corresponding record for this reference.
- 103Xiao, H.; Murakami, H.; Suga, H.; Ferré-D’Amaré, A. R. Structural Basis of Specific tRNA Aminoacylation by a Small in vitro Selected Ribozyme. Nature 2008, 454 (7202), 358– 361, DOI: 10.1038/nature07033Google Scholar103Structural basis of specific tRNA aminoacylation by a small in vitro selected ribozymeXiao, Hong; Murakami, Hiroshi; Suga, Hiroaki; Ferre-D'Amare, Adrian R.Nature (London, United Kingdom) (2008), 454 (7202), 358-361CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)In modern organisms, protein enzymes are solely responsible for the aminoacylation of tRNA. However, the evolution of protein synthesis in the RNA world required RNAs capable of catalyzing this reaction. Ribozymes that aminoacylate RNA by using activated amino acids have been discovered through selection in vitro. Flexizyme is a 45-nucleotide ribozyme capable of charging tRNA in trans with various activated L-phenylalanine derivs. In addn. to a more than 105 rate enhancement and more than 104-fold discrimination against some non-cognate amino acids, this ribozyme achieves good regioselectivity: of all the hydroxyl groups of a tRNA, it exclusively aminoacylates the terminal 3'-OH. Here we report the 2.8-Å resoln. structure of flexizyme fused to a substrate RNA. Together with randomization of ribozyme core residues and reselection, this structure shows that very few nucleotides are needed for the aminoacylation of specific tRNAs. Although it primarily recognizes tRNA through base-pairing with the CCA terminus of the tRNA mol., flexizyme makes numerous local interactions to position the acceptor end of tRNA precisely. A comparison of two crystallog. independent flexizyme conformations, only one of which appears capable of binding activated phenylalanine, suggests that this ribozyme may achieve enhanced specificity by coupling active-site folding to tRNA docking. Such a mechanism would be reminiscent of the mutually induced fit of tRNA and protein employed by some aminoacyl-tRNA synthetases to increase specificity.
- 104Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. A General Method for Site-specific Incorporation of Unnatural Amino Acids into Proteins. Science 1989, 244 (4901), 182– 188, DOI: 10.1126/science.2649980Google Scholar104A general method for site-specific incorporation of unnatural amino acids into proteinsNoren, Christopher J.; Anthony-Cahill, Spencer J.; Griffith, Michael C.; Schultz, Peter G.Science (Washington, DC, United States) (1989), 244 (4901), 182-8CODEN: SCIEAS; ISSN:0036-8075.A new method has been developed that makes it possible to site-specifically incorporate unnatural amino acids into proteins. Synthetic amino acids were incorporated into the enzyme β-lactamase by the use of a chem. acylated suppressor tRNA that inserted the amino acid in response to a stop codon substituted for the codon encoding residue of interest. Peptide mapping localized the inserted amino acid to a single peptide, and enough enzyme could be generated for purifn. to homogeneity. The catalytic properties of several mutants at the conserved Phe66 were characterized. The ability to selectively replace amino acids in a protein with a wide variety of structural and electronic variants should provide a more detailed understanding of protein structure and function.
- 105Goto, Y.; Katoh, T.; Suga, H. Flexizymes for Genetic Code Reprogramming. Nat. Protoc. 2011, 6 (6), 779– 790, DOI: 10.1038/nprot.2011.331Google Scholar105Flexizymes for genetic code reprogrammingGoto, Yuki; Katoh, Takayuki; Suga, HiroakiNature Protocols (2011), 6 (6), 779-790CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Genetic code reprogramming is a method for the reassignment of arbitrary codons from proteinogenic amino acids to nonproteinogenic ones; thus, specific sequences of nonstandard peptides can be ribosomally expressed according to their mRNA templates. Here we describe a protocol that facilitates genetic code reprogramming using flexizymes integrated with a custom-made in vitro translation app., referred to as the flexible in vitro translation (FIT) system. Flexizymes are flexible tRNA acylation ribozymes that enable the prepn. of a diverse array of nonproteinogenic acyl-tRNAs. These acyl-tRNAs read vacant codons created in the FIT system, yielding the desired nonstandard peptides with diverse exotic structures, such as N-Me amino acids, D-amino acids and physiol. stable macrocyclic scaffolds. The facility of the protocol allows a wide variety of applications in the synthesis of new classes of nonstandard peptides with biol. functions. Prepn. of flexizymes and tRNA used for genetic code reprogramming, optimization of flexizyme reaction conditions and expression of nonstandard peptides using the FIT system can be completed by one person in ∼1 wk. However, once the flexizymes and tRNAs are in hand and reaction conditions are fixed, synthesis of acyl-tRNAs and peptide expression is generally completed in 1 d, and alteration of a peptide sequence can be achieved by simply changing the corresponding mRNA template.
- 106Shimizu, Y.; Inoue, A.; Tomari, Y.; Suzuki, T.; Yokogawa, T.; Nishikawa, K.; Ueda, T. Cell-Free Translation Reconstituted with Purified Components. Nat. Biotechnol. 2001, 19 (8), 751– 755, DOI: 10.1038/90802Google Scholar106Cell-free translation reconstituted with purified componentsShimizu, Yoshihiro; Inoue, Akio; Tomari, Yukihide; Suzuki, Tsutomu; Yokogawa, Takashi; Nishikawa, Kazuya; Ueda, TakuyaNature Biotechnology (2001), 19 (8), 751-755CODEN: NABIF9; ISSN:1087-0156. (Nature America Inc.)We have developed a protein-synthesizing system reconstituted from recombinant tagged protein factors purified to homogeneity. The system was able to produce protein at a rate of about 160 μg/mL/h in a batch mode without the need for any supplementary app. The protein products were easily purified within 1 h using affinity chromatog. to remove the tagged protein factors. Moreover, omission of a release factor allowed efficient incorporation of an unnatural amino acid using suppressor tRNA.
- 107Chapeville, F.; Lipmann, F.; Ehrenstein, G. v.; Weisblum, B.; Ray, W. J.; Benzer, S. On the Role of Soluble Ribonucleic Acid in Coding for Amino Acids. Proc. Natl. Acad. Sci. U.S.A. 1962, 48 (6), 1086– 1092, DOI: 10.1073/pnas.48.6.1086Google ScholarThere is no corresponding record for this reference.
- 108Fahnestock, S.; Rich, A. Ribosome-Catalyzed Polyester Formation. Science 1971, 173 (3994), 340– 343, DOI: 10.1126/science.173.3994.340Google Scholar108Ribosome-catalyzed polyester formationFahnestock, Stephen; Rich, AlexanderScience (Washington, DC, United States) (1971), 173 (3994), 340-3CODEN: SCIEAS; ISSN:0036-8075.The deamination of phenylalanyl-tRNA with HNO2 gave the α-hydroxyacyl analog phenyllactyl-tRNA, which incubated in a protein-synthesizing system directed by polyuridylic acid yielded an acid-precipitable, alkali-labile phenyllactic acid polyester. Similarities with polyphenylalanine formation suggested the existence of the same ribosomal mechanism. The polymer consisted 70-80% of phenyllactic acid residues, the remaining residues being probably phenylalanine.
- 109Hecht, S. M.; Alford, B. L.; Kuroda, Y.; Kitano, S. “Chemical Aminoacylation” of tRNA’s. J. Biol. Chem. 1978, 253 (13), 4517– 4520, DOI: 10.1016/S0021-9258(17)30417-9Google ScholarThere is no corresponding record for this reference.
- 110Katoh, T.; Goto, Y.; Passioura, T.; Suga, H. Development of Flexizyme Aminoacylation Ribozymes and Their Applications. Ribozymes 2021, 519– 543, DOI: 10.1002/9783527814527.ch20Google ScholarThere is no corresponding record for this reference.
- 111Morimoto, J.; Hayashi, Y.; Iwasaki, K.; Suga, H. Flexizymes: Their Evolutionary History and the Origin of Catalytic Function. Acc. Chem. Res. 2011, 44 (12), 1359– 1368, DOI: 10.1021/ar2000953Google Scholar111Flexizymes: Their Evolutionary History and the Origin of Catalytic FunctionMorimoto, Jumpei; Hayashi, Yuuki; Iwasaki, Kazuhiro; Suga, HiroakiAccounts of Chemical Research (2011), 44 (12), 1359-1368CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. TRNA is an essential component of the cell's translation app. These RNA strands contain the anticodon for a given amino acid, and when "charged" with that amino acid are termed aminoacyl-tRNA. Aminoacylation, which occurs exclusively at one of the 3'-terminal hydroxyl groups of tRNA, is catalyzed by a family of enzymes called aminoacyl-tRNA synthetases (ARSs). In a primitive translation system, before the advent of sophisticated protein-based enzymes, this chem. event could conceivably have been catalyzed solely by RNA enzymes. Given the evolutionary implications, our group attempted in vitro selection of artificial ARS-like ribozymes, successfully uncovering a functional ribozyme (r24) from an RNA pool of random sequences attached to the 5'-leader region of tRNA. This ribozyme preferentially charges arom. amino acids (such as phenylalanine) activated with cyanomethyl ester (CME) onto specific kinds of tRNA. During the course of our studies, we became interested in developing a versatile, rather than a specific, aminoacylation catalyst. Such a ribozyme could facilitate the prepn. of intentionally misacylated tRNAs and thus serve a convenient tool for manipulating the genetic code. On the basis of biochem. studies of r24, we constructed a truncated version of r24 (r24mini) that was 57 nucleotides long. This r24mini was then further shortened to 45 nucleotides. This ribozyme could charge various tRNAs through very simple three-base-pair interactions between the ribozyme's 3'-end and the tRNA's 3'-end. We termed this ribozyme a "flexizyme" (Fx3 for this particular construct) owing to its flexibility in addressing tRNAs. To devise an even more flexible tool for tRNA acylation, we attempted to eliminate the amino acid specificity from Fx3. This attempt yielded an Fx3 variant, termed dFx, which accepts amino acid substrates having 3,5-dinitrobenzyl ester instead of CME as a leaving group. Similar selection attempts with the original phenylalanine-CME and a substrate activated by (2-aminoethyl)amidocarboxybenzyl thioester yielded the variants eFx and aFx (e and a denote enhanced and amino, resp.). In this Account, we describe the history and development of these flexizymes and their appropriate substrates, which provide a versatile and easy-to-use tRNA acylation system. Their use permits the synthesis of a wide array of acyl-tRNAs charged with artificial amino and hydroxy acids. In parallel with these efforts, we initiated a crystn. study of Fx3 covalently conjugated to a microhelix RNA, which is an analog of tRNA. The X-ray crystal structure, solved as a co-complex with phenylalanine Et ester and U1A-binding protein, revealed the structural basis of this enzyme. Most importantly, many biochem. observations were consistent with the crystal structure. Along with the predicted three regular-helix regions, however, the flexizyme has a unique irregular helix that was unexpected. This irregular helix constitutes a recognition pocket for the arom. ring of the amino acid side chain and precisely brings the carbonyl group to the 3'-hydroxyl group of the tRNA 3'-end. This study has clearly defined the mol. interactions between Fx3, tRNA, and the amino acid substrate, revealing the fundamental basis of this unique catalytic system.
- 112Murakami, H.; Ohta, A.; Ashigai, H.; Suga, H. A Highly Flexible tRNA Acylation Method for Non-Natural Polypeptide Synthesis. Nat. Methods 2006, 3 (5), 357– 359, DOI: 10.1038/nmeth877Google Scholar112A highly flexible tRNA acylation method for non-natural polypeptide synthesisMurakami, Hiroshi; Ohta, Atsushi; Ashigai, Hiroshi; Suga, HiroakiNature Methods (2006), 3 (5), 357-359CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Here the authors describe a de novo tRNA acylation system, the flexizyme (Fx) system, for the prepn. of acyl tRNAs with nearly unlimited selection of amino and hydroxy acids and tRNAs. The combination of the Fx system with an appropriate cell-free translation system allows the authors to readily perform mRNA-encoded synthesis of proteins and short polypeptides involving multiple nonnatural amino acids.
- 113Katoh, T.; Suga, H. In Vitro Genetic Code Reprogramming for the Expansion of Usable Noncanonical Amino Acids. Annu. Rev. Biochem. 2022, 91 (1), 221– 243, DOI: 10.1146/annurev-biochem-040320-103817Google ScholarThere is no corresponding record for this reference.
- 114Goto, Y.; Suga, H. The RaPID Platform for the Discovery of Pseudo-Natural Macrocyclic Peptides. Acc. Chem. Res. 2021, 54 (18), 3604– 3617, DOI: 10.1021/acs.accounts.1c00391Google Scholar114The RaPID Platform for the Discovery of Pseudo-Natural Macrocyclic PeptidesGoto, Yuki; Suga, HiroakiAccounts of Chemical Research (2021), 54 (18), 3604-3617CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. Conspectus: Although macrocyclic peptides bearing exotic building blocks have proven their utility as pharmaceuticals, the sources of macrocyclic peptide drugs have been largely limited to mimetics of native peptides or natural product peptides. However, the recent emergence of technologies for discovering de novo bioactive peptides has led to their reconceptualization as a promising therapeutic modality. For the construction and screening of libraries of such macrocyclic peptides, our group has devised a platform to conduct affinity-based selection of massive libraries (>1012 unique sequences) of in vitro expressed macrocyclic peptides, which is referred to as the random nonstandard peptides integrated discovery (RaPID) system. The RaPID system integrates genetic code reprogramming using the FIT (flexible in vitro translation) system, which is largely facilitated by flexizymes (flexible tRNA-aminoacylating ribozymes), with mRNA display technol. We have demonstrated that the RaPID system enables rapid discovery of various de novo pseudo-natural peptide ligands for protein targets of interest. Many examples discussed in this Account prove that thioether-closed macrocyclic peptides (teMPs) obtained by the RaPID system generally exhibit remarkably high affinity and specificity, thereby potently inhibiting or activating a specific function(s) of the target. Moreover, such teMPs are used for a wide range of biochem. applications, for example, as crystn. chaperones for intractable transmembrane proteins and for in vivo recognition of specific cell types. Furthermore, recent studies demonstrate that some teMPs exhibit pharmacol. activities in animal models and that even intracellular proteins can be inhibited by teMPs, illustrating the potential of this class of peptides as drug leads. Besides the ring-closing thioether linkage in the teMPs, genetic code reprogramming by the FIT system allows for incorporation of a variety of other exotic building blocks. For instance, diverse nonproteinogenic amino acids, hydroxy acids (ester linkage), amino carbothioic acid (thioamide linkage), and abiotic foldamer units have been successfully incorporated into ribosomally synthesized peptides. Despite such enormous successes in the conventional FIT system, multiple or consecutive incorporation of highly exotic amino acids, such as D- and β-amino acids, is yet challenging, and particularly the synthesis of peptides bearing non-carbonyl backbone structures remains a demanding task. To upgrade the RaPID system to the next generation, we have engaged in intensive manipulation of the FIT system to expand the structural diversity of peptides accessible by our in vitro biosynthesis strategy. Semilogical engineering of tRNA body sequences led to a new suppressor tRNA (tRNAPro1E2) capable of effectively recruiting translation factors, particularly EF-Tu and EF-P. The use of tRNAPro1E2 in the FIT system allows for not only single but also consecutive and multiple elongation of exotic amino acids, such as D-, β-, and γ-amino acids as well as aminobenzoic acids. Moreover, the integration of the FIT system with various chem. or enzymic posttranslational modifications enables us to expand the range of accessible backbone structures to non-carbonyl moieties prominent in natural products and peptidomimetics. In such systems, FIT-expressed peptides undergo multistep backbone conversions in a one-pot manner to yield designer peptides composed of modified backbones such as azolines, azoles, and ring-closing pyridines. Our current research endeavors focus on applying such in vitro biosynthesis systems for the discovery of bioactive de novo pseudo-natural products.
- 115Schultz, P. Expanding the Genetic Code. Protein Sci. 2023, 32 (1), e4488 DOI: 10.1002/pro.4488Google ScholarThere is no corresponding record for this reference.
- 116Neumann, H.; Wang, K.; Davis, L.; Garcia-Alai, M.; Chin, J. W. Encoding Multiple Unnatural Amino Acids via Evolution of a Quadruplet-Decoding Ribosome. Nature 2010, 464 (7287), 441– 444, DOI: 10.1038/nature08817Google Scholar116Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosomeNeumann, Heinz; Wang, Kaihang; Davis, Lloyd; Garcia-Alai, Maria; Chin, Jason W.Nature (London, United Kingdom) (2010), 464 (7287), 441-444CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The in vivo, genetically programmed incorporation of designer amino acids allows the properties of proteins to be tailored with mol. precision. The Methanococcus jannaschii tyrosyl-transfer-RNA synthetase-tRNACUA (MjTyrRS-tRNACUA) and the Methanosarcina barkeri pyrrolysyl-tRNA synthetase-tRNACUA (MbPylRS-tRNACUA) orthogonal pairs have been evolved to incorporate a range of unnatural amino acids in response to the amber codon in Escherichia coli. However, the potential of synthetic genetic code expansion is generally limited to the low efficiency incorporation of a single type of unnatural amino acid at a time, because every triplet codon in the universal genetic code is used in encoding the synthesis of the proteome. To encode efficiently many distinct unnatural amino acids into proteins we require blank codons and mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs that recognize unnatural amino acids and decode the new codons. Here we synthetically evolve an orthogonal ribosome (ribo-Q1) that efficiently decodes a series of quadruplet codons and the amber codon, providing several blank codons on an orthogonal mRNA, which it specifically translates. By creating mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs and combining them with ribo-Q1 we direct the incorporation of distinct unnatural amino acids in response to two of the new blank codons on the orthogonal mRNA. Using this code, we genetically direct the formation of a specific, redox-insensitive, nanoscale protein cross-link by the bio-orthogonal cycloaddn. of encoded azide- and alkyne-contg. amino acids. Because the synthetase-tRNA pairs used have been evolved to incorporate numerous unnatural amino acids, it will be possible to encode more than 200 unnatural amino acid combinations using this approach. As ribo-Q1 independently decodes a series of quadruplet codons, this work provides foundational technologies for the encoded synthesis and synthetic evolution of unnatural polymers in cells.
- 117Robertson, W. E.; Funke, L. F. H.; de la Torre, D.; Fredens, J.; Elliott, T. S.; Spinck, M.; Christova, Y.; Cervettini, D.; Böge, F. L.; Liu, K. C. Sense Codon Reassignment Enables Viral Resistance and Encoded Polymer Synthesis. Science 2021, 372 (6546), 1057– 1062, DOI: 10.1126/science.abg3029Google Scholar117Sense codon reassignment enables viral resistance and encoded polymer synthesisRobertson, Wesley E.; Funke, Louise F. H.; de la Torre, Daniel; Fredens, Julius; Elliott, Thomas S.; Spinck, Martin; Christova, Yonka; Cervettini, Daniele; Boge, Franz L.; Liu, Kim C.; Buse, Salvador; Maslen, Sarah; Salmond, George P. C.; Chin, Jason W.Science (Washington, DC, United States) (2021), 372 (6546), 1057-1062CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)It is widely hypothesized that removing cellular tRNAs (tRNAs)-making their cognate codons unreadable-might create a genetic firewall to viral infection and enable sense codon reassignment. However, it has been impossible to test these hypotheses. In this work, following synonymous codon compression and lab. evolution in Escherichia coli, we deleted the tRNAs and release factor 1, which normally decode two sense codons and a stop codon; the resulting cells could not read the canonical genetic code and were completely resistant to a cocktail of viruses. We reassigned these codons to enable the efficient synthesis of proteins contg. three distinct noncanonical amino acids. Notably, we demonstrate the facile reprogramming of our cells for the encoded translation of diverse noncanonical heteropolymers and macrocycles.
- 118Wang, L.; Brock, A.; Herberich, B.; Schultz, P. G. Expanding the Genetic Code of Escherichia coli. Science 2001, 292 (5516), 498– 500, DOI: 10.1126/science.1060077Google Scholar118Expanding the genetic code of Escherichia coliWang, Lei; Brock, Ansgar; Herberich, Brad; Schultz, Peter G.Science (Washington, DC, United States) (2001), 292 (5516), 498-500CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A unique tRNA/aminoacyl-tRNA synthetase pair has been generated that expands the no. of genetically encoded amino acids in Escherichia coli. When introduced into E. coli, this pair leads to the in vivo incorporation of the synthetic amino acid O-methyl-L-tyrosine into protein in response to an amber nonsense codon. The fidelity of translation is greater than 99%, as detd. by anal. of dihydrofolate reductase contg. the unnatural amino acid. This approach should provide a general method for increasing the genetic repertoire of living cells to include a variety of amino acids with novel structural, chem., and phys. properties not found in the common 20 amino acids.
- 119Dunkelmann, D. L.; Piedrafita, C.; Dickson, A.; Liu, K. C.; Elliott, T. S.; Fiedler, M.; Bellini, D.; Zhou, A.; Cervettini, D.; Chin, J. W. Adding α,α-Disubstituted and β-Linked Monomers to the Genetic Code of an Organism. Nature 2024, 625 (7995), 603– 610, DOI: 10.1038/s41586-023-06897-6Google ScholarThere is no corresponding record for this reference.
- 120Bryson, D. I. Continuous Directed Evolution of Aminoacyl-tRNA Synthetases. Nat. Chem. Biol. 2017, 13, 1253, DOI: 10.1038/nchembio.2474Google Scholar120Continuous directed evolution of aminoacyl-tRNA synthetasesBryson, David I.; Fan, Chenguang; Guo, Li-Tao; Miller, Corwin; Soll, Dieter; Liu, David R.Nature Chemical Biology (2017), 13 (12), 1253-1260CODEN: NCBABT; ISSN:1552-4450. (Nature Research)Directed evolution of orthogonal aminoacyl-tRNA synthetases (AARSs) enables site-specific installation of noncanonical amino acids (ncAAs) into proteins. Traditional evolution techniques typically produce AARSs with greatly reduced activity and selectivity compared to their wild-type counterparts. We designed phage-assisted continuous evolution (PACE) selections to rapidly produce highly active and selective orthogonal AARSs through hundreds of generations of evolution. PACE of a chimeric Methanosarcina spp. pyrrolysyl-tRNA synthetase (PylRS) improved its enzymic efficiency (kcat/KMtRNA) 45-fold compared to the parent enzyme. Transplantation of the evolved mutations into other PylRS-derived synthetases improved yields of proteins contg. noncanonical residues up to 9.7-fold. Simultaneous pos. and neg. selection PACE over 48 h greatly improved the selectivity of a promiscuous Methanocaldococcus jannaschii tyrosyl-tRNA synthetase variant for site-specific incorporation of p-iodo-L-phenylalanine. These findings offer new AARSs that increase the utility of orthogonal translation systems and establish the capability of PACE to efficiently evolve orthogonal AARSs with high activity and amino acid specificity.
- 121Krahn, N.; Tharp, J. M.; Crnković, A.; Söll, D. Chapter Twelve - Engineering Aminoacyl-tRNA Synthetases for Use in Synthetic Biology. In The Enzymes; Ribas de Pouplana, L., Kaguni, L. S., Eds.; Academic Press, 2020; Vol. 48, pp 351– 395. DOI: 10.1016/bs.enz.2020.06.004Google ScholarThere is no corresponding record for this reference.
- 122Amiram, M.; Haimovich, A. D.; Fan, C.; Wang, Y.-S.; Aerni, H.-R.; Ntai, I.; Moonan, D. W.; Ma, N. J.; Rovner, A. J.; Hong, S. H. Evolution of Translation Machinery in Recoded Bacteria Enables Multi-Site Incorporation of Nonstandard Amino Acids. Nat. Biotechnol. 2015, 33 (12), 1272– 1279, DOI: 10.1038/nbt.3372Google Scholar122Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acidsAmiram, Miriam; Haimovich, Adrian D.; Fan, Chenguang; Wang, Yane-Shih; Aerni, Hans-Rudolf; Ntai, Ioanna; Moonan, Daniel W.; Ma, Natalie J.; Rovner, Alexis J.; Hong, Seok Hoon; Kelleher, Neil L.; Goodman, Andrew L.; Jewett, Michael C.; Soll, Dieter; Rinehart, Jesse; Isaacs, Farren J.Nature Biotechnology (2015), 33 (12), 1272-1279CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Expansion of the genetic code with nonstandard amino acids (nsAAs) has enabled biosynthesis of proteins with diverse new chemistries. However, this technol. has been largely restricted to proteins contg. a single or few nsAA instances. Here we describe an in vivo evolution approach in a genomically recoded Escherichia coli strain for the selection of orthogonal translation systems capable of multi-site nsAA incorporation. We evolved chromosomal aminoacyl-tRNA synthetases (aaRSs) with up to 25-fold increased protein prodn. for p-acetyl-L-phenylalanine and p-azido-L-phenylalanine (pAzF). We also evolved aaRSs with tunable specificities for 14 nsAAs, including an enzyme that efficiently charges pAzF while excluding 237 other nsAAs. These variants enabled prodn. of elastin-like-polypeptides with 30 nsAA residues at high yields (∼50 mg/L) and high accuracy of incorporation (>95%). This approach to aaRS evolution should accelerate and expand our ability to produce functionalized proteins and sequence-defined polymers with diverse chemistries.
- 123Hohl, A.; Karan, R.; Akal, A.; Renn, D.; Liu, X.; Ghorpade, S.; Groll, M.; Rueping, M.; Eppinger, J. Engineering a Polyspecific Pyrrolysyl-tRNA Synthetase by a High Throughput FACS Screen. Sci. Rep. 2019, 9 (1), 11971, DOI: 10.1038/s41598-019-48357-0Google Scholar123Engineering a Polyspecific Pyrrolysyl-tRNA Synthetase by a High Throughput FACS ScreenHohl Adrian; Karan Ram; Akal Anastassja; Renn Dominik; Liu Xuechao; Ghorpade Seema; Rueping Magnus; Eppinger Jorg; Hohl Adrian; Akal Anastassja; Renn Dominik; Groll MichaelScientific reports (2019), 9 (1), 11971 ISSN:.The Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA(Pyl) are extensively used to add non-canonical amino acids (ncAAs) to the genetic code of bacterial and eukaryotic cells. However, new ncAAs often require a cumbersome de novo engineering process to generate an appropriate PylRS/tRNA(Pyl) pair. We here report a strategy to predict a PylRS variant with novel properties. The designed polyspecific PylRS variant HpRS catalyzes the aminoacylation of 31 structurally diverse ncAAs bearing clickable, fluorinated, fluorescent, and for the first time biotinylated entities. Moreover, we demonstrated a site-specific and copper-free conjugation strategy of a nanobody by the incorporation of biotin. The design of polyspecific PylRS variants offers an attractive alternative to existing screening approaches and provides insights into the complex PylRS-substrate interactions.
- 124Wang, L.; Xie, J.; Schultz, P. G. Expanding the Genetic Code. Annu. Rev. Biophys. Biomol. Struct. 2006, 35, 225– 249, DOI: 10.1146/annurev.biophys.35.101105.121507Google Scholar124Expanding the genetic codeWang, Lei; Xie, Jianming; Schultz, Peter G.Annual Review of Biophysics and Biomolecular Structure (2006), 35 (), 225-249CODEN: ABBSE4; ISSN:1056-8700. (Annual Reviews Inc.)A review. Recently, a general method was developed that makes it possible to genetically encode unnatural amino acids with diverse phys., chem., or biol. properties in Escherichia coli, yeast, and mammalian cells. More than 30 unnatural amino acids have been incorporated into proteins with high fidelity and efficiency by means of a unique codon and corresponding tRNA/aminoacyl-tRNA synthetase pair. These include fluorescent, glycosylated, metal-ion-binding, and redox-active amino acids, as well as amino acids with unique chem. and photochem. reactivity. This methodol. provides a powerful tool both for exploring protein structure and function in vitro and in vivo and for generating proteins with new or enhanced properties.
- 125Santoro, S.; Wang, L.; Herberich, B.; King, D. S.; Schultz, P. G. An Efficient System for the Evolution of Aminoacyl-tRNA Synthetase Specificity. Nat. Biotechnol. 2002, 20, 1044– 1048, DOI: 10.1038/nbt742Google Scholar125An efficient system for the evolution of aminoacyl-tRNA synthetase specificitySantoro, Stephen W.; Wang, Lei; Herberich, Brad; King, David S.; Schultz, Peter G.Nature Biotechnology (2002), 20 (10), 1044-1048CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)A variety of strategies to incorporate unnatural amino acids into proteins have been pursued, but all have limitations with respect to tech. accessibility, scalability, applicability to in vivo studies, or site specificity of amino acid incorporation. The ability to selectively introduce unnatural functional groups into specific sites within proteins, in vivo, provides a potentially powerful approach to the study of protein function and to large-scale prodn. of novel proteins. Here the authors describe a combined genetic selection and screen that allows the rapid evolution of aminoacyl-tRNA synthetase substrate specificity. The authors' strategy involves the use of an "orthogonal" aminoacyl-tRNA synthetase and tRNA pair that cannot interact with any of the endogenous synthetase-tRNA pairs in Escherichia coli. A chloramphenicol-resistance (Cmr) reporter is used to select highly active synthetase variants, and an amplifiable fluorescence reporter is used together with fluorescence-activated cell sorting (FACS) to screen for variants with the desired change in amino acid specificity. Both reporters are contained within a single genetic construct, eliminating the need for plasmid shuttling and allowing the evolution to be completed in a matter of days. Following evolution, the amplifiable fluorescence reporter allows visual and fluorimetric evaluation of synthetase activity and selectivity. Using this system to explore the evolvability of an amino acid binding pocket of a tyrosyl-tRNA synthetase, the authors identified three new variants that allow the selective incorporation of amino-, isopropyl-, and allyl-contg. tyrosine analogs into a desired protein. The new enzymes can be used to produce milligram-per-liter quantities of unnatural amino acid-contg. protein in E. coli.
- 126Dumas, A.; Lercher, L.; Spicer, C. D.; Davis, B. G. Designing Logical Codon Reassignment - Expanding the Chemistry in Biology. Chem. Sci. 2015, 6 (1), 50– 69, DOI: 10.1039/C4SC01534GGoogle Scholar126Designing logical codon reassignment - Expanding the chemistry in biologyDumas, Anaelle; Lercher, Lukas; Spicer, Christopher D.; Davis, Benjamin G.Chemical Science (2015), 6 (1), 50-69CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)A review. Over the last decade, the ability to genetically encode unnatural amino acids (UAAs) has evolved rapidly. The programmed incorporation of UAAs into recombinant proteins relies on the reassignment or suppression of canonical codons with an amino-acyl tRNA synthetase/tRNA (aaRS/tRNA) pair, selective for the UAA of choice. In order to achieve selective incorporation, the aaRS should be selective for the designed tRNA and UAA over the endogenous amino acids and tRNAs. Enhanced selectivity has been achieved by transferring an aaRS/tRNA pair from another kingdom to the organism of interest, and subsequent aaRS evolution to acquire enhanced selectivity for the desired UAA. Today, over 150 non-canonical amino acids have been incorporated using such methods. This enables the introduction of a large variety of structures into proteins, in organisms ranging from prokaryote, yeast and mammalian cells lines to whole animals, enabling the study of protein function at a level that could not previously be achieved. While most research to date has focused on the suppression of 'non-sense' codons, recent developments are beginning to open up the possibility of quadruplet codon decoding and the more selective reassignment of sense codons, offering a potentially powerful tool for incorporating multiple amino acids. Here, we aim to provide a focused review of methods for UAA incorporation with an emphasis in particular on the different tRNA synthetase/tRNA pairs exploited or developed, focusing upon the different UAA structures that have been incorporated and the logic behind the design and future creation of such systems. Our hope is that this will help rationalize the design of systems for incorporation of unexplored unnatural amino acids, as well as novel applications for those already known.
- 127Wan, W.; Tharp, J. M.; Liu, W. R. Pyrrolysyl-tRNA Synthetase: An Ordinary Enzyme but an Outstanding Genetic Code Expansion Tool. Biochim. Biophys. Acta Proteins Proteomics 2014, 1844 (6), 1059– 1070, DOI: 10.1016/j.bbapap.2014.03.002Google Scholar127Pyrrolysyl-tRNA synthetase: An ordinary enzyme but an outstanding genetic code expansion toolWan, Wei; Tharp, Jeffery M.; Liu, Wenshe R.Biochimica et Biophysica Acta, Proteins and Proteomics (2014), 1844 (6), 1059-1070CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B. V.)A review. The genetic incorporation of the 22nd proteinogenic amino acid, pyrrolysine (Pyl) at the amber codon is achieved by the action of pyrrolysyl-tRNA synthetase (PylRS) together with its cognate tRNAPyl. Unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side-chain promiscuity, low selectivity toward its substrate α-amine, and low selectivity toward the anticodon of tRNAPyl. These unique but ordinary features of PylRS as an aminoacyl-tRNA synthetase allow the Pyl incorporation machinery to be easily engineered for the genetic incorporation of >100 noncanonical amino acids (NCAAs) or α-hydroxy acids into proteins at the amber codon and the reassignment of other codons such as ochre UAA, opal UGA, and 4-base AGGA codons to code NCAAs.
- 128Ryu, Y.; Schultz, P. G. Efficient Incorporation of Unnatural Amino Acids into Proteins in Escherichia coli. Nat. Methods 2006, 3 (4), 263– 265, DOI: 10.1038/nmeth864Google Scholar128Efficient incorporation of unnatural amino acids into proteins in Escherichia coliRyu, Youngha; Schultz, Peter G.Nature Methods (2006), 3 (4), 263-265CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)We have developed a single-plasmid system for the efficient bacterial expression of mutant proteins contg. unnatural amino acids at specific sites designated by amber nonsense codons. In this system, multiple copies of a gene encoding an amber suppressor tRNA derived from a Methanocaldococcus jannaschii tyrosyl-tRNA (MjtRNATyrCUA) are expressed under control of the proK promoter and terminator, and a gene encoding the desired mutant M. jannaschii tyrosyl-tRNA synthetase (MjTyrRS) is expressed under control of a mutant glnS (glnS') promoter.
- 129Srinivasan, G.; James, C. M.; Krzycki, J. A. Pyrrolysine Encoded by UAG in Archaea: Charging of a UAG-Decoding Specialized tRNA. Science 2002, 296 (5572), 1459– 1462, DOI: 10.1126/science.1069588Google Scholar129Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNASrinivasan, Gayathri; James, Carey M.; Krzycki, Joseph A.Science (Washington, DC, United States) (2002), 296 (5572), 1459-1462CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Pyrrolysine is a lysine deriv. encoded by the UAG codon in methylamine methyltransferase genes of Methanosarcina barkeri. Near a methyltransferase gene cluster is the pylT gene, which encodes an unusual tRNA with a CUA anticodon. The adjacent pylS gene encodes a class II aminoacyl-tRNA synthetase that charges the pylT-derived tRNA with lysine but is not closely related to known lysyl-tRNA synthetases. Homologs of pylS and pylT are found in a Gram-pos. bacterium. Charging a tRNACUA with lysine is a likely first step in translating UAG amber codons as pyrrolysine in certain methanogens. Our results indicate that pyrrolysine is the 22nd genetically encoded natural amino acid.
- 130Hao, B.; Gong, W.; Ferguson, T. K.; James, C. M.; Krzycki, J. A.; Chan, M. K. A New UAG-Encoded Residue in the Structure of a Methanogen Methyltransferase. Science 2002, 296 (5572), 1462– 1466, DOI: 10.1126/science.1069556Google Scholar130A new UAG-encoded residue in the structure of a methanogen methyltransferaseHao, Bing; Gong, Weimin; Ferguson, Tsuneo K.; James, Carey M.; Krzycki, Joseph A.; Chan, Michael K.Science (Washington, DC, United States) (2002), 296 (5572), 1462-1466CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Genes encoding methanogenic methylamine methyltransferases all contain an in-frame amber (UAG) codon that is read through during translation. We have identified the UAG-encoded residue in a 1.55 angstrom resoln. structure of the Methanosarcina barkeri monomethylamine methyltransferase (MtmB). This structure reveals a homohexamer comprised of individual subunits with a TIM barrel fold. The electron d. for the UAG-encoded residue is distinct from any of the 21 natural amino acids. Instead it appears consistent with a lysine in amide-linkage to (4R,5R)-4-substituted-pyrroline-5-carboxylate. We suggest that this amino acid be named L-pyrrolysine.
- 131Chin, J. W. Expanding and Reprogramming the Genetic Code of Cells and Animals. Annu. Rev. Biochem. 2014, 83 (1), 379– 408, DOI: 10.1146/annurev-biochem-060713-035737Google Scholar131Expanding and reprogramming the genetic code of cells and animalsChin, Jason W.Annual Review of Biochemistry (2014), 83 (), 379-408CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)Genetic code expansion and reprogramming enable the site-specific incorporation of diverse designer amino acids into proteins produced in cells and animals. Recent advances are enhancing the efficiency of unnatural amino acid incorporation by creating and evolving orthogonal ribosomes and manipulating the genome. Increasing the no. of distinct amino acids that can be site-specifically encoded has been facilitated by the evolution of orthogonal quadruplet decoding ribosomes and the discovery of mutually orthogonal synthetase/tRNA pairs. Rapid progress in moving genetic code expansion from bacteria to eukaryotic cells and animals (C. elegans and D. melanogaster) and the incorporation of useful unnatural amino acids has been aided by the development and application of the pyrrolysyl-tRNA (tRNA) synthetase/tRNA pair for unnatural amino acid incorporation. Combining chemoselective reactions with encoded amino acids has facilitated the installation of posttranslational modifications, as well as rapid derivatization with diverse fluorophores for imaging.
- 132Ranaghan, K. E.; Hung, J. E.; Bartlett, G. J.; Mooibroek, T. J.; Harvey, J. N.; Woolfson, D. N.; van der Donk, W. A.; Mulholland, A. J. A Catalytic Role for Methionine Revealed by a Combination of Computation and Experiments on Phosphite Dehydrogenase. Chem. Sci. 2014, 5 (6), 2191– 2199, DOI: 10.1039/C3SC53009DGoogle Scholar132A catalytic role for methionine revealed by a combination of computation and experiments on phosphite dehydrogenaseRanaghan, Kara E.; Hung, John E.; Bartlett, Gail J.; Mooibroek, Tiddo J.; Harvey, Jeremy N.; Woolfson, Derek N.; van der Donk, Wilfred A.; Mulholland, Adrian J.Chemical Science (2014), 5 (6), 2191-2199CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Combined quantum mechanics/mol. mechanics (QM/MM) simulations of the reaction catalyzed by phosphite dehydrogenase (PTDH) identify Met53 as important for catalysis. This catalytic role is verified by expts. (including replacement by norleucine and selenomethionine), which show that mutation of this residue significantly affects kcat, without changing KM for phosphite. QM/MM and ab initio QM calcns. show that the catalytic effect is electrostatic in nature. The side chain of Met53 specifically stabilizes the transition state for the hydride transfer step of the reaction catalyzed by PTDH, forming a 'face-on' interaction with His292. To our knowledge, a defined catalytic role for methionine in an enzyme (as opposed to a steric or binding effect, or interaction with a metal ion) has not previously been identified. Analyses of the Protein Data Bank and Cambridge Structural Database indicate that this type of interaction may be relatively widespread, with implications for enzyme-catalyzed reaction mechanisms and protein structure.
- 133Ekanayake, K. B.; Mahawaththa, M. C.; Qianzhu, H.; Abdelkader, E. H.; George, J.; Ullrich, S.; Murphy, R. B.; Fry, S. E.; Johansen-Leete, J.; Payne, R. J. Probing Ligand Binding Sites on Large Proteins by Nuclear Magnetic Resonance Spectroscopy of Genetically Encoded Non-Canonical Amino Acids. J. Med. Chem. 2023, 66 (7), 5289– 5304, DOI: 10.1021/acs.jmedchem.3c00222Google ScholarThere is no corresponding record for this reference.
- 134Ellman, J. A.; Volkman, B. F.; Mendel, D.; Schulz, P. G.; Wemmer, D. E. Site-Specific Isotopic Labeling of Proteins for NMR Studies. J. Am. Chem. Soc. 1992, 114 (20), 7959– 7961, DOI: 10.1021/ja00046a080Google Scholar134Site-specific isotopic labeling of proteins for NMR studiesEllman, Jonathan A.; Volkman, Brian F.; Mendel, David; Schulz, Peter G.; Wemmer, David E.Journal of the American Chemical Society (1992), 114 (20), 7959-61CODEN: JACSAT; ISSN:0002-7863.A single 13C-labeled alanine was site-specifically incorporated at position 82 of T4 lysozyme by in vitro suppression of an Ala 82 → TAG nonsense mutation with a chem. aminoacylated suppressor tRNA. The 13C-filtered proton NMR spectra obtained for this protein in both the native and denatured states clearly shows the Cα proton and Me group. The general methodol. described here should make possible a variety of detailed NMR studies of larger proteins, including the detn. of chem. shifts, pKA values, and relaxation parameters for individual amino acids in both the native and denatured states.
- 135Schmidt, M. J.; Borbas, J.; Drescher, M.; Summerer, D. A Genetically Encoded Spin Label for Electron Paramagnetic Resonance Distance Measurements. J. Am. Chem. Soc. 2014, 136 (4), 1238– 1241, DOI: 10.1021/ja411535qGoogle Scholar135A Genetically Encoded Spin Label for Electron Paramagnetic Resonance Distance MeasurementsSchmidt, Moritz J.; Borbas, Julia; Drescher, Malte; Summerer, DanielJournal of the American Chemical Society (2014), 136 (4), 1238-1241CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The authors report the genetic encoding of a noncanonical, spin-labeled amino acid in Escherichia coli. This enables the intracellular biosynthesis of spin-labeled proteins and obviates the need for any chem. labeling step usually required for protein EPR studies. The amino acid can be introduced at multiple, user-defined sites of a protein and is stable in E. coli even for prolonged expression times. It can report intramol. distance distributions in proteins by double-electron electron resonance measurements. Moreover, the signal of spin-labeled protein can be selectively detected in cells. This provides elegant new perspectives for in-cell EPR studies of endogenous proteins.
- 136Fafarman, A. T.; Boxer, S. G. Nitrile Bonds as Infrared Probes of Electrostatics in Ribonuclease S. J. Phys. Chem. B 2010, 114 (42), 13536– 13544, DOI: 10.1021/jp106406pGoogle Scholar136Nitrile Bonds as Infrared Probes of Electrostatics in Ribonuclease SFafarman, Aaron T.; Boxer, Steven G.Journal of Physical Chemistry B (2010), 114 (42), 13536-13544CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)Three different nitrile-contg. amino acids, p-cyanophenylalanine, m-cyanophenylalanine, and S-cyanohomocysteine, have been introduced near the active site of the semisynthetic enzyme RNase S to serve as probes of electrostatic fields. Vibrational Stark spectra, measured directly on the probe-modified proteins, confirm the predominance of the linear Stark tuning rate in describing the sensitivity of the nitrile stretch to external elec. fields, a necessary property for interpreting obsd. frequency shifts as a quant. measure of local elec. fields that can be compared with simulations. The x-ray structures of these nitrile-modified RNase variants and enzymic assays demonstrate minimal perturbation to the structure and function, resp., by the probes and provide a context for understanding the influence of the environment on the nitrile stretching frequency. The authors examine the ability of simulation techniques to recapitulate the spectroscopic properties of these nitriles as a means to directly test a computational electrostatic model for proteins, specifically that in the ubiquitous Amber-99 force field. Although qual. agreement between theory and expt. is obsd. for the largest shifts, substantial discrepancies are obsd. in some cases, highlighting the ongoing need for exptl. metrics to inform the development of theor. models of electrostatic fields in proteins.
- 137Xie, J.; Wang, L.; Wu, N.; Brock, A.; Spraggon, G.; Schultz, P. G. The Site-Specific Incorporation of p-Iodo-L-Phenylalanine into Proteins for Structure Determination. Nat. Biotechnol. 2004, 22 (10), 1297– 1301, DOI: 10.1038/nbt1013Google ScholarThere is no corresponding record for this reference.
- 138Summerer, D.; Chen, S.; Wu, N.; Deiters, A.; Chin, J. W.; Schultz, P. G. A Genetically Encoded Fluorescent Amino Acid. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (26), 9785– 9789, DOI: 10.1073/pnas.0603965103Google Scholar138A genetically encoded fluorescent amino acidSummerer, Daniel; Chen, Shuo; Wu, Ning; Deiters, Alexander; Chin, Jason W.; Schultz, Peter G.Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (26), 9785-9789CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The ability to introduce fluorophores selectively into proteins provides a powerful tool to study protein structure, dynamics, localization, and biomol. interactions both in vitro and in vivo. Here, we report a strategy for the selective and efficient biosynthetic incorporation of a low-mol.-wt. fluorophore into proteins at defined sites. The fluorescent amino acid 2-amino-3-(5-(dimethylamino)naphthalene-1-sulfonamide)propanoic acid (dansylalanine) was genetically encoded in Saccharomyces cerevisiae by using an amber nonsense codon and corresponding orthogonal tRNA/aminoacyl-tRNA synthetase pair. This environmentally sensitive fluorophore was selectively introduced into human superoxide dismutase and used to monitor unfolding of the protein in the presence of guanidinium chloride. The strategy described here should be applicable to a no. of different fluorophores in both prokaryotic and eukaryotic organisms, and it should facilitate both biochem. and cellular studies of protein structure and function.
- 139Lang, K.; Davis, L.; Wallace, S.; Mahesh, M.; Cox, D. J.; Blackman, M. L.; Fox, J. M.; Chin, J. W. Genetic Encoding of Bicyclononynes and trans-Cyclooctenes for Site-Specific Protein Labeling in Vitro and in Live Mammalian Cells via Rapid Fluorogenic Diels-Alder Reactions. J. Am. Chem. Soc. 2012, 134 (25), 10317– 10320, DOI: 10.1021/ja302832gGoogle Scholar139Genetic Encoding of Bicyclononynes and trans-Cyclooctenes for Site-Specific Protein Labeling in Vitro and in Live Mammalian Cells via Rapid Fluorogenic Diels-Alder ReactionsLang, Kathrin; Davis, Lloyd; Wallace, Stephen; Mahesh, Mohan; Cox, Daniel J.; Blackman, Melissa L.; Fox, Joseph M.; Chin, Jason W.Journal of the American Chemical Society (2012), 134 (25), 10317-10320CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Rapid, site-specific labeling of proteins with diverse probes remains an outstanding challenge for chem. biologists. Enzyme-mediated labeling approaches may be rapid but use protein or peptide fusions that introduce perturbations into the protein under study and may limit the sites that can be labeled, while many "bioorthogonal" reactions for which a component can be genetically encoded are too slow to effect quant. site-specific labeling of proteins on a time scale that is useful for studying many biol. processes. We report a fluorogenic reaction between bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) and tetrazines that is 3-7 orders of magnitude faster than many bioorthogonal reactions. Unlike the reactions of strained alkenes, including trans-cyclooctenes and norbornenes, with tetrazines, the BCN-tetrazine reaction gives a single product of defined stereochem. We have discovered aminoacyl-tRNA synthetase/tRNA pairs for the efficient site-specific incorporation of a BCN-contg. amino acid, 1, and a trans-cyclooctene-contg. amino acid 2 (which also reacts extremely rapidly with tetrazines) into proteins expressed in Escherichia coli and mammalian cells. We demonstrate the rapid fluorogenic labeling of proteins contg. 1 and 2 in vitro, in E. coli, and in live mammalian cells. These approaches may be extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on labeling and imaging studies.
- 140Plass, T.; Milles, S.; Koehler, C.; Schultz, C.; Lemke, E. A. Genetically Encoded Copper-Free Click Chemistry. Angew. Chem. Int. Ed. 2011, 50 (17), 3878– 3881, DOI: 10.1002/anie.201008178Google Scholar140Genetically Encoded Copper-Free Click ChemistryPlass, Tilman; Milles, Sigrid; Koehler, Christine; Schultz, Carsten; Lemke, Edward A.Angewandte Chemie, International Edition (2011), 50 (17), 3878-3881, S3878/1-S3878/13CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)One of the most potent functional groups for in vivo chem. has been genetically encoded into E. coli, and its basic utility for in vivo labeling as well as high-resoln. single-mol. measurements has been demonstrated. SPAAC (strain-promoted azide-alkyne cycloaddn.) chem. is now available to site-specifically and noninvasively modify proteins in living cells. As the tRNA'/pylRSA showed no obvious dependence on linker length (1 vs. 2), it is conceivable that slightly altered derivs., such as mono- and difluorinated cyclooctynes, and possibly bicyclonones, could be directly used in this system. Other enhanced cyclooctynes, such as dibenzocycloctynes, could pose substantial challenges to the synthetase and/or the host translational machinery owing to their larger size. As pylRS from M. mazei is orthogonal in a variety of eukaryotic organisms, we are now evaluating the transfer of this system to mammalian cells, where the technique would not only greatly expand our abilities to track proteins in living specimen but also to introduce other type of functional groups, such as cross-linkers or spin-labels for NMR spectroscopy and magnetic resonance imaging (MRI) in living specimens.
- 141Brustad, E. M.; Lemke, E. A.; Schultz, P. G.; Deniz, A. A. A General and Efficient Method for the Site-Specific Dual-Labeling of Proteins for Single Molecule Fluorescence Resonance Energy Transfer. J. Am. Chem. Soc. 2008, 130 (52), 17664– 17665, DOI: 10.1021/ja807430hGoogle Scholar141A General and Efficient Method for the Site-Specific Dual-Labeling of Proteins for Single Molecule Fluorescence Resonance Energy TransferBrustad, Eric M.; Lemke, Edward A.; Schultz, Peter G.; Deniz, Ashok A.Journal of the American Chemical Society (2008), 130 (52), 17664-17665CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A general strategy for the site-specific dual-labeling of proteins for single-mol. fluorescence resonance energy transfer is presented. A genetically encoded unnatural ketone amino acid was labeled with a hydroxylamine-contg. fluorophore with high yield (>95%) and specificity. This methodol. was used to construct dual-labeled T4 lysozyme variants, allowing the study of T4 lysozyme folding at single-mol. resoln. The presented strategy is anticipated to expand the scope of single-mol. protein structure and function studies.
- 142Loving, G.; Imperiali, B. A Versatile Amino Acid Analogue of the Solvatochromic Fluorophore 4-N,N-Dimethylamino-1,8-naphthalimide: A Powerful Tool for the Study of Dynamic Protein Interactions. J. Am. Chem. Soc. 2008, 130 (41), 13630– 13638, DOI: 10.1021/ja804754yGoogle Scholar142A Versatile Amino Acid Analogue of the Solvatochromic Fluorophore 4-N,N-Dimethylamino-1,8-naphthalimide: A Powerful Tool for the Study of Dynamic Protein InteractionsLoving, Galen; Imperiali, BarbaraJournal of the American Chemical Society (2008), 130 (41), 13630-13638CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The authors have developed a new unnatural amino acid based on the solvatochromic fluorophore 4-N,N-dimethylamino-1,8-naphthalimide (4-DMN) for application in the study of protein-protein interactions. The fluorescence quantum yield of this chromophore is highly sensitive to changes in the local solvent environment, demonstrating "switch-like" emission properties characteristic of the dimethylaminophthalimide family of fluorophores. In particular, this new species possesses a no. of significant advantages over related fluorophores, including greater chem. stability under a wide range of conditions, a longer wavelength of excitation (408 nm), and improved synthetic accessibility. This amino acid has been prepd. as an Fmoc-protected building block and may readily be incorporated into peptides via std. solid-phase peptide synthesis. A series of comparative studies are presented to demonstrate the advantageous properties of the 4-DMN amino acid relative to those of the previously reported 4-N,N-dimethylaminophthalimidoalanine and 6-N,N-dimethylamino-2,3-naphthalimidoalanine amino acids. Other com. available solvatochromic fluorophores are also include in these studies. The potential of this new probe as a tool for the study of protein-protein interactions is demonstrated by introducing it into a peptide that is recognized by calcium-activated calmodulin. The binding interaction between these two components yields an increase in fluorescence emission greater than 900-fold.
- 143Mendes, K. R.; Martinez, J. A.; Kantrowitz, E. R. Asymmetric Allosteric Signaling in Aspartate Transcarbamoylase. ACS Chem. Biol. 2010, 5 (5), 499– 506, DOI: 10.1021/cb9003207Google Scholar143Asymmetric Allosteric Signaling in Aspartate TranscarbamoylaseMendes, Kimberly R.; Martinez, Jessica A.; Kantrowitz, Evan R.ACS Chemical Biology (2010), 5 (5), 499-506CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Here we use the fluorescence from a genetically encoded unnatural amino acid, L-(7-hydroxycoumarin-4-yl)ethylglycine (HCE-Gly), replacing an amino acid in the regulatory site of Escherichia coli aspartate transcarbamoylase (ATCase) to decipher the mol. details of regulation of this allosteric enzyme. The fluorescence of HCE-Gly is exquisitely sensitive to the binding of all four nucleotide effectors. Although ATP and CTP are primarily responsible for influencing enzyme activity, the results of our fluorescent binding studies indicate that UTP and GTP bind with similar affinities, suggesting a dissocn. between nucleotide binding and control of enzyme activity. Furthermore, while CTP is the strongest regulator of enzyme activity, it binds selectively to only a fraction of regulatory sites, allowing UTP to effectively fill the residual ones. Our results suggest that CTP and UTP are not competing for the same binding sites, but instead reveal an asymmetry between the two allosteric sites on the regulatory subunit of the enzyme. Correlation of binding and activity measurements explain how ATCase uses asym. allosteric sites to achieve regulatory sensitivity over a broad range of heterotropic effector concns.
- 144Dean, S. F.; Whalen, K. L.; Spies, M. A. Biosynthesis of a Novel Glutamate Racemase Containing a Site-Specific 7-Hydroxycoumarin Amino Acid: Enzyme-Ligand Promiscuity Revealed at the Atomistic Level. ACS Cent. Sci. 2015, 1 (7), 364– 373, DOI: 10.1021/acscentsci.5b00211Google Scholar144Biosynthesis of a Novel Glutamate Racemase Containing a Site-Specific 7-Hydroxycoumarin Amino Acid: Enzyme-Ligand Promiscuity Revealed at the Atomistic LevelDean, Sondra F.; Whalen, Katie L.; Spies, M. AshleyACS Central Science (2015), 1 (7), 364-373CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)Glutamate racemase (GR) catalyzes the cofactor independent stereoinversion of L- to D-glutamate for biosynthesis of bacterial cell walls. Because of its essential nature, this enzyme is under intense scrutiny as a drug target for the design of novel antimicrobial agents. However, the flexibility of the enzyme has made inhibitor design challenging. Previous steered mol. dynamics (MD), docking, and exptl. studies have suggested that the enzyme forms highly varied complexes with different competitive inhibitor scaffolds. The current study employs a mutant orthogonal tRNA/aminoacyl-tRNA synthetase pair to genetically encode a non-natural fluorescent amino acid, L-(7-hydroxycoumarin-4-yl) ethylglycine (7HC), into a region (Tyr53) remote from the active site (previously identified by MD studies as undergoing ligand-assocd. changes) to generate an active mutant enzyme (GRY53/7HC). The GRY53/7HC enzyme is an active racemase, which permitted us to examine the nature of these idiosyncratic ligand-assocd. phenomena. One type of competitive inhibitor resulted in a dose-dependent quenching of the fluorescence of GRY53/7HC, while another type of competitive inhibitor resulted in a dose-dependent increase in fluorescence of GRY53/7HC. In order to investigate the environmental changes of the 7HC ring system that are distinctly assocd. with each of the GRY53/7HC-ligand complexes, and thus the source of the disparate quenching phenomena, a parallel computational study is described, which includes essential dynamics, ensemble docking and MD simulations of the relevant GRY53/7HC-ligand complexes. The changes in the solvent exposure of the 7HC ring system due to ligand-assocd. GR changes are consistent with the exptl. obsd. quenching phenomena. This study describes an approach for rationally predicting global protein allostery resulting from enzyme ligation to distinctive inhibitor scaffolds. The implications for fragment-based drug discovery and high throughput screening are discussed.
- 145Wang, J.; Xie, J.; Schultz, P. G. A Genetically Encoded Fluorescent Amino Acid. J. Am. Chem. Soc. 2006, 128 (27), 8738– 8739, DOI: 10.1021/ja062666kGoogle Scholar145A Genetically Encoded Fluorescent Amino AcidWang, Jiangyun; Xie, Jianming; Schultz, Peter G.Journal of the American Chemical Society (2006), 128 (27), 8738-8739CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The fluorescent amino acid L-(7-hydroxycoumarin-4-yl) ethylglycine 1 has been genetically encoded in E. coli in response to the amber TAG codon. Because of its high fluorescence quantum yield, relatively large Stoke's shift, and sensitivity to both pH and polarity, this amino acid should provide a useful probe of protein localization and trafficking, protein conformation changes, and protein-protein interactions.
- 146Li, M.; Peng, T. Genetic Encoding of a Fluorescent Noncanonical Amino Acid as a FRET Donor for the Analysis of Deubiquitinase Activities. In Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols; Tsai, Y.-H., Elsässer, S. J., Eds.; Springer: US, 2023; pp 55- 67. DOI: 10.1007/978-1-0716-3251-2_4Google ScholarThere is no corresponding record for this reference.
- 147Miyake-Stoner, S. J.; Miller, A. M.; Hammill, J. T.; Peeler, J. C.; Hess, K. R.; Mehl, R. A.; Brewer, S. H. Probing Protein Folding Using Site-Specifically Encoded Unnatural Amino Acids as FRET Donors with Tryptophan. Biochemistry 2009, 48 (25), 5953– 5962, DOI: 10.1021/bi900426dGoogle Scholar147Probing Protein Folding Using Site-Specifically Encoded Unnatural Amino Acids as FRET Donors with TryptophanMiyake-Stoner, Shigeki J.; Miller, Andrew M.; Hammill, Jared T.; Peeler, Jennifer C.; Hess, Kenneth R.; Mehl, Ryan A.; Brewer, Scott H.Biochemistry (2009), 48 (25), 5953-5962CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The exptl. study of protein folding is enhanced by the use of nonintrusive probes that are sensitive to local conformational changes in the protein structure. Here, we report the selection of an aminoacyl-tRNA synthetase/tRNA pair for the cotranslational, site-specific incorporation of two unnatural amino acids that can function as fluorescence resonance energy transfer (FRET) donors with Trp to probe the disruption of the hydrophobic core upon protein unfolding. L-4-Cyanophenylalanine (pCNPhe) and 4-ethynylphenylalanine (pENPhe) were incorporated into the hydrophobic core of the 171-residue protein, T4 lysozyme. The FRET donor ability of pCNPhe and pENPhe is evident by the overlap of the emission spectra of pCNPhe and pENPhe with the absorbance spectrum of Trp. The incorporation of both unnatural amino acids in place of a phenylalanine in the hydrophobic core of T4 lysozyme was well tolerated by the protein, due in part to the small size of the cyano and ethynyl groups. The hydrophobic nature of the ethynyl group of pENPhe suggests that this unnatural amino acid is a more conservative substitution into the hydrophobic core of the protein compared to pCNPhe. The urea-induced disruption of the hydrophobic core of the protein was probed by the change in FRET efficiency between either pCNPhe or pENPhe and the Trp residues in T4 lysozyme. The methodol. for the study of protein conformational changes using FRET presented here is of general applicability to the study of protein structural changes, since the incorporation of the unnatural amino acids is not inherently limited by the size of the protein.
- 148Bergfors, T. M. Protein crystallization. Internat’l University Line 2009. ISBN: 978–0-9720774–4-6.Google ScholarThere is no corresponding record for this reference.
- 149Sakamoto, K.; Murayama, K.; Oki, K.; Iraha, F.; Kato-Murayama, M.; Takahashi, M.; Ohtake, K.; Kobayashi, T.; Kuramitsu, S.; Shirouzu, M. Genetic Encoding of 3-Iodo-l-Tyrosine in Escherichia coli for Single-Wavelength Anomalous Dispersion Phasing in Protein Crystallography. Structure 2009, 17 (3), 335– 344, DOI: 10.1016/j.str.2009.01.008Google ScholarThere is no corresponding record for this reference.
- 150Lee, H. S.; Spraggon, G.; Schultz, P. G.; Wang, F. Genetic Incorporation of a Metal-Ion Chelating Amino Acid into Proteins as a Biophysical Probe. J. Am. Chem. Soc. 2009, 131 (7), 2481– 2483, DOI: 10.1021/ja808340bGoogle Scholar150Genetic Incorporation of a Metal-Ion Chelating Amino Acid into Proteins as a Biophysical ProbeLee, Hyun Soo; Spraggon, Glen; Schultz, Peter G.; Wang, FengJournal of the American Chemical Society (2009), 131 (7), 2481-2483CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A metal-ion chelating amino acid, (8-hydroxyquinolin-3-yl)alanine, was genetically encoded in Escherichia coli by an amber nonsense codon and corresponding orthogonal tRNA/aminoacyl-tRNA synthetase pair. The amino acid was incorporated into TM0665 protein, and the mutant protein was cocrystd. with Zn2+ to det. the structure by SAD phasing. The structure showed a high occupancy of the heavy metal bound to the HQ-Ala residue, and the heavy metal provided excellent phasing power to det. the structure. This method also facilitates the de novo design of metalloproteins with novel structures and functions, including fluorescent sensors.
- 151Nogly, P.; Weinert, T.; James, D.; Carbajo, S.; Ozerov, D.; Furrer, A.; Gashi, D.; Borin, V.; Skopintsev, P.; Jaeger, K. Retinal Isomerization in Bacteriorhodopsin Captured by a Femtosecond X-Ray Laser. Science 2018, 361 (6398), eaat0094 DOI: 10.1126/science.aat0094Google ScholarThere is no corresponding record for this reference.
- 152Tenboer, J.; Basu, S.; Zatsepin, N.; Pande, K.; Milathianaki, D.; Frank, M.; Hunter, M.; Boutet, S.; Williams, G. J.; Koglin, J. E. Time-Resolved Serial Crystallography Captures High-Resolution Intermediates of Photoactive Yellow Protein. Science 2014, 346 (6214), 1242– 1246, DOI: 10.1126/science.1259357Google Scholar152Time-resolved serial crystallography captures high-resolution intermediates of photoactive yellow proteinTenboer, Jason; Basu, Shibom; Zatsepin, Nadia; Pande, Kanupriya; Milathianaki, Despina; Frank, Matthias; Hunter, Mark; Boutet, Sebastien; Williams, Garth J.; Koglin, Jason E.; Oberthuer, Dominik; Heymann, Michael; Kupitz, Christopher; Conrad, Chelsie; Coe, Jesse; Roy-Chowdhury, Shatabdi; Weierstall, Uwe; James, Daniel; Wang, Dingjie; Grant, Thomas; Barty, Anton; Yefanov, Oleksandr; Scales, Jennifer; Gati, Cornelius; Seuring, Carolin; Srajer, Vukica; Henning, Robert; Schwander, Peter; Fromme, Raimund; Ourmazd, Abbas; Moffat, Keith; Van Thor, Jasper J.; Spence, John C. H.; Fromme, Petra; Chapman, Henry N.; Schmidt, MariusScience (Washington, DC, United States) (2014), 346 (6214), 1242-1246CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Serial femtosecond crystallog. using ultrashort pulses from x-ray free electron lasers (XFELs) enables studies of the light-triggered dynamics of biomols. The authors used microcrystals of photoactive yellow protein (a bacterial blue light photoreceptor) as a model system and obtained high-resoln., time-resolved difference electron d. maps of excellent quality with strong features; these allowed the detn. of structures of reaction intermediates to a resoln. of 1.6 Å. The authors' results open the way to the study of reversible and nonreversible biol. reactions on time scales as short as femtoseconds under conditions that maximize the extent of reaction initiation throughout the crystal.
- 153Suga, M.; Akita, F.; Sugahara, M.; Kubo, M.; Nakajima, Y.; Nakane, T.; Yamashita, K.; Umena, Y.; Nakabayashi, M.; Yamane, T. Light-Induced Structural Changes and the Site of O = O Bond Formation in PSII Caught by XFEL. Nature 2017, 543 (7643), 131– 135, DOI: 10.1038/nature21400Google Scholar153Light-induced structural changes and the site of O=O bond formation in PSII caught by XFELSuga, Michihiro; Akita, Fusamichi; Sugahara, Michihiro; Kubo, Minoru; Nakajima, Yoshiki; Nakane, Takanori; Yamashita, Keitaro; Umena, Yasufumi; Nakabayashi, Makoto; Yamane, Takahiro; Nakano, Takamitsu; Suzuki, Mamoru; Masuda, Tetsuya; Inoue, Shigeyuki; Kimura, Tetsunari; Nomura, Takashi; Yonekura, Shinichiro; Yu, Long-Jiang; Sakamoto, Tomohiro; Motomura, Taiki; Chen, Jing-Hua; Kato, Yuki; Noguchi, Takumi; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Hatsui, Takaki; Nango, Eriko; Tanaka, Rie; Naitow, Hisashi; Matsuura, Yoshinori; Yamashita, Ayumi; Yamamoto, Masaki; Nureki, Osamu; Yabashi, Makina; Ishikawa, Tetsuya; Iwata, So; Shen, Jian-RenNature (London, United Kingdom) (2017), 543 (7643), 131-135CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total mol. mass of 350 kDa for a monomer. It catalyzes light-driven water oxidn. at its catalytic center, the oxygen-evolving complex (OEC). The structure of PSII has been analyzed at 1.9 Å resoln. by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asym., 'distorted-chair' form. This structure was further analyzed with femtosecond X-ray free electron lasers (XFEL), providing the 'radiation damage-free' structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temp. at a resoln. of 2.35 Å using time-resolved serial femtosecond crystallog. with an XFEL provided by the SPring-8 angstrom compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water mol. located 3.5 Å from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water mol. and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent pos. peak around O5, a unique μ4-oxo-bridge located in the quasi-center of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously.
- 154Dods, R.; Båth, P.; Morozov, D.; Gagnér, V. A.; Arnlund, D.; Luk, H. L.; Kübel, J.; Maj, M.; Vallejos, A.; Wickstrand, C. Ultrafast Structural Changes within a Photosynthetic Reaction Centre. Nature 2021, 589 (7841), 310– 314, DOI: 10.1038/s41586-020-3000-7Google ScholarThere is no corresponding record for this reference.
- 155Chapman, H. N. X-Ray Free-Electron Lasers for the Structure and Dynamics of Macromolecules. Annu. Rev. Biochem. 2019, 88 (1), 35– 58, DOI: 10.1146/annurev-biochem-013118-110744Google ScholarThere is no corresponding record for this reference.
- 156Kern, J.; Chatterjee, R.; Young, I. D.; Fuller, F. D.; Lassalle, L.; Ibrahim, M.; Gul, S.; Fransson, T.; Brewster, A. S.; Alonso-Mori, R. Structures of the Intermediates of Kok’s Photosynthetic Water Oxidation Clock. Nature 2018, 563 (7731), 421– 425, DOI: 10.1038/s41586-018-0681-2Google Scholar156Structures of the intermediates of Kok's photosynthetic water oxidation clockKern, Jan; Chatterjee, Ruchira; Young, Iris D.; Fuller, Franklin D.; Lassalle, Louise; Ibrahim, Mohamed; Gul, Sheraz; Fransson, Thomas; Brewster, Aaron S.; Alonso-Mori, Roberto; Hussein, Rana; Zhang, Miao; Douthit, Lacey; de Lichtenberg, Casper; Cheah, Mun Hon; Shevela, Dmitry; Wersig, Julia; Seuffert, Ina; Sokaras, Dimosthenis; Pastor, Ernest; Weninger, Clemens; Kroll, Thomas; Sierra, Raymond G.; Aller, Pierre; Butryn, Agata; Orville, Allen M.; Liang, Mengning; Batyuk, Alexander; Koglin, Jason E.; Carbajo, Sergio; Boutet, Sebastien; Moriarty, Nigel W.; Holton, James M.; Dobbek, Holger; Adams, Paul D.; Bergmann, Uwe; Sauter, Nicholas K.; Zouni, Athina; Messinger, Johannes; Yano, Junko; Yachandra, Vittal K.Nature (London, United Kingdom) (2018), 563 (7731), 421-425CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed addnl. expts. and proposed a five-state kinetic model for photosynthetic oxygen evolution, known as Kok's S-state clock or cycle1,2. The model comprises four (meta)stable intermediates (S0, S1, S2 and S3) and one transient S4 state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn4CaO5) cluster in the oxygen-evolving complex. This reaction is coupled to the two-step redn. and protonation of the mobile plastoquinone QB at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallog. and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temp., we visualize all (meta)stable states of Kok's cycle as high-resoln. structures (2.04-2.08 Å). In addn., we report structures of two transient states at 150 and 400 μs, revealing notable structural changes including the binding of one addnl. 'water', Ox, during the S2→S3 state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the addnl. oxygen Ox in the S3 state between Ca and Mn1 supports O-O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O2 release. Thus, our results exclude peroxo-bond formation in the S3 state, and the nucleophilic attack of W3 onto W2 is unlikely.
- 157Nango, E.; Royant, A.; Kubo, M.; Nakane, T.; Wickstrand, C.; Kimura, T.; Tanaka, T.; Tono, K.; Song, C.; Tanaka, R. A Three-Dimensional Movie of Structural Changes in Bacteriorhodopsin. Science 2016, 354 (6319), 1552– 1557, DOI: 10.1126/science.aah3497Google Scholar157A three-dimensional movie of structural changes in bacteriorhodopsinNango, Eriko; Royant, Antoine; Kubo, Minoru; Nakane, Takanori; Wickstrand, Cecilia; Kimura, Tetsunari; Tanaka, Tomoyuki; Tono, Kensuke; Song, Changyong; Tanaka, Rie; Arima, Toshi; Yamashita, Ayumi; Kobayashi, Jun; Hosaka, Toshiaki; Mizohata, Eiichi; Nogly, Przemyslaw; Sugahara, Michihiro; Nam, Daewoong; Nomura, Takashi; Shimamura, Tatsuro; Im, Dohyun; Fujiwara, Takaaki; Yamanaka, Yasuaki; Jeon, Byeonghyun; Nishizawa, Tomohiro; Oda, Kazumasa; Fukuda, Masahiro; Andersson, Rebecka; Bath, Petra; Dods, Robert; Davidsson, Jan; Matsuoka, Shigeru; Kawatake, Satoshi; Murata, Michio; Nureki, Osamu; Owada, Shigeki; Kameshima, Takashi; Hatsui, Takaki; Joti, Yasumasa; Schertler, Gebhard; Yabashi, Makina; Bondar, Ana-Nicoleta; Standfuss, Joerg; Neutze, Richard; Iwata, SoScience (Washington, DC, United States) (2016), 354 (6319), 1552-1557CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Bacteriorhodopsin is a membrane protein that harvests the energy content from light to transport protons out of the cell against a transmembrane potential. Here, the authors used time-resolved serial femtosecond crystallog. at an x-ray free electron laser to provide 13 structural snapshots of the conformational changes that occur in the nanoseconds to milliseconds after photoactivation. These changes began at the active site, propagated toward the extracellular side of the protein, and mediated internal protonation exchanges that achieved proton transport.
- 158Liu, X.; Liu, P.; Li, H.; Xu, Z.; Jia, L.; Xia, Y.; Yu, M.; Tang, W.; Zhu, X.; Chen, C. Excited-State Intermediates in a Designer Protein Encoding a Phototrigger Caught by an X-Ray Free-Electron Laser. Nat. Chem. 2022, 14 (9), 1054– 1060, DOI: 10.1038/s41557-022-00992-3Google ScholarThere is no corresponding record for this reference.
- 159Hosaka, T.; Katsura, K.; Ishizuka-Katsura, Y.; Hanada, K.; Ito, K.; Tomabechi, Y.; Inoue, M.; Akasaka, R.; Takemoto, C.; Shirouzu, M. Crystal Structure of an Archaeal Tyrosyl-tRNA Synthetase Bound to Photocaged L-Tyrosine and Its Potential Application to Time-Resolved X-ray Crystallography. Int. J. Mol. Sci. 2022, 23 (18), 10399, DOI: 10.3390/ijms231810399Google ScholarThere is no corresponding record for this reference.
- 160Markley, J. L.; Putter, I.; Jardetzky, O. High-Resolution Nuclear Magnetic Resonance Spectra of Selectively Deuterated Staphylococcal Nuclease. Science 1968, 161 (3847), 1249– 1251, DOI: 10.1126/science.161.3847.1249Google ScholarThere is no corresponding record for this reference.
- 161Deiters, A.; Geierstanger, B. H.; Schultz, P. G. Site-Specific in vivo Labeling of Proteins for NMR Studies. ChemBioChem 2005, 6 (1), 55– 58, DOI: 10.1002/cbic.200400319Google ScholarThere is no corresponding record for this reference.
- 162Jones, D. H.; Cellitti, S. E.; Hao, X.; Zhang, Q.; Jahnz, M.; Summerer, D.; Schultz, P. G.; Uno, T.; Geierstanger, B. H. Site-Specific Labeling of Proteins with NMR-Active Unnatural Amino Acids. J. Biomol. NMR 2010, 46 (1), 89– 100, DOI: 10.1007/s10858-009-9365-4Google ScholarThere is no corresponding record for this reference.
- 163Abdelkader, E. H.; Qianzhu, H.; Huber, T.; Otting, G. Genetic Encoding of 7-Aza-l-tryptophan: Isoelectronic Substitution of a Single CH-Group in a Protein for a Nitrogen Atom for Site-Selective Isotope Labeling. ACS Sensors 2023, 8 (11), 4402– 4406, DOI: 10.1021/acssensors.3c01904Google ScholarThere is no corresponding record for this reference.
- 164Cellitti, S. E.; Jones, D. H.; Lagpacan, L.; Hao, X.; Zhang, Q.; Hu, H.; Brittain, S. M.; Brinker, A.; Caldwell, J.; Bursulaya, B. In vivo Incorporation of Unnatural Amino Acids to Probe Structure, Dynamics, and Ligand Binding in a Large Protein by Nuclear Magnetic Resonance Spectroscopy. J. Am. Chem. Soc. 2008, 130 (29), 9268– 9281, DOI: 10.1021/ja801602qGoogle Scholar164In vivo Incorporation of Unnatural Amino Acids to Probe Structure, Dynamics, and Ligand Binding in a Large Protein by Nuclear Magnetic Resonance SpectroscopyCellitti, Susan E.; Jones, David H.; Lagpacan, Leanna; Hao, Xueshi; Zhang, Qiong; Hu, Huiyong; Brittain, Scott M.; Brinker, Achim; Caldwell, Jeremy; Bursulaya, Badry; Spraggon, Glen; Brock, Ansgar; Ryu, Youngha; Uno, Tetsuo; Schultz, Peter G.; Geierstanger, Bernhard H.Journal of the American Chemical Society (2008), 130 (29), 9268-9281CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of NMR spectra. Incorporation is accomplished by coexpressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (OCF3Phe), 13C/15N-labeled p-methoxyphenylalanine (OMePhe), and 15N-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF3Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the resp. aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of Escherichia coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compd. Chem. shift changes in 1H-15N HSQC, 1H-13C HSQC, and 19F NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF3Phe mutants of an active site tyrosine inhibited binding; incorporating 15N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr reestablished binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with any other method.
- 165Lampe, J. N.; Floor, S. N.; Gross, J. D.; Nishida, C. R.; Jiang, Y.; Trnka, M. J.; Ortiz de Montellano, P. R. Ligand-Induced Conformational Heterogeneity of Cytochrome P450 CYP119 Identified by 2D NMR Spectroscopy with the Unnatural Amino Acid (13)C-p-Methoxyphenylalanine. J. Am. Chem. Soc. 2008, 130 (48), 16168– 16169, DOI: 10.1021/ja8071463Google Scholar165Ligand-Induced Conformational Heterogeneity of Cytochrome P450 CYP119 Identified by 2D NMR Spectroscopy with the Unnatural Amino Acid 13C-p-MethoxyphenylalanineLampe, Jed N.; Floor, Stephen N.; Gross, John D.; Nishida, Clinton R.; Jiang, Yongying; Trnka, Michael J.; Ortiz de Montellano, Paul R.Journal of the American Chemical Society (2008), 130 (48), 16168-16169CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Conformational dynamics are thought to play an important role in ligand binding and catalysis by cytochrome P 450 enzymes, but few techniques exist to examine them in mol. detail. Using a unique isotopic labeling strategy, we have site specifically inserted a 13C-labeled unnatural amino acid residue, 13C-p-methoxyphenylalanine (MeOF), into two different locations in the substrate binding region of the thermophilic cytochrome P 450 enzyme CYP119. Surprisingly, in both cases the resonance signal from the ligand-free protein is represented by a doublet in the 1H,13C-HSQC spectrum. Upon binding of 4-phenylimidazole, the signals from the initial resonances are reduced in favor of a single new resonance, in the case of the F162MeOF mutant, or two new resonances, in the case of the F153MeOF mutant. This represents the first direct phys. evidence for the ligand-dependent existence of multiple P 450 conformers simultaneously in soln. This general approach may be used to further illuminate the role that conformational dynamics plays in the complex enzymic phenomena exhibited by P 450 enzymes.
- 166Hull, W. E.; Sykes, B. D. Fluorotyrosine Alkaline Phosphatase. Fluorine-19 Nuclear Magnetic Resonance Relaxation Times and Molecular Motion of the Individual Fluorotyrosines. Biochemistry 1974, 13 (17), 3431– 3437, DOI: 10.1021/bi00714a002Google Scholar166Fluorotyrosine alkaline phosphatase. Fluorine-19 nuclear magnetic resonance relaxation times and molecular motion of the individual fluorotyrosinesHull, William E.; Sykes, Brian D.Biochemistry (1974), 13 (17), 3431-7CODEN: BICHAW; ISSN:0006-2960.Alk. phosphatase from Escherichia coli was labeled in vivo withm-fluorotyrosine and the 19F NMR spectrum of the fully activelabeled protein showed 11 resolvable resonances corresponding to the 11 known tyrosines/subunit. Nuclear spin relaxation times T1 and T2 were detd. for each 19F resonance. Consideration of the theory of dipole-dipole relaxation between unlike spins (1H and 19F) results in the following conclusions. First, the relaxation times are insensitive to internal rotation about the Cβ-arom. ring bond. Secondly, the data require that motion about the Cα-Cβ bond have a correlation time of ≥10-6 sec; hence, such motion does not contribute significantly to relaxation. All of the relaxation data are well represented by a model which assumes (1) isotropic motion of the protein as a whole with a rotational correlation time τc ≃ 70 nsec and (2) a varying degree of intermol. contribution to the 19F relaxation in tyrosine residues by protons on nearby residues. Finally, the intermol. relaxation exhibited a strong correlation with the 19F chem. shift; the contribution of intermol. relaxation was roughly proportional to the shift of a tyrosine from the position of the denatured protein resonance. Thus, 19F NMR is a very useful tool for studying the general tertiary or quaternary structure of a protein, its motional properties, and differences in the local environments of particular residues.
- 167Gamcsik, M. P.; Gerig, J. T. NMR Studies of Fluorophenylalanine-Containing Carbonic Anhydrase. FEBS Lett. 1986, 196 (1), 71– 74, DOI: 10.1016/0014-5793(86)80216-2Google ScholarThere is no corresponding record for this reference.
- 168Jackson, J. C.; Hammill, J. T.; Mehl, R. A. Site-Specific Incorporation of a 19F-Amino Acid into Proteins as an NMR Probe for Characterizing Protein Structure and Reactivity. J. Am. Chem. Soc. 2007, 129 (5), 1160– 1166, DOI: 10.1021/ja064661tGoogle Scholar168Site-Specific Incorporation of a 19F-Amino Acid into Proteins as an NMR Probe for Characterizing Protein Structure and ReactivityJackson, Jennifer C.; Hammill, Jared T.; Mehl, Ryan A.Journal of the American Chemical Society (2007), 129 (5), 1160-1166CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)19F NMR is a powerful tool for monitoring protein conformational changes and interactions; however, the inability to site-specifically introduce fluorine labels into proteins of biol. interest severely limits its applicability. Using methods for genetically directing incorporation of unnatural amino acids, the authors have inserted trifluoromethyl-L-phenylalanine (tfm-Phe) into proteins in vivo at TAG nonsense codons with high translational efficiency and fidelity. The binding of substrates, inhibitors, and cofactors, as well as reactions in enzymes, were studied by selective introduction of tfm-Phe and subsequent monitoring of the 19F NMR chem. shifts. Subtle protein conformational changes were detected near the active site and at long distances (25 Å). 19F signal sensitivity and resoln. was also sufficient to differentiate protein environments in vivo. Since there has been interest in using 19F-labeled proteins in solid-state membrane protein studies, folding studies, and in vivo studies, this general method for genetically incorporating a 19F-label into proteins of any size in Escherichia coli should have broad application beyond that of monitoring protein conformational changes.
- 169Hammill, J. T.; Miyake-Stoner, S.; Hazen, J. L.; Jackson, J. C.; Mehl, R. A. Preparation of Site-Specifically Labeled Fluorinated Proteins for 19F-NMR Structural Characterization. Nat. Protoc. 2007, 2 (10), 2601– 2607, DOI: 10.1038/nprot.2007.379Google ScholarThere is no corresponding record for this reference.
- 170Wacks, D. B.; Schachman, H. K. 19F Nuclear Magnetic Resonance Studies of Fluorotyrosine-Labeled Aspartate Transcarbamoylase. Properties of the Enzyme and Its Catalytic and Regulatory Subunits. J. Biol. Chem. 1985, 260 (21), 11651– 11658, DOI: 10.1016/S0021-9258(17)39080-4Google ScholarThere is no corresponding record for this reference.
- 171Gerig, J. T. Fluorine NMR of Proteins. Prog. Nucl. Magn. Reson. Spectrosc. 1994, 26, 293– 370, DOI: 10.1016/0079-6565(94)80009-XGoogle Scholar171Fluorine NMR of proteinsGerig, J. T.Progress in Nuclear Magnetic Resonance Spectroscopy (1994), 26 (4), 293-370CODEN: PNMRAT; ISSN:0079-6565.A review with 409 refs. demonstrating the scope of current applications of fluorine NMR to studies of protein structure and function. The authors focus on work that has been done over the past 10 yr. Topics covered include: receptor proteins, enzymes, reactions of proteins with fluorinated reagents, protein-fluorinated small mol. complexes, etc.
- 172Furter, R. Expansion of the Genetic Code: Site-Directed p-Fluoro-Phenylalanine Incorporation in Escherichia coli. Protein Sci. 1998, 7 (2), 419– 426, DOI: 10.1002/pro.5560070223Google Scholar172Expansion of the genetic code: site-directed p-fluoro-phenylalanine incorporation in Escherichia coliFurter, RolfProtein Science (1998), 7 (2), 419-426CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)Site-directed incorporation of the amino acid analog p-fluoro-phenylalanine (p-F-Phe) was achieved in Escherichia coli. A yeast suppressor tRNAamberPhe/phenylalanyl-tRNA synthetase pair was expressed in an analog-resistant E. coli strain to direct analog incorporation at a programmed amber stop codon in the DHFR marker protein. The programmed position was translated to 64-75% as p-F-Phe and the remainder as phenylalanine and lysine. Depending on the expression conditions, the p-F-Phe incorporation was 11-21-fold higher at the programmed position than the background incorporation at phenylalanine codons, showing high specificity of analog incorporation. Protein expression yields of 8-12 mg/L of culture, corresponding to about two thirds of the expression level of the wild-type DHFR protein, are sufficient to provide fluorinated proteins suitable for 19F-NMR spectroscopy and other sample-intensive methods. The use of a nonessential "21st" tRNA/synthetase pair will permit incorporation of a wide range of analogs, once the synthetase specificity has been modified accordingly.
- 173Kim, H.-W.; Perez, J. A.; Ferguson, S. J.; Campbell, I. D. The Specific Incorporation of Labelled Aromatic Amino Acids into Proteins through Growth of Bacteria in the Presence of Glyphosate. FEBS Lett. 1990, 272 (1–2), 34– 36, DOI: 10.1016/0014-5793(90)80442-LGoogle ScholarThere is no corresponding record for this reference.
- 174Niu, W.; Shu, Q.; Chen, Z.; Mathews, S.; Di Cera, E.; Frieden, C. The Role of Zn2+ on the Structure and Stability of Murine Adenosine Deaminase. J. Phys. Chem. B 2010, 114 (49), 16156– 16165, DOI: 10.1021/jp106041vGoogle Scholar174The role of Zn2+ on the structure and stability of murine adenosine deaminaseNiu, Weiling; Shu, Qin; Chen, Zhiwei; Mathews, Scott; Di Cera, Enrico; Frieden, CarlJournal of Physical Chemistry B (2010), 114 (49), 16156-16165CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)Adenosine deaminase (ADA) is a key enzyme in purine metab. and crucial for normal immune competence. It is a 40-kDa monomeric TIM-barrel protein contg. a tightly bound Zn2+, which is required for activity. Here, the authors investigated the role of Zn2+ with respect to ADA structure and stability. After removing Zn2+, the crystallog. structure of the protein remained highly ordered and similar to that of the holoprotein with structural changes limited to regions capping the active site pocket. The stability of the protein, however, was decreased significantly in the absence of Zn2+. Denaturation with urea showed the midpoint to be about 3.5M for the apoenzyme, compared with 6.4M for the holoenzyme. ADA contained 4 Trp residues distant from the Zn2+site; 19F NMR studies in the presence and absence of Zn2+ were carried out after incorporation of 6-19F-tryptophan. Chem. shift differences were obsd. for 3 of the 4 Trp residues, suggesting that, in contrast to the x-ray data, Zn2+-induced structural changes are propagated throughout the protein. Changes throughout the structure as suggested by the NMR data may explain the lower stability of the Zn2+-free protein. Real-time 19F NMR spectroscopy measuring the loss of Zn2+ showed that structural changes correlated with the loss of enzymic activity.
- 175Ruben, E. A.; Gandhi, P. S.; Chen, Z.; Koester, S. K.; DeKoster, G. T.; Frieden, C.; Di Cera, E. 19F NMR Reveals the Conformational Properties of Free Thrombin and Its Zymogen Precursor Prethrombin-2. J. Biol. Chem. 2020, 295 (24), 8227– 8235, DOI: 10.1074/jbc.RA120.013419Google ScholarThere is no corresponding record for this reference.
- 176Duewel, H.; Daub, E.; Robinson, V.; Honek, J. F. Incorporation of Trifluoromethionine into a Phage Lysozyme: Implications and a New Marker for Use in Protein 19F NMR. Biochemistry 1997, 36 (11), 3404– 3416, DOI: 10.1021/bi9617973Google Scholar176Incorporation of Trifluoromethionine into a Phage Lysozyme: Implications and a New Marker for Use in Protein 19F NMRDuewel, Henry; Daub, Elisabeth; Robinson, Valerie; Honek, John F.Biochemistry (1997), 36 (11), 3404-3416CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Much interest is currently focused on understanding the detailed contribution that particular amino acid residues make in protein structure and function. Although the use of site-directed mutagenesis has greatly contributed to this goal, the approach is limited to the std. repertoire of twenty amino acids. Fluorinated amino acids have been utilized successfully to probe protein structure and dynamics as well as point to the importance of specific residues to biol. function. In our continuing investigations on the importance of the amino acid methionine in biol. systems, the successful incorporation of L-S-trifluoromethylhomocysteine (L-trifluoromethionine; L-TFM) into bacteriophage λ lysozyme (LaL), an enzyme contg. three methionine residues, is reported. The L isomer of TFM was synthesized in an overall yield of 33% from N-acetyl-D,L-homocysteine thiolactone and trifluoromethyl iodide. An expression plasmid giving strong overprodn. of LaL was prepd. and transformed into an Escherichia coli strain auxotrophic for methionine permitting the expression of LaL in the presence of L-TFM. The analog would not support growth of the auxotroph and was found to be inhibitory to cell growth. However, cells that were initially grown in a Met-rich media followed by protein induction under careful control of the resp. concns. of L-Met and L-TFM in the media, were able to overexpress TFM-labeled LaL (TFM-LaL) at both high (70%) and low (31%) levels of TFM incorporation. TFM-LaL at both levels of incorporation exhibited analogous activity to the wild type enzyme and were inhibited by chitooligosaccharides indicating that incorporation of the analog did not hinder enzyme function. Interestingly, the 19F soln. NMR spectra of the TFM-labeled enzymes consisted of four sharp resonances spanning a chem. shift range of 0.9 ppm, with three of the resonances showing very modest shielding changes on binding of chitopentaose. The 19F NMR anal. of TFM-LaL at both high and low levels of incorporation suggested that one of the methionine positions gives rise to two sep. resonances. The intensities of these two resonances were influenced by the extent of incorporation which was interpreted as an indication that subtle conformational changes in protein structure are induced by incorporated TFM. The similarities and differences between Met and TFM were analyzed using ab initio MO calcns. The methodol. presented offers promise as a new approach to the study of protein-ligand interactions as well as for future investigations into the functional importance of methionine in proteins.
- 177Cleve, P.; Robinson, V.; Duewel, H. S.; Honek, J. F. Difluoromethionine as a Novel 19F NMR Structural Probe for Internal Amino Acid Packing in Proteins. J. Am. Chem. Soc. 1999, 121 (37), 8475– 8478, DOI: 10.1021/ja9911418Google Scholar177Difluoromethionine as a Novel 19F NMR Structural Probe for Internal Amino Acid Packing in ProteinsVaughan, Mark D.; Cleve, Paul; Robinson, Valerie; Duewel, Henry S.; Honek, John F.Journal of the American Chemical Society (1999), 121 (37), 8475-8478CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The successful incorporation of difluoromethionine (DFM), a novel 19F NMR probe of internal amino acid packing, into the three methionine positions (1, 14, and 107) of a recombinant protein, the lysozyme from bacteriophage λ (LaL), is reported. The anisochronous 19F NMR signals of the diastereotopic fluorines showed a variation in the degree of chem. shift difference when present at relatively free surface positions (Met1 and Met107) vs. the tightly packed protein core (Met14), with the anisochronicity greatly enhanced for DFM incorporated at this latter position. The increased magnetic nonequivalence of the two fluorines at position 14 is thought to be a consequence of the restricted environment of DFM at this position. The anisochronicity of these two fluorines is further manifested in a differential chem. shift change for these two fluorines upon binding of an oligosaccharide inhibitor to LaL, with one of the two fluorines experiencing a significant upfield shift compared to the other. This differential variation is thought to be assocd. with a very subtle change in the protein conformation surrounding one fluorine at position 14, which is not significantly translated to the environment of the other fluorine.
- 178Holzberger, B.; Rubini, M.; Möller, H. M.; Marx, A. A Highly Active DNA Polymerase with a Fluorous Core. Angew. Chem. Int. Ed. 2010, 49 (7), 1324– 1327, DOI: 10.1002/anie.200905978Google Scholar178A highly active DNA polymerase with a fluorous coreHolzberger, Bastian; Rubini, Marina; Moeller, Heiko M.; Marx, AndreasAngewandte Chemie, International Edition (2010), 49 (7), 1324-1327, S1324/1-S1324/8CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)DNA polymerases catalyze all DNA synthesis in the cell and are key tools in important mol. biol. core technologies. Apart from naturally available DNA polymerases, several modified DNA polymerases with new characteristics have been developed. To date, directed evolution using the 20 natural amino acids is a promising method for the creation of nucleic acid polymerases with modified properties. Yet, the incorporation of non-natural amino acids may lead to enhanced chem. and biol. diversity of protein structures and properties by introduction of functional groups that are not represented by the natural amino acids. Herein, the authors present the generation of a multifluorinated DNA polymerase. The N-terminally truncated version of DNA polymerase I from Thermus aquaticus (KlenTaq) is a thermophilic DNA polymerase composed of 540 amino acids (63 kDa), including 13 methionine (Met) residues that were globally replaced by trifluoromethionine (TFM) with a substitution level of approx. 82%. The multifluorinated KlenTaq was highly active and exhibited a similar selectivity as the wild-type (wt.) enzyme. Moreover, the introduction of the NMR-active nucleus 19F offers the possibility to study DNA polymerase dynamics by 19F NMR spectroscopy. Despite its large size of 63 kDa, at least nine individual 19F resonances are obsd., which allow us to distinguish different states of the DNA polymerase on the way to incorporating a canonical or a noncanonical nucleotide. To our knowledge, this is by far the largest enzymically active protein with Met globally replaced by TFM.
- 179Orton, H. W.; Qianzhu, H.; Abdelkader, E. H.; Habel, E. I.; Tan, Y. J.; Frkic, R. L.; Jackson, C. J.; Huber, T.; Otting, G. Through-Space Scalar 19F-19F Couplings between Fluorinated Noncanonical Amino Acids for the Detection of Specific Contacts in Proteins. J. Am. Chem. Soc. 2021, 143 (46), 19587– 19598, DOI: 10.1021/jacs.1c10104Google ScholarThere is no corresponding record for this reference.
- 180Miao, Q.; Nitsche, C.; Orton, H.; Overhand, M.; Otting, G.; Ubbink, M. Paramagnetic Chemical Probes for Studying Biological Macromolecules. Chem. Rev. 2022, 122 (10), 9571– 9642, DOI: 10.1021/acs.chemrev.1c00708Google Scholar180Paramagnetic Chemical Probes for Studying Biological MacromoleculesMiao, Qing; Nitsche, Christoph; Orton, Henry; Overhand, Mark; Otting, Gottfried; Ubbink, MarcellusChemical Reviews (Washington, DC, United States) (2022), 122 (10), 9571-9642CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Paramagnetic chem. probes have been used in ESR (EPR) and NMR (NMR) spectroscopy for more than four decades. Recent years witnessed a great increase in the variety of probes for the study of biol. macromols. (proteins, nucleic acids, and oligosaccharides). This Review aims to provide a comprehensive overview of the existing paramagnetic chem. probes, including chem. synthetic approaches, functional properties, and selected applications. Recent developments have seen, in particular, a rapid expansion of the range of lanthanoid probes with anisotropic magnetic susceptibilities for the generation of structural restraints based on residual dipolar couplings and pseudocontact shifts in soln. and solid state NMR spectroscopy, mostly for protein studies. Also many new isotropic paramagnetic probes, suitable for NMR measurements of paramagnetic relaxation enhancements, as well as EPR spectroscopic studies (in particular double resonance techniques) have been developed and employed to investigate biol. macromols. Notwithstanding the large no. of reported probes, only few have found broad application and further development of probes for dedicated applications is foreseen.
- 181Fanucci, G. E.; Cafiso, D. S. Recent advances and applications of site-directed spin labeling. Curr. Opin. Struct. Biol. 2006, 16 (5), 644– 653, DOI: 10.1016/j.sbi.2006.08.008Google Scholar181Recent advances and applications of site-directed spin labelingFanucci, Gail E.; Cafiso, David S.Current Opinion in Structural Biology (2006), 16 (5), 644-653CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. Site-directed spin labeling has become a popular biophys. tool for the characterization of protein structure, dynamics and conformational change. This method is well suited and widely used to study small sol. proteins, membrane proteins and large protein complexes. Recent advances in site-directed spin labeling methodol. have occurred in two areas. The first involves an understanding of the conformations and local dynamics of the spin-labeled side chain, including the features of proteins that influence ESR lineshape. The second advance is the application of pulse techniques to det. long-range distances and distance distributions in proteins. During the past two years, these tech. developments have been used to address several important problems concerning the mol. function of proteins.
- 182Braun, T.; Drescher, M.; Summerer, D. Expanding the Genetic Code for Site-Directed Spin-Labeling. Int. J. Mol. Sci. 2019, 20 (2), 373, DOI: 10.3390/ijms20020373Google ScholarThere is no corresponding record for this reference.
- 183Kálai, T.; Fleissner, M. R.; Jeko, J.; Hubbell, W. L.; Hideg, K. Synthesis of New Spin Labels for Cu-Free Click Conjugation. Tetrahedron Lett. 2011, 52 (21), 2747– 2749, DOI: 10.1016/j.tetlet.2011.03.077Google ScholarThere is no corresponding record for this reference.
- 184Kugele, A.; Braun, T. S.; Widder, P.; Williams, L.; Schmidt, M. J.; Summerer, D.; Drescher, M. Site-Directed Spin Labelling of Proteins by Suzuki-Miyaura Coupling via a Genetically Encoded Aryliodide Amino Acid. Chem. Commun. 2019, 55 (13), 1923– 1926, DOI: 10.1039/C8CC09325CGoogle Scholar184Site-directed spin labelling of proteins by Suzuki-Miyaura coupling via a genetically encoded aryliodide amino acidKugele Anandi; Braun Theresa Sophie; Widder Pia; Williams Lara; Schmidt Moritz Johannes; Summerer Daniel; Drescher MalteChemical communications (Cambridge, England) (2019), 55 (13), 1923-1926 ISSN:.We report site-directed protein spin labelling via Suzuki-Miyaura coupling of a nitroxide boronic acid label with the genetically encoded amino acid 4-iodo-l-phenylalanine. The resulting spin label bears a rigid biphenyl linkage with lower flexibility than spin label R1. It is suitable to obtain defined electron paramagnetic resonance distance distributions and to report protein-membrane interactions and conformational transitions of α-synuclein.
- 185Jana, S.; Evans, E. G. B.; Jang, H. S.; Zhang, S.; Zhang, H.; Rajca, A.; Gordon, S. E.; Zagotta, W. N.; Stoll, S.; Mehl, R. A. Ultrafast Bioorthogonal Spin-Labeling and Distance Measurements in Mammalian Cells Using Small, Genetically Encoded Tetrazine Amino Acids. J. Am. Chem. Soc. 2023, 145 (27), 14608– 14620, DOI: 10.1021/jacs.3c00967Google Scholar185Ultrafast Bioorthogonal Spin-Labeling and Distance Measurements in Mammalian Cells Using Small, Genetically Encoded Tetrazine Amino AcidsJana, Subhashis; Evans, Eric G. B.; Jang, Hyo Sang; Zhang, Shuyang; Zhang, Hui; Rajca, Andrzej; Gordon, Sharona E.; Zagotta, William N.; Stoll, Stefan; Mehl, Ryan A.Journal of the American Chemical Society (2023), 145 (27), 14608-14620CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Site-directed spin-labeling (SDSL)-in combination with double electron-electron resonance (DEER) spectroscopy-has emerged as a powerful technique for detg. both the structural states and the conformational equil. of biomacromols. DEER combined with in situ SDSL in live cells is challenging since current bioorthogonal labeling approaches are too slow to allow for complete labeling with low concns. of spin label prior to loss of signal from cellular redn. Here, we overcome this limitation by genetically encoding a novel family of small, tetrazine-bearing noncanonical amino acids (Tet-v4.0) at multiple sites in proteins expressed in Escherichia coli and in human HEK293T cells. We achieved specific and quant. spin-labeling of Tet-v4.0-contg. proteins by developing a series of strained trans-cyclooctene (sTCO)-functionalized nitroxides-including a gem-diethyl-substituted nitroxide with enhanced stability in cells-with rate consts. that can exceed 106 M-1 s-1. The remarkable speed of the Tet-v4.0/sTCO reaction allowed efficient spin-labeling of proteins in live cells within minutes, requiring only sub-micromolar concns. of sTCO-nitroxide. DEER recorded from intact cells revealed distance distributions in good agreement with those measured from proteins purified and labeled in vitro. Furthermore, DEER was able to resolve the maltose-dependent conformational change of Tet-v4.0-incorporated and spin-labeled MBP in vitro and support assignment of the conformational state of an MBP mutant within HEK293T cells. We anticipate the exceptional reaction rates of this system, combined with the relatively short and rigid side chains of the resulting spin labels, will enable structure/function studies of proteins directly in cells, without any requirements for protein purifn.
- 186Fleissner, M. R.; Brustad, E. M.; Kálai, T.; Altenbach, C.; Cascio, D.; Peters, F. B.; Hideg, K.; Peuker, S.; Schultz, P. G.; Hubbell, W. L. Site-Directed Spin Labeling of a Genetically Encoded Unnatural Amino Acid. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (51), 21637– 21642, DOI: 10.1073/pnas.0912009106Google Scholar186Site-directed spin labeling of a genetically encoded unnatural amino acidFleissner, Mark R.; Brustad, Eric M.; Kalai, Tamas; Altenbach, Christian; Cascio, Duilio; Peters, Francis B.; Hideg, Kalman; Schultz, Peter G.; Hubbell, Wayne L.Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (51), 21637-21642, S21637/1-S21637/10CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The traditional site-directed spin labeling (SDSL) method, which utilizes cysteine residues and sulfhydryl-reactive nitroxide reagents, can be challenging for proteins that contain functionally important native cysteine residues or disulfide bonds. To make SDSL amenable to any protein, we introduce an orthogonal labeling strategy, i.e., one that does not rely on any of the functional groups found in the common 20 amino acids. In this method, the genetically encoded unnatural amino acid p-acetyl-L-phenylalanine (p-AcPhe) is reacted with a hydroxylamine reagent to generate a nitroxide side chain (K1). The utility of this scheme was demonstrated with seven mutants of T4 lysozyme, each contg. a single p-AcPhe at a solvent-exposed helix site; the mutants were expressed in amts. qual. similar to the wild-type protein. In general, the EPR spectra of the resulting K1 mutants reflect higher nitroxide mobilities than the spectra of analogous mutants contg. the more constrained disulfide-linked side chain (R1) commonly used in SDSL. Despite this increased flexibility, site dependence of the EPR spectra suggests that K1 will be a useful sensor of local structure and of conformational changes in soln. Distance measurements between pairs of K1 residues using double electron electron resonance (DEER) spectroscopy indicate that K1 will also be useful for distance mapping.
- 187Nguyen, D. P.; Lusic, H.; Neumann, H.; Kapadnis, P. B.; Deiters, A.; Chin, J. W. Genetic Encoding and Labeling of Aliphatic Azides and Alkynes in Recombinant Proteins via a Pyrrolysyl-tRNA Synthetase/tRNACUA Pair and Click Chemistry. J. Am. Chem. Soc. 2009, 131 (25), 8720– 8721, DOI: 10.1021/ja900553wGoogle Scholar187Genetic Encoding and Labeling of Aliphatic Azides and Alkynes in Recombinant Proteins via a Pyrrolysyl-tRNA Synthetase/tRNACUA Pair and Click ChemistryNguyen, Duy P.; Lusic, Hrvoje; Neumann, Heinz; Kapadnis, Prashant B.; Deiters, Alexander; Chin, Jason W.Journal of the American Chemical Society (2009), 131 (25), 8720-8721CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We demonstrate that an orthogonal Methanosarcina barkeri MS pyrrolysyl-tRNA synthetase/tRNACUA pair directs the efficient, site-specific incorporation of N6-[(2-propynyloxy)carbonyl]-L-lysine, contg. a carbon-carbon triple bond, and N6-[(2-azidoethoxy)carbonyl]-L-lysine, contg. an azido group, into recombinant proteins in Escherichia coli. Proteins contg. the alkyne functional group are labeled with an azido biotin and an azido fluorophore, via copper catalyzed [3+2] cycloaddn. reactions, to produce the corresponding triazoles in good yield. The methods reported are useful for the site-specific labeling of recombinant proteins and may be combined with mutually orthogonal methods of introducing unnatural amino acids into proteins as well as with chem. orthogonal methods of protein labeling. This should allow the site specific incorporation of multiple distinct probes into proteins and the control of protein topol. and structure by intramol. orthogonal conjugation reactions.
- 188Chin, J. W.; Santoro, S. W.; Martin, A. B.; King, D. S.; Wang, L.; Schultz, P. G. Addition of p-Azido-l-phenylalanine to the Genetic Code of Escherichia coli. J. Am. Chem. Soc. 2002, 124 (31), 9026– 9027, DOI: 10.1021/ja027007wGoogle Scholar188Addition of p-Azido-L-phenylalanine to the Genetic Code of Escherichia coliChin, Jason W.; Santoro, Stephen W.; Martin, Andrew B.; King, David S.; Wang, Lei; Schultz, Peter G.Journal of the American Chemical Society (2002), 124 (31), 9026-9027CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We report the selection of a new orthogonal aminoacyl tRNA synthetase/tRNA pair for the in vivo incorporation of a photocrosslinker, p-azido-L-phenylalanine, into proteins in response to the amber codon, TAG. The amino acid is incorporated in good yield with high fidelity and can be used to crosslink interacting proteins.
- 189Kucher, S.; Korneev, S.; Tyagi, S.; Apfelbaum, R.; Grohmann, D.; Lemke, E. A.; Klare, J. P.; Steinhoff, H.-J.; Klose, D. Orthogonal Spin Labeling Using Click Chemistry for in vitro and in vivo Applications. J. Magn. Reson. 2017, 275, 38– 45, DOI: 10.1016/j.jmr.2016.12.001Google Scholar189Orthogonal spin labeling using click chemistry for in vitro and in vivo applicationsKucher, Svetlana; Korneev, Sergei; Tyagi, Swati; Apfelbaum, Ronja; Grohmann, Dina; Lemke, Edward A.; Klare, Johann P.; Steinhoff, Heinz-Juergen; Klose, DanielJournal of Magnetic Resonance (2017), 275 (), 38-45CODEN: JMARF3; ISSN:1090-7807. (Elsevier B.V.)Site-directed spin labeling for EPR- and NMR spectroscopy has mainly been achieved exploiting the specific reactivity of cysteines. For proteins with native cysteines or for in vivo applications, an alternative coupling strategy is required. In these cases click chem. offers major benefits by providing a fast and highly selective, biocompatible reaction between azide and alkyne groups. Here, we establish click chem. as a tool to target unnatural amino acids in vitro and in vivo using azide- and alkyne-functionalized spin labels. The approach is compatible with a variety of labels including redn.-sensitive nitroxides. Comparing spin labeling efficiencies from the copper-free with the strongly reducing copper(I)-catalyzed azide-alkyne click reaction, we find that the faster kinetics for the catalyzed reaction outrun redn. of the labile nitroxide spin labels and allow quant. labeling yields within short reaction times. Inter-spin distance measurements demonstrate that the novel side chain is suitable for paramagnetic NMR- or EPR-based conformational studies of macromol. complexes.
- 190Abdelkader, E. H.; Feintuch, A.; Yao, X.; Adams, L. A.; Aurelio, L.; Graham, B.; Goldfarb, D.; Otting, G. Protein Conformation by EPR Spectroscopy Using Gadolinium Tags Clicked to Genetically Encoded p-Azido-L-Phenylalanine. Chem. Commun. 2015, 51 (88), 15898– 15901, DOI: 10.1039/C5CC07121FGoogle ScholarThere is no corresponding record for this reference.
- 191Loh, C. T.; Ozawa, K.; Tuck, K. L.; Barlow, N.; Huber, T.; Otting, G.; Graham, B. Lanthanide Tags for Site-Specific Ligation to an Unnatural Amino Acid and Generation of Pseudocontact Shifts in Proteins. Bioconjug. Chem. 2013, 24 (2), 260– 268, DOI: 10.1021/bc300631zGoogle ScholarThere is no corresponding record for this reference.
- 192Mahawaththa, M. C.; Lee, M. D.; Giannoulis, A.; Adams, L. A.; Feintuch, A.; Swarbrick, J. D.; Graham, B.; Nitsche, C.; Goldfarb, D.; Otting, G. Small Neutral Gd(iii) Tags for Distance Measurements in Proteins by Double Electron-Electron Resonance Experiments. Phys. Chem. Chem. Phys. 2018, 20 (36), 23535– 23545, DOI: 10.1039/C8CP03532FGoogle ScholarThere is no corresponding record for this reference.
- 193Yang, H.; Yang, S.; Kong, J.; Dong, A.; Yu, S. Obtaining Information About Protein Secondary Structures in Aqueous Solution Using Fourier Transform IR Spectroscopy. Nat. Protoc. 2015, 10 (3), 382– 396, DOI: 10.1038/nprot.2015.024Google Scholar193Obtaining information about protein secondary structures in aqueous solution using Fourier transform IR spectroscopyYang, Huayan; Yang, Shouning; Kong, Jilie; Dong, Aichun; Yu, ShaoningNature Protocols (2015), 10 (3), 382-396CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Fourier transform IR (FTIR) spectroscopy is a nondestructive technique for structural characterization of proteins and polypeptides. The IR spectral data of polymers are usually interpreted in terms of the vibrations of a structural repeat. The repeat units in proteins give rise to nine characteristic IR absorption bands (amides A, B and I-VII). Amide I bands (1,700-1,600 cm-1) are the most prominent and sensitive vibrational bands of the protein backbone, and they relate to protein secondary structural components. In this protocol, we have detailed the principles that underlie the detn. of protein secondary structure by FTIR spectroscopy, as well as the basic steps involved in protein sample prepn., instrument operation, FTIR spectra collection and spectra anal. in order to est. protein secondary-structural components in aq. (both H2O and deuterium oxide (D2O)) soln. using algorithms, such as second-deriv., deconvolution and curve fitting. Small amts. of high-purity (>95%) proteins at high concns. (>3 mg ml-1) are needed in this protocol; typically, the procedure can be completed in 1-2 d.
- 194Lorenz-Fonfria, V. A. Infrared Difference Spectroscopy of Proteins: From Bands to Bonds. Chem. Rev. 2020, 120 (7), 3466– 3576, DOI: 10.1021/acs.chemrev.9b00449Google Scholar194Infrared Difference Spectroscopy of Proteins: From Bands to BondsLorenz-Fonfria, Victor A.Chemical Reviews (Washington, DC, United States) (2020), 120 (7), 3466-3576CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. IR difference spectroscopy probes vibrational changes of proteins upon their perturbation. Compared with other spectroscopic methods it stands out by its sensitivity to the protonation state, H-bonding and the conformation of different groups in proteins, including the peptide backbone, amino acid side chains, internal water mols. or cofactors. In particular, the detection of protonation and H-bonding changes in a time-resolved manner, not easily obtained by other techniques, is one of the most successful applications of IR difference spectroscopy. The present review deals with the use of perturbations designed to specifically change the protein between two (or more) functionally relevant states, a strategy often referred to as reaction-induced IR difference spectroscopy. In the first half of this contribution I review the technique of reaction-induced IR difference spectroscopy of proteins, with special emphasis given to the prepn. of suitable samples and their characterization; strategies for protein perturbations; and methodologies for time-resolved measurements (from nanoseconds to minutes). The second half of this contribution focus on the spectral interpretation. It starts by reviewing how changes in H-bonding, medium polarity, and vibrational coupling affect vibrational frequencies, intensities and bandwidths. It is followed by band assignments, a crucial aspect mostly performed with the help of isotopic labeling and site-directed mutagenesis, and completed by integrating and interpreting the results in the context of the studied protein, an aspect increasingly supported by spectral calcns. Selected examples from the literature, predominately but not exclusively from retinal proteins, were used to illustrate the topics covered in this review.
- 195Chung, J. K.; Thielges, M. C.; Fayer, M. D. Dynamics of the Folded and Unfolded Villin Headpiece (HP35) Measured with Ultrafast 2D IR Vibrational Echo Spectroscopy. Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (9), 3578– 3583, DOI: 10.1073/pnas.1100587108Google Scholar195Dynamics of the folded and unfolded villin headpiece (HP35) measured with ultrafast 2D IR vibrational echo spectroscopyChung, Jean K.; Thielges, Megan C.; Fayer, Michael D.Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (9), 3578-3583, S3578/1-S3578/5CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A series of two-dimensional IR vibrational echo expts. performed on nitrile-labeled villin headpiece [HP35-(CN)2] is described. HP35 is a small peptide composed of three alpha helixes in the folded configuration. The dynamics of the folded HP35-(CN)2 are compared to that of the guanidine-induced unfolded peptide, as well as the nitrile-functionalized phenylalanine (PheCN), which is used to differentiate the peptide dynamic contributions to the observables from those of the water solvent. Because the viscosity of solvent has a significant effect on fast dynamics, the viscosity of the solvent is held const. by adding glycerol. For the folded peptide, the addn. of glycerol to the water solvent causes observable slowing of the peptide's dynamics. Holding the viscosity const. as GuHCl is added, the dynamics of unfolded peptide are much faster than those of the folded peptide, and they are very similar to that of PheCN. These observations indicate that the local environment of the nitrile in the unfolded peptide resembles that of PheCN, and the dynamics probed by the CN are dominated by the fluctuations of the solvent mols., in contrast to the observations on the folded peptide.
- 196Chung, J. K.; Thielges, M. C.; Fayer, M. D. Conformational Dynamics and Stability of HP35 Studied with 2D IR Vibrational Echoes. J. Am. Chem. Soc. 2012, 134 (29), 12118– 12124, DOI: 10.1021/ja303017dGoogle Scholar196Conformational Dynamics and Stability of HP35 Studied with 2D IR Vibrational EchoesChung, Jean K.; Thielges, Megan C.; Fayer, Michael D.Journal of the American Chemical Society (2012), 134 (29), 12118-12124CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Two-dimensional IR (2D IR) vibrational echo spectroscopy was used to measure the fast dynamics of two variants of chicken villin headpiece 35 (HP35). The CN of cyanophenylalanine residues inserted in the hydrophobic core was used as a vibrational probe. Expts. were performed on both singly (HP35-P) and doubly CN-labeled peptide (HP35-P2) within the wild-type sequence, as well as on HP-35 contg. a singly labeled cyanophenylalanine and two norleucine mutations (HP35-P NleNle). There is a remarkable similarity between the dynamics measured in singly and doubly CN-labeled HP35, demonstrating that the presence of an addnl. CN vibrational probe does not significantly alter the dynamics of the small peptide. The substitution of two lysine residues by norleucines markedly improves the stability of HP35 by replacing charged with nonpolar residues, stabilizing the hydrophobic core. The results of the 2D IR expts. reveal that the dynamics of HP35-P are significantly faster than those of HP35-P NleNle. These observations suggest that the slower structural fluctuations in the hydrophobic core, indicating a more tightly structured core, may be an important contributing factor to HP35-P NleNle's increased stability.
- 197Urbanek, D. C.; Vorobyev, D. Y.; Serrano, A. L.; Gai, F.; Hochstrasser, R. M. The Two-Dimensional Vibrational Echo of a Nitrile Probe of the Villin HP35 Protein. J. Phys. Chem. Lett. 2010, 1 (23), 3311– 3315, DOI: 10.1021/jz101367dGoogle Scholar197The Two-Dimensional Vibrational Echo of a Nitrile Probe of the Villin HP35 ProteinUrbanek, Diana C.; Vorobyev, Dmitriy Yu.; Serrano, Arnaldo L.; Gai, Feng; Hochstrasser, Robin M.Journal of Physical Chemistry Letters (2010), 1 (23), 3311-3315CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)Two-dimensional (2D) IR spectroscopy was used to probe the hydrophobic core structure of the 35-residue villin headpiece subdomain, HP35, by monitoring the C≡N vibrational stretching band of a cyano-substituted phenylalanine (Phe). The presence of two humps in the vibrational frequency distribution in the folded equil. state is revealed. They represent two states that exchange more slowly than ca. 10 ps. The two CN stretch mode peak frequencies (and their equil. populations) are 2228.7 (44%) and 2234.5 cm-1 (56%). The two CN modes have different frequency-frequency correlation times of 7.4 and 1.6 ps, resp. These results suggest that the population with the higher frequency CN group is partly exposed, whereas the other CN mode experiences a hydrophobic-like environment.
- 198Bagchi, S.; Boxer, S. G.; Fayer, M. D. Ribonuclease S Dynamics Measured Using a Nitrile Label with 2D IR Vibrational Echo Spectroscopy. J. Phys. Chem. B 2012, 116 (13), 4034– 4042, DOI: 10.1021/jp2122856Google Scholar198Ribonuclease S dynamics measured using a nitrile label with 2D IR vibrational echo spectroscopyBagchi, Sayan; Boxer, Steven G.; Fayer, Michael D.Journal of Physical Chemistry B (2012), 116 (13), 4034-4042CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)A nitrile-labeled amino acid, p-cyanophenylalanine, is introduced near the active site of the semisynthetic enzyme, RNase S, to serve as a probe of protein dynamics and fluctuations. RNase S is the limited proteolysis product of subtilisin acting on RNase A, and consists of a small fragment including amino acids 1-20 (the S-peptide) and a larger fragment including residues 21-124 (the S-protein). A series of 2-dimensional vibrational echo expts. performed on the nitrile-labeled S-peptide and RNase S are described. The time-dependent changes in the 2-dimensional IR vibrational echo line shapes were analyzed using the center line slope method to obtain the frequency-frequency correlation function (FFCF). The observations showed that the nitrile probe in the S-peptide had dynamics that were similar to, but faster than, those of the single amino acid p-cyanophenylalanine in water. In contrast, the dynamics of the nitrile label when the peptide was bound to form RNase S were dominated by homogeneous dephasing (motionally narrowed) contributions with only a small contribution from very fast inhomogeneous structural dynamics. These results provided insights into the nature of the structural dynamics of the RNase S complex. The equil. dynamics of the nitrile-labeled S-peptide and the RNase S complex were also investigated by mol. dynamics (MD) simulations. The exptl. detd. FFCFs are compared to the FFCFs obtained from the MD simulations, thereby testing the capacity of simulations to det. the amplitudes and time scales of protein structural fluctuations on fast time scales under thermal equil. conditions.
- 199Tharp, J. M.; Wang, Y.-S.; Lee, Y.-J.; Yang, Y.; Liu, W. R. Genetic Incorporation of Seven ortho-Substituted Phenylalanine Derivatives. ACS Chem. Biol. 2014, 9 (4), 884– 890, DOI: 10.1021/cb400917aGoogle Scholar199Genetic incorporation of seven ortho-substituted phenylalanine derivativesTharp, Jeffery M.; Wang, Yane-Shih; Lee, Yan-Jiun; Yang, Yanyan; Liu, Wenshe R.ACS Chemical Biology (2014), 9 (4), 884-890CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Seven phenylalanine derivs. with small ortho substitutions were genetically encoded in Escherichia coli and mammalian cells at an amber codon using a previously reported, rationally designed pyrrolysyl-tRNA synthetase mutant (PylRS(N346A/C348A)) coupled with tRNACUAPyl. Ortho substitutions of the phenylalanine derivs. reported here included 3 halides, Me, methoxy, nitro, and nitrile. These compds. have the potential for use in multiple biochem. and biophys. applications. Specifically, the authors demonstrated that o-cyanophenylalanine could be used as a selective sensor to probe the local environment of proteins and applied this to study protein folding/unfolding. For 6 of these compds. this constitutes the 1st report of their genetic incorporation in living cells. With these compds. the total no. of substrates available for PylRS(N346A/C348A) is increased to nearly 40, which demonstrates that PylRS(N346A/C348A) is able to recognize phenylalanine with a substitution at any side-chain arom. position as a substrate. To the authors' knowledge, PylRS(N346A/C348A) is the only aminoacyl-tRNA synthetase with such a high substrate promiscuity.
- 200van Wilderen, L. J. G. W.; Kern-Michler, D.; Müller-Werkmeister, H. M.; Bredenbeck, J. Vibrational Dynamics and Solvatochromism of the Label SCN in Various Solvents and Hemoglobin by Time Dependent IR and 2D-IR Spectroscopy. Phys. Chem. Chem. Phys. 2014, 16 (36), 19643– 19653, DOI: 10.1039/C4CP01498GGoogle Scholar200Vibrational dynamics and solvatochromism of the label SCN in various solvents and hemoglobin by time dependent IR and 2D-IR spectroscopyvan Wilderen, Luuk J. G. W.; Kern-Michler, Daniela; Mueller-Werkmeister, Henrike M.; Bredenbeck, JensPhysical Chemistry Chemical Physics (2014), 16 (36), 19643-19653CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)We investigated the characteristics of the thiocyanate (SCN) functional group as a probe of local structural dynamics for 2D-IR spectroscopy of proteins, exploiting the dependence of vibrational frequency on the environment of the label. Steady-state and time-resolved IR spectroscopy are performed on the model compd. methylthiocyanate (MeSCN) in solvents of different polarity, and compared to data obtained on SCN as a local probe introduced as cyanylated cysteine in the protein bovine Hb. The vibrational lifetime of the protein label is detd. to be 37 ps, and its anharmonicity is obsd. to be lower than that of the model compd. (which itself exhibits solvent-independent anharmonicity). The vibrational lifetime of MeSCN generally correlates with the solvent polarity, i.e. longer lifetimes in less polar solvents, with the longest lifetime being 158 ps. However, the capacity of the solvent to form hydrogen bonds complicates this simplified picture. The long lifetime of the SCN vibration is in contrast to commonly used azide labels or isotopically-labeled amide I and better suited to monitor structural rearrangements by 2D-IR spectroscopy. We present time-dependent 2D-IR data on the labeled protein which reveal an initially inhomogeneous structure around the CN oscillator. The distribution becomes homogeneous after 5 ps so that spectral diffusion has effectively erased the 'memory' of the CN stretching frequency. Therefore, the 2D-IR data of the label incorporated in Hb demonstrate how SCN can be utilized to sense rearrangements in the local structure on a picosecond timescale.
- 201Bloem, R.; Koziol, K.; Waldauer, S. A.; Buchli, B.; Walser, R.; Samatanga, B.; Jelesarov, I.; Hamm, P. Ligand Binding Studied by 2D IR Spectroscopy Using the Azidohomoalanine Label. J. Phys. Chem. B 2012, 116 (46), 13705– 13712, DOI: 10.1021/jp3095209Google Scholar201Ligand Binding Studied by 2D IR Spectroscopy Using the Azidohomoalanine LabelBloem, Robbert; Koziol, Klemens; Waldauer, Steven A.; Buchli, Brigitte; Walser, Reto; Samatanga, Brighton; Jelesarov, Ilian; Hamm, PeterJournal of Physical Chemistry B (2012), 116 (46), 13705-13712CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)We explore the capability of the azidohomoalanine (Aha) as a vibrational label for 2D IR spectroscopy to study the binding of the target peptide to the PDZ2 domain. The Aha label responds sensitively to its local environment and its peak extinction coeff. of 350-400 M-1 cm-1 is high enough to routinely measure it in the low millimolar concn. regime. The central frequency, inhomogeneous width and spectral diffusion times deduced from the 2D IR line shapes of the Aha label at various positions in the peptide sequence is discussed in relationship to the known X-ray structure of the peptide bound to the PDZ2 domain. The results suggest that the Aha label introduces only a small perturbation to the overall structure of the peptide in the binding pocket. Finally, Aha is a methionine analog that can be incorporated also into larger proteins at essentially any position using protein expression. Altogether, Aha thus fulfills the requirements a versatile label should have for studies of protein structure and dynamics by 2D IR spectroscopy.
- 202Thielges, M. C.; Axup, J. Y.; Wong, D.; Lee, H. S.; Chung, J. K.; Schultz, P. G.; Fayer, M. D. Two-Dimensional IR Spectroscopy of Protein Dynamics Using Two Vibrational Labels: A Site-Specific Genetically Encoded Unnatural Amino Acid and an Active Site Ligand. J. Phys. Chem. B 2011, 115 (38), 11294– 11304, DOI: 10.1021/jp206986vGoogle Scholar202Two-Dimensional IR Spectroscopy of Protein Dynamics Using Two Vibrational Labels: A Site-Specific Genetically Encoded Unnatural Amino Acid and an Active Site LigandThielges, Megan C.; Axup, Jun Y.; Wong, Daryl; Lee, Hyun Soo; Chung, Jean K.; Schultz, Peter G.; Fayer, Michael D.Journal of Physical Chemistry B (2011), 115 (38), 11294-11304CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)Protein dynamics and interactions in myoglobin (Mb) were characterized via two vibrational dynamics labels (VDLs): a genetically incorporated site-specific azide (Az) bearing unnatural amino acid (AzPhe43) and an active site CO ligand. The Az-labeled protein was studied using ultrafast two-dimensional IR (2D IR) vibrational echo spectroscopy. CO bound at the active site of the heme serves as a second VDL located nearby. Therefore, it was possible to use Fourier transform IR (FT-IR) and 2D IR spectroscopic expts. on the Az in unligated Mb and in Mb bound to CO (MbAzCO) and on the CO in MbCO and MbAzCO to investigate the environment and motions of different states of one protein from the perspective of two spectrally resolved VDLs. A very broad bandwidth 2D IR spectrum, encompassing both the Az and CO spectral regions, found no evidence of direct coupling between the two VDLs. In MbAzCO, both VDLs reported similar time scale motions: very fast homogeneous dynamics, fast, ∼1 ps dynamics, and dynamics on a much slower time scale. Therefore, each VDL reports independently on the protein dynamics and interactions, and the measured dynamics are reflective of the protein motions rather than intrinsic to the chem. nature of the VDL. The AzPhe VDL also permitted study of oxidized Mb dynamics, which could not be accessed previously with 2D IR spectroscopy. The expts. demonstrate that the combined application of 2D IR spectroscopy and site-specific incorporation of VDLs can provide information on dynamics, structure, and interactions at virtually any site throughout any protein.
- 203Ye, S.; Zaitseva, E.; Caltabiano, G.; Schertler, G. F. X.; Sakmar, T. P.; Deupi, X.; Vogel, R. Tracking G-Protein-Coupled Receptor Activation Using Genetically Encoded Infrared Probes. Nature 2010, 464 (7293), 1386– 1389, DOI: 10.1038/nature08948Google Scholar203Tracking G-protein-coupled receptor activation using genetically encoded infrared probesYe, Shixin; Zaitseva, Ekaterina; Caltabiano, Gianluigi; Schertler, Gebhard F. X.; Sakmar, Thomas P.; Deupi, Xavier; Vogel, ReinerNature (London, United Kingdom) (2010), 464 (7293), 1386-1389CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Rhodopsin is a prototypical heptahelical family A G-protein-coupled receptor (GPCR) responsible for dim-light vision. Light isomerizes rhodopsin's retinal chromophore and triggers concerted movements of transmembrane helixes, including an outward tilting of helix 6 (H6) and a smaller movement of H5, to create a site for G-protein binding and activation. However, the precise temporal sequence and mechanism underlying these helix rearrangements is unclear. We used site-directed non-natural amino acid mutagenesis to engineer rhodopsin with p-azido-l-phenylalanine residues incorporated at selected sites, and monitored the azido vibrational signatures using IR spectroscopy as rhodopsin proceeded along its activation pathway. Here we report significant changes in electrostatic environments of the azido probes even in the inactive photoproduct Meta I, well before the active receptor state was formed. These early changes suggest a significant rotation of H6 and movement of the cytoplasmic part of H5 away from H3. Subsequently, a large outward tilt of H6 leads to opening of the cytoplasmic surface to form the active receptor photoproduct Meta II. Thus, our results reveal early conformational changes that precede larger rigid-body helix movements, and provide a basis to interpret recent GPCR crystal structures and to understand conformational sub-states obsd. during the activation of other GPCRs.
- 204Le Sueur, A. L.; Horness, R. E.; Thielges, M. C. Applications of Two-Dimensional Infrared Spectroscopy. Analyst 2015, 140 (13), 4336– 4349, DOI: 10.1039/C5AN00558BGoogle Scholar204Applications of two-dimensional infrared spectroscopyLe Sueur, Amanda L.; Horness, Rachel E.; Thielges, Megan C.Analyst (Cambridge, United Kingdom) (2015), 140 (13), 4336-4349CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)A review. Two-dimensional IR (2D IR) spectroscopy has recently emerged as a powerful tool with applications in many areas of scientific research. The inherent high time resoln. coupled with bond-specific spatial resoln. of IR spectroscopy enable direct characterization of rapidly interconverting species and fast processes, even in complex systems found in chem. and biol. In this minireview, we briefly outline the fundamental principles and exptl. procedures of 2D IR spectroscopy. Using illustrative example studies, we explain the important features of 2D IR spectra and their capability to elucidate mol. structure and dynamics. Primarily, this minireview aims to convey the scope and potential of 2D IR spectroscopy by highlighting select examples of recent applications including the use of innate or introduced vibrational probes for the study of nucleic acids, peptides/proteins, and materials.
- 205Smith, E. E.; Linderman, B. Y.; Luskin, A. C.; Brewer, S. H. Probing Local Environments with the Infrared Probe: l-4-Nitrophenylalanine. J. Phys. Chem. B 2011, 115 (10), 2380– 2385, DOI: 10.1021/jp109288jGoogle Scholar205Probing Local Environments with the Infrared Probe: L-4-NitrophenylalanineSmith, Emily E.; Linderman, Barton Y.; Luskin, Austin C.; Brewer, Scott H.Journal of Physical Chemistry B (2011), 115 (10), 2380-2385CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)The genetic incorporation of unnatural amino acids (UAAs) with high efficiency and fidelity is a powerful tool for the study of protein structure and dynamics with site-specificity in a relatively nonintrusive manner. Here, we illustrate the ability of L-4-nitrophenylalanine to serve as a sensitive IR probe of local protein environments in the 247 residue superfolder green fluorescent protein (sfGFP). Specifically, the nitro sym. stretching frequency of L-4-nitrophenylalanine was shown to be sensitive to both solvents that mimic different protein environments and 15N isotopic labeling of the three-atom nitro group of this UAA. 14NO2 and 15NO2 variants of this UAA were incorporated utilizing an engineered orthogonal aminoacyl-tRNA synthetase/tRNA pair into a solvent exposed and a partially buried position in sfGFP with high efficiency and fidelity. The combination of isotopic labeling and difference FTIR spectroscopy permitted the nitro sym. stretching frequency of L-4-nitrophenylalanine to be exptl. measured at either site in sfGFP. The 14NO2 sym. stretching frequency red-shifted 7.7 cm-1 between the solvent exposed and partially buried position, thus illustrating the ability of this UAA to serve as an effective IR probe of local protein environments.
- 206Kooter, I. M.; Moguilevsky, N.; Bollen, A.; van der Veen, L. A.; Otto, C.; Dekker, H. L.; Wever, R. The Sulfonium Ion Linkage in Myeloperoxidase: direct spectroscopic detection by isotopic labeling and effect of mutation. J. Biol. Chem. 1999, 274 (38), 26794– 26802, DOI: 10.1074/jbc.274.38.26794Google ScholarThere is no corresponding record for this reference.
- 207Thielges, M. C.; Case, D. A.; Romesberg, F. E. Carbon-Deuterium Bonds as Probes of Dihydrofolate Reductase. J. Am. Chem. Soc. 2008, 130 (20), 6597– 6603, DOI: 10.1021/ja0779607Google Scholar207Carbon-Deuterium Bonds as Probes of Dihydrofolate ReductaseThielges, Megan C.; Case, David A.; Romesberg, Floyd E.Journal of the American Chemical Society (2008), 130 (20), 6597-6603CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Much effort has been directed toward understanding the contributions of electrostatics and dynamics to protein function and esp. to enzyme catalysis. Unfortunately, these studies have been limited by the absence of direct exptl. probes. We have been developing the use of carbon-deuterium bonds as probes of proteins and now report the application of the technique to the enzyme dihydrofolate reductase, which catalyzes a hydride transfer and has served as a paradigm for biol. catalysis. We observe that the stretching absorption frequency of (methyl-d3) methionine carbon-deuterium bonds shows an approx. linear dependence on solvent dielec. Solvent and computational studies support the empirical interpretation of the stretching frequency in terms of local polarity. To begin to explore the use of this technique to study enzyme function and mechanism, we report a preliminary anal. of (methyl-d3) methionine residues within dihydrofolate reductase. Specifically, we characterize the IR absorptions at Met16 and Met20, within the catalytically important Met20 loop, and Met42, which is located within the hydrophobic core of the enzyme. The results confirm the sensitivity of the carbon-deuterium bonds to their local protein environment, demonstrate that dihydrofolate reductase is electrostatically and dynamically heterogeneous, and lay the foundation for the direct characterization protein electrostatics and dynamics and, potentially, their contribution to catalysis.
- 208Le Sueur, A. L.; Schaugaard, R. N.; Baik, M.-H.; Thielges, M. C. Methionine Ligand Interaction in a Blue Copper Protein Characterized by Site-Selective Infrared Spectroscopy. J. Am. Chem. Soc. 2016, 138 (22), 7187– 7193, DOI: 10.1021/jacs.6b03916Google Scholar208Methionine Ligand Interaction in a Blue Copper Protein Characterized by Site-Selective Infrared SpectroscopyLe Sueur, Amanda L.; Schaugaard, Richard N.; Baik, Mu-Hyun; Thielges, Megan C.Journal of the American Chemical Society (2016), 138 (22), 7187-7193CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The reactivity of metal sites in proteins is tuned by protein-based ligands. For example, in blue copper proteins such as plastocyanin (Pc), the structure imparts a highly elongated bond between the Cu and a methionine (Met) axial ligand to modulate its redox properties. Despite extensive study, a complete understanding of the contribution of the protein to redox activity is challenged by exptl. accessing both redox states of metalloproteins. Using IR spectroscopy in combination with site-selective labeling with carbon-deuterium (C-D) vibrational probes, we characterized the localized changes at the Cu ligand Met97 in the oxidized and reduced states, as well as the Zn(II) or Co(II)-substituted, the pH-induced low-coordinate, the apoprotein, and the unfolded states. The IR absorptions of (d3-methyl)Met97 are highly sensitive to interaction of the sulfur-based orbitals with the metal center and are demonstrated to be useful reporters of its modulation in the different states. Unrestricted Kohn-Sham d. functional theory calcns. performed on a model of the Cu site of Pc confirm the obsd. dependence. IR spectroscopy was then applied to characterize the impact of binding to the physiol. redox partner cytochrome (cyt) f. The spectral changes suggest a slightly stronger Cu-S(Met97) interaction in the complex with cyt f that has potential to modulate the electron transfer properties. Besides providing direct, mol.-level comparison of the oxidized and reduced states of Pc from the perspective of the axial Met ligand and evidence for perturbation of the Cu site properties by redox partner binding, this study demonstrates the localized spatial information afforded by IR spectroscopy of selectively incorporated C-D probes.
- 209Horness, R. E.; Basom, E. J.; Thielges, M. C. Site-Selective Characterization of Src Homology 3 Domain Molecular Recognition with Cyanophenylalanine Infrared Probes. Anal. Methods 2015, 7 (17), 7234– 7241, DOI: 10.1039/C5AY00523JGoogle Scholar209Site-selective characterization of Src homology 3 domain molecular recognition with cyanophenylalanine infrared probesHorness, Rachel E.; Basom, Edward J.; Thielges, Megan C.Analytical Methods (2015), 7 (17), 7234-7241CODEN: AMNEGX; ISSN:1759-9679. (Royal Society of Chemistry)Local heterogeneity of microenvironments in proteins is important in biol. function, but difficult to characterize exptl. One approach is the combination of IR spectroscopy and site-selective incorporation of probe moieties with spectrally resolved IR absorptions that enable characterization within inherently congested protein IR spectra. We employed this method to study mol. recognition of a Src homol. 3 (SH3) domain from the yeast protein Sho1 for a peptide contg. the proline-rich recognition sequence of its physiol. binding partner Pbs2. Nitrile IR probes were introduced at four distinct sites in the protein by selective incorporation of p-cyanophenylalanine via the amber codon suppressor method and characterized by IR spectroscopy. Variation among the IR absorption bands reports on heterogeneity in local residue environments dictated by the protein structure, as well as on residue-dependent changes upon peptide binding. The study informs on the mol. recognition of SH3Sho1 and illustrates the speed and simplicity of this approach for characterization of select microenvironments within proteins.
- 210Basom, E. J.; Maj, M.; Cho, M.; Thielges, M. C. Site-Specific Characterization of Cytochrome P450cam Conformations by Infrared Spectroscopy. Anal. Chem. 2016, 88 (12), 6598– 6606, DOI: 10.1021/acs.analchem.6b01520Google ScholarThere is no corresponding record for this reference.
- 211Bagchi, S.; Fried, S. D.; Boxer, S. G. A Solvatochromic Model Calibrates Nitriles’ Vibrational Frequencies to Electrostatic Fields. J. Am. Chem. Soc. 2012, 134 (25), 10373– 10376, DOI: 10.1021/ja303895kGoogle Scholar211A Solvatochromic Model Calibrates Nitriles' Vibrational Frequencies to Electrostatic FieldsBagchi, Sayan; Fried, Stephen D.; Boxer, Steven G.Journal of the American Chemical Society (2012), 134 (25), 10373-10376CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Electrostatic interactions provide a primary connection between a protein's three-dimensional structure and its function. IR probes are useful because vibrational frequencies of certain chem. groups, such as nitriles, are linearly sensitive to local electrostatic field and can serve as a mol. elec. field meter. IR spectroscopy has been used to study electrostatic changes or fluctuations in proteins, but measured peak frequencies have not been previously mapped to total elec. fields, because of the absence of a field-frequency calibration and the complication of local chem. effects such as H-bonds. The authors report a solvatochromic model that provides a means to assess the H-bonding status of arom. nitrile vibrational probes and calibrates their vibrational frequencies to electrostatic field. The anal. involves correlations between the nitrile's IR frequency and its 13C chem. shift, whose observation is facilitated by a robust method for introducing isotopes into arom. nitriles. The method is tested on the model protein RNase S contg. a labeled p-CN-Phe near the active site. Comparison of the measurements in RNase S against solvatochromic data gives an est. of the av. total electrostatic field at this location. The value detd. agrees quant. with mol. dynamics simulations, suggesting broader potential for the use of IR probes in the study of protein electrostatics.
- 212Wang, L.; Zhang, J.; Han, M.-J.; Zhang, L.; Chen, C.; Huang, A.; Xie, R.; Wang, G.; Zhu, J.; Wang, Y. A Genetically Encoded Two-Dimensional Infrared Probe for Enzyme Active-Site Dynamics. Angew. Chem. Int. Ed. 2021, 60 (20), 11143– 11147, DOI: 10.1002/anie.202016880Google ScholarThere is no corresponding record for this reference.
- 213Huguenin-Dezot, N.; Alonzo, D. A.; Heberlig, G. W.; Mahesh, M.; Nguyen, D. P.; Dornan, M. H.; Boddy, C. N.; Schmeing, T. M.; Chin, J. W. Trapping Biosynthetic Acyl-Enzyme Intermediates with Encoded 2,3-Diaminopropionic Acid. Nature 2019, 565 (7737), 112– 117, DOI: 10.1038/s41586-018-0781-zGoogle Scholar213Trapping biosynthetic acyl-enzyme intermediates with encoded 2,3-diaminopropionic acidHuguenin-Dezot, Nicolas; Alonzo, Diego A.; Heberlig, Graham W.; Mahesh, Mohan; Nguyen, Duy P.; Dornan, Mark H.; Boddy, Christopher N.; Schmeing, T. Martin; Chin, Jason W.Nature (London, United Kingdom) (2019), 565 (7737), 112-117CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Many enzymes catalyze reactions that proceed through covalent acyl-enzyme (ester or thioester) intermediates. These enzymes include serine hydrolases (encoded by one per cent of human genes, and including serine proteases and thioesterases), cysteine proteases (including caspases), and many components of the ubiquitination machinery. Their important acyl-enzyme intermediates are unstable, commonly having half-lives of minutes to hours. In some cases, acyl-enzyme complexes can be stabilized using substrate analogs or active-site mutations but, although these approaches can provide valuable insight, they often result in complexes that are substantially non-native. Here we develop a strategy for incorporating 2,3-diaminopropionic acid (DAP) into recombinant proteins, via expansion of the genetic code. We show that replacing catalytic cysteine or serine residues of enzymes with DAP permits their first-step reaction with native substrates, allowing the efficient capture of acyl-enzyme complexes that are linked through a stable amide bond. For one of these enzymes, the thioesterase (TE) domain of valinomycin synthetase (Vlm), we elucidate the biosynthetic pathway by which it progressively oligomerizes tetradepsipeptidyl substrates to a dodecadepsipeptidyl intermediate, which it then cyclizes to produce valinomycin. By trapping the first and last acyl-thioesterase intermediates in the catalytic cycle as DAP conjugates, we provide structural insight into how conformational changes in thioesterase domains of such nonribosomal peptide synthetases (NRPSs) control the oligomerization and cyclization of linear substrates. The encoding of DAP will facilitate the characterization of diverse acyl-enzyme complexes, and may be extended to capturing the native substrates of transiently acylated proteins of unknown function.
- 214Tang, S.; Beattie, A. T.; Kafkova, L.; Petris, G.; Huguenin-Dezot, N.; Fiedler, M.; Freeman, M.; Chin, J. W. Mechanism-Based Traps Enable Protease and Hydrolase Substrate Discovery. Nature 2022, 602 (7898), 701– 707, DOI: 10.1038/s41586-022-04414-9Google Scholar214Mechanism-based traps enable protease and hydrolase substrate discoveryTang, Shan; Beattie, Adam T.; Kafkova, Lucie; Petris, Gianluca; Huguenin-Dezot, Nicolas; Fiedler, Marc; Freeman, Matthew; Chin, Jason W.Nature (London, United Kingdom) (2022), 602 (7898), 701-707CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)Hydrolase enzymes, including proteases, are encoded by 2-3% of the genes in the human genome and 14% of these enzymes are active drug targets1. However, the activities and substrate specificities of many proteases-esp. those embedded in membranes-and other hydrolases remain unknown. Here we report a strategy for creating mechanism-based, light-activated protease and hydrolase substrate traps in complex mixts. and live mammalian cells. The traps capture substrates of hydrolases, which normally use a serine or cysteine nucleophile. Replacing the catalytic nucleophile with genetically encoded 2,3-diaminopropionic acid allows the first step reaction to form an acyl-enzyme intermediate in which a substrate fragment is covalently linked to the enzyme through a stable amide bond2; this enables stringent purifn. and identification of substrates. We identify new substrates for proteases, including an intramembrane mammalian rhomboid protease RHBDL4 (refs. 3,4). We demonstrate that RHBDL4 can shed luminal fragments of endoplasmic reticulum-resident type I transmembrane proteins to the extracellular space, as well as promoting non-canonical secretion of endogenous sol. endoplasmic reticulum-resident chaperones. We also discover that the putative serine hydrolase retinoblastoma binding protein 9 (ref. 5) is an aminopeptidase with a preference for removing arom. amino acids in human cells. Our results exemplify a powerful paradigm for identifying the substrates and activities of hydrolase enzymes.
- 215Dorman, G.; Prestwich, G. D. Benzophenone Photophores in Biochemistry. Biochemistry 1994, 33 (19), 5661– 5673, DOI: 10.1021/bi00185a001Google Scholar215Benzophenone Photophores in BiochemistryDorman, Gyorgy; Prestwich, Glenn D.Biochemistry (1994), 33 (19), 5661-73CODEN: BICHAW; ISSN:0006-2960.A review, with ∼85 refs. The photoactivatable aryl ketone derivs. have been rediscovered as biochem. probes in the last 5 yr. The expanding use of benzophenone (BP) photoprobes can be attributed to three distinct chem. and biochem. advantages. First, BPs are chem. more stable than diazo esters, aryl azides, and diazirines. Second, BPs can be manipulated in ambient light and can be activated at 350-360 nm, avoiding protein-damaging wavelengths. Third, BPs react preferentially with unreactive C-H bonds, even in the presence of solvent water and bulk nucleophiles. These three properties combine to produce highly efficient covalent modifications of macromols., frequently with remarkable site specificity. This perspective includes a brief review of BP photochem. and a selection of specific applications of these photoprobes, which address questions in protein, nucleic acid, and lipid biochem.
- 216Chin, J. W.; Martin, A. B.; King, D. S.; Wang, L.; Schultz, P. G. Addition of a Photocrosslinking Amino Acid to the Genetic Code of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (17), 11020– 11024, DOI: 10.1073/pnas.172226299Google Scholar216Addition of a photocrosslinking amino acid to the genetic code of Escherichia coliChin, Jason W.; Martin, Andrew B.; King, David S.; Wang, Lei; Schultz, Peter G.Proceedings of the National Academy of Sciences of the United States of America (2002), 99 (17), 11020-11024CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Benzophenones are among the most useful photocrosslinking agents in biol. We have evolved an orthogonal aminoacyl-tRNA synthetase/tRNA pair that makes possible the in vivo incorporation of p-benzoyl-L-phenylalanine into proteins in Escherichia coli in response to the amber codon, TAG. This unnatural amino acid was incorporated with high translational efficiency and fidelity into the dimeric protein glutathione S-transferase. Irradn. resulted in efficient crosslinking (>50%) of the protein subunits. This methodol. may prove useful for discovering and defining protein interactions in vitro and in vivo.
- 217Farrell, I. S.; Toroney, R.; Hazen, J. L.; Mehl, R. A.; Chin, J. W. Photo-Cross-Linking Interacting Proteins with a Genetically Encoded Benzophenone. Nat. Methods 2005, 2 (5), 377– 384, DOI: 10.1038/nmeth0505-377Google Scholar217Photo-cross-linking interacting proteins with a genetically encoded benzophenoneFarrell, Ian S.; Toroney, Rebecca; Hazen, Jennifer L.; Mehl, Ryan A.; Chin, Jason W.Nature Methods (2005), 2 (5), 377-384CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A major challenge in understanding the networks of interactions that control cell and organism function is the definition of protein interactions. Solid-phase peptide synthesis has allowed the photo-crosslinkable amino acid p-benzoyl-L-phenylalanine to be site-specifically incorporated into peptide chains, to facilitate the definition of peptide-ligand complexes. The method, however, is limited to the in vitro study of peptides and small proteins. An innovative develpoment allows the incorporation of a site-specific photo-cross-linker into virtually any protein that can be expressed in Escherichia coli, thereby promoting in vivo or in vitro crosslinking of proteins. The method relies on an orthogonal aminoacyl tRNA synthetase-tRnACUA pair that incorporates pBpa at the position encoded by the amber codon (UAG) in any gene transformed into E. coli. The system described in this protocol uses two plasmids: a p15A-based plasmid to express the orthogonal tRNA and synthetase pair (pDULE) and a second plasmid contg. an amber mutant of the gene of interest. To produce the photo-cross-linker-contg. protein, cultures of E. coli carrying both plasmids are grown in the presence of the unnatural amino acid. To photo-cross-link the protein to its binding partner in vivo or in vitro, cells or purified proteins, resp., are exposed to UV light.
- 218Chin, J. W.; Schultz, P. G. In Vivo Photocrosslinking with Unnatural Amino Acid Mutagenesis. ChemBioChem 2002, 3 (11), 1135– 1137, DOI: 10.1002/1439-7633(20021104)3:11<1135::AID-CBIC1135>3.0.CO;2-MGoogle Scholar218In vivo photocrosslinking with unnatural amino acid mutagenesisChin, Jason W.; Schultz, Peter G.ChemBioChem (2002), 3 (11), 1135-1137CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)A method for the characterization of protein interactions in vivo by photocrosslinking with unnatural amino acid mutagenesis was developed. The method involves the replacement of a single amino acid in a protein with amino acid p-benzoyl-L-phenylalanine (pβpa) in vivo; irradn. of the cell with near UV-light to crosslink proteins proximal to the surface of the pβpa- contg. protein; cell lysis, purifn., and identification of the complex or complexes formed.
- 219Mori, H.; Ito, K. Different Modes of SecY-SecA Interactions Revealed by Site-Directed in vivo Photo-Cross-Linking. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (44), 16159– 16164, DOI: 10.1073/pnas.0606390103Google ScholarThere is no corresponding record for this reference.
- 220Majmudar, C. Y.; Lee, L. W.; Lancia, J. K.; Nwokoye, A.; Wang, Q.; Wands, A. M.; Wang, L.; Mapp, A. K. Impact of Nonnatural Amino Acid Mutagenesis on the in Vivo Function and Binding Modes of a Transcriptional Activator. J. Am. Chem. Soc. 2009, 131 (40), 14240– 14242, DOI: 10.1021/ja904378zGoogle Scholar220Impact of Nonnatural Amino Acid Mutagenesis on the in Vivo Function and Binding Modes of a Transcriptional ActivatorMajmudar, Chinmay Y.; Lee, Lori W.; Lancia, Jody K.; Nwokoye, Adaora; Wang, Qian; Wands, Amberlyn M.; Wang, Lei; Mapp, Anna K.Journal of the American Chemical Society (2009), 131 (40), 14240-14242CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein-protein interactions play an essential role in cellular function, and methods to discover and characterize them in their native context are of paramount importance for gaining a deeper understanding of biol. networks. In this study, an enhanced nonsense suppression system was utilized to incorporate the non-natural amino acid p-benzoyl-L-phenylalanine (pBpa) throughout the transcriptional activation domain of the prototypical eukaryotic transcriptional activator Gal4 in vivo (Saccharomyces cerevisiae). Functional studies of the pBpa-contg. Gal4 mutants suggest that this essential binding interface of Gal4 is minimally impacted by these substitutions, with both transcriptional activity and sensitivity to growth conditions maintained. Further supporting this are in vivo crosslinking studies, including the detection of a key binding partner of Gal4, the inhibitor protein Gal80. Crosslinking with a range of pBpa-contg. mutants revealed a Gal4·Gal80 binding interface that extends beyond that previously predicted by conventional strategies. Thus, this approach can be broadened to the discovery of novel binding partners of transcription factors, information that will be crit. for the development of therapeutically useful small mol. modulators of these protein-protein interactions.
- 221Liu, C.; Young, A. L.; Starling-Windhof, A.; Bracher, A.; Saschenbrecker, S.; Rao, B. V.; Rao, K. V.; Berninghausen, O.; Mielke, T.; Hartl, F. U. Coupled Chaperone Action in Folding and Assembly of Hexadecameric Rubisco. Nature 2010, 463 (7278), 197– 202, DOI: 10.1038/nature08651Google ScholarThere is no corresponding record for this reference.
- 222Ye, Z.; Bair, M.; Desai, H.; Williams, G. J. A Photocrosslinking Assay for Reporting Protein Interactions in Polyketide and Fatty Acid Synthases. Mol. BioSyst. 2011, 7 (11), 3152– 3156, DOI: 10.1039/c1mb05270eGoogle ScholarThere is no corresponding record for this reference.
- 223Ye, Z.; Williams, G. J. Mapping a Ketosynthase:Acyl Carrier Protein Binding Interface via Unnatural Amino Acid-Mediated Photo-Cross-Linking. Biochemistry 2014, 53 (48), 7494– 7502, DOI: 10.1021/bi500936uGoogle Scholar223Mapping a ketosynthase:acyl carrier protein binding interface via unnatural amino acid-mediated Photo-Cross-LinkingYe, Zhixia; Williams, Gavin J.Biochemistry (2014), 53 (48), 7494-7502CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Probing and interrogating protein interactions that involve acyl carrier proteins (ACP's) in fatty acid synthases and polyketide synthases are crit. to understanding the mol. basis for the programmed assembly of complex natural products. Here, we have used unnatural amino acid mutagenesis to site specifically install photo-crosslinking functionality into acyl carrier proteins from diverse systems and the ketosynthase FabF from the Escherichia coli type II fatty acid synthase. Subsequently, a photo-crosslinking assay was employed to systematically probe the ability of FabF to interact with a broad panel of ACP's, illustrating the expected orthogonality of ACP:FabF interactions and the role of charged residues in helix II of the ACP. In addn., FabF residues involved in the binding interaction with the cognate carrier protein were identified via surface scanning mutagenesis and photo-crosslinking. Furthermore, the ability to install the photo-crosslinking amino acid at virtually any position allowed interrogation of the role that carrier protein acylation plays in detg. the binding interface with FabF. A conserved carrier protein motif that includes the phosphopantetheinylation site was also shown to play an integral role in maintenance of the AcpP:FabF binding interaction. Our results provide unprecedented insight into the mol. details that describe the AcpP:FabF binding interface and demonstrate that unnatural amino acid based photo-crosslinking is a powerful tool for probing and interrogating protein interactions in complex biosynthetic systems.
- 224Pavic, K.; Rios, P.; Dzeyk, K.; Koehler, C.; Lemke, E. A.; Köhn, M. Unnatural Amino Acid Mutagenesis Reveals Dimerization As a Negative Regulatory Mechanism of VHR’s Phosphatase Activity. ACS Chem. Biol. 2014, 9 (7), 1451– 1459, DOI: 10.1021/cb500240nGoogle Scholar224Unnatural Amino Acid Mutagenesis Reveals Dimerization As a Negative Regulatory Mechanism of VHR's Phosphatase ActivityPavic, Karolina; Rios, Pablo; Dzeyk, Kristina; Koehler, Christine; Lemke, Edward A.; Koehn, MajaACS Chemical Biology (2014), 9 (7), 1451-1459CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Vaccinia H1-related (VHR) phosphatase (also known as DUSP3) is a dual specificity phosphatase that is required for cell-cycle progression and plays a role in cell growth of certain cancers. Therefore, it represents a potential drug target. VHR is structurally and biochem. well characterized, yet its regulatory principles are still poorly understood. Understanding its regulation is important, not only to comprehend VHR's biol. mechanisms and roles but also to det. its potential and druggability as a target in cancer. Here, we investigated the functional role of the unique "variable insert" region in VHR by selectively introducing the photo-cross-linkable amino acid para-benzoylphenylalanine (pBPA) using the amber suppression method. This approach led to the discovery of VHR dimerization, which was further confirmed using traditional chem. cross-linkers. Phe68 in VHR was discovered as a residue involved in the dimerization. We demonstrate that VHR can dimerize inside cells, and that VHR catalytic activity is reduced upon dimerization. Our results suggest that dimerization could occlude the active site of VHR, thereby blocking its accessibility to substrates. These findings indicate that the previously unknown transient self-assocn. of VHR acts as a means for the neg. regulation of its catalytic activity.
- 225Morozov, Y. I.; Agaronyan, K.; Cheung, A. C. M.; Anikin, M.; Cramer, P.; Temiakov, D. A Novel Intermediate in Transcription Initiation by Human Mitochondrial RNA Polymerase. Nucleic Acids Res. 2014, 42 (6), 3884– 3893, DOI: 10.1093/nar/gkt1356Google ScholarThere is no corresponding record for this reference.
- 226Wong, H. E.; Kwon, I. Effects of Non-Natural Amino Acid Incorporation into the Enzyme Core Region on Enzyme Structure and Function. Int. J. Mol. Sci. 2015, 16 (9), 22735– 22753, DOI: 10.3390/ijms160922735Google Scholar226Effects of non-natural amino acid incorporation into the enzyme core region on enzyme structure and functionWong, H. Edward; Kwon, InchanInternational Journal of Molecular Sciences (2015), 16 (9), 22735-22753CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)Techniques to incorporate non-natural amino acids (NNAAs) have enabled biosynthesis of proteins contg. new building blocks with unique structures, chem., and reactivity that are not found in natural amino acids. It is crucial to understand how incorporation of NNAAs affects protein function because NNAA incorporation may perturb crit. function of a target protein. This study investigated how the site-specific incorporation of NNAAs affects the catalytic properties of an enzyme. L-3-(2-Naphthyl)alanine (2Nal), a NNAA with a hydrophobic and bulky side-chain, , was site-specifically incorporated at 6 different positions in the hydrophobic core of a model enzyme, murine dihydrofolate reductase (mDHFR). The mDHFR variants with a greater change in van der Waals vol. upon 2Nal incorporation exhibited a greater redn. in catalytic efficiency. Similarly, the steric incompatibility calcd. using RosettaDesign, a protein stability calcn. program, correlated with the changes in catalytic efficiency.
- 227Rappaport, F.; Boussac, A.; Force, D. A.; Peloquin, J.; Brynda, M.; Sugiura, M.; Un, S.; Britt, R. D.; Diner, B. A. Probing the Coupling between Proton and Electron Transfer in Photosystem II Core Complexes Containing a 3-Fluorotyrosine. J. Am. Chem. Soc. 2009, 131 (12), 4425– 4433, DOI: 10.1021/ja808604hGoogle Scholar227Probing the coupling between proton and electron transfer in photosystem II core complexes containing a 3-fluorotyrosineRappaport, Fabrice; Boussac, Alain; Force, Dee Ann; Peloquin, Jeffrey; Brynda, Marcin; Sugiura, Miwa; Un, Sun; Britt, R. David; Diner, Bruce A.Journal of the American Chemical Society (2009), 131 (12), 4425-4433CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The catalytic cycle of numerous enzymes involves the coupling between proton transfer and electron transfer. Yet, the understanding of this coordinated transfer in biol. systems remains limited, likely because its characterization relies on the controlled but exptl. challenging modifications of the free energy changes assocd. with either the electron or proton transfer. The authors have performed such a study here in photosystem II. The driving force for electron transfer from TyrZ to P680•+ has been decreased by ∼80 meV by mutating the axial ligand of P680, and that for proton transfer upon oxidn. of TyrZ by substituting a 3-fluorotyrosine (3F-TyrZ) for TyrZ. In Mn-depleted photosystem II, the dependence upon pH of the oxidn. rates of TyrZ and 3F-TyrZ were found to be similar. However, in the pH range where the phenolic hydroxyl of TyrZ is involved in a H-bond with a proton acceptor, the activation energy of the oxidn. of 3F-TyrZ is decreased by 110 meV, a value which correlates with the in vitro finding of a 90 meV stabilization energy to the phenolate form of 3F-Tyr when compared to Tyr. Thus, when the phenol of YZ acts as a H-bond donor, its oxidn. by P680•+ is controlled by its prior deprotonation. This contrasts with the situation prevailing at lower pH, where the proton acceptor is protonated and therefore unavailable, in which the oxidn.-induced proton transfer from the phenolic hydroxyl of TyrZ has been proposed to occur concertedly with the electron transfer to P680•+. This suggests a switch between a concerted proton/electron transfer at pHs < 7.5 to a sequential one at pHs > 7.5 and illustrates the roles of the H-bond and of the likely salt-bridge existing between the phenolate and the nearby proton acceptor in detg. the coupling between proton and electron transfer.
- 228Schlesinger, S.; Schlesinger, M. J. The Effect of Amino Acid Analogues on Alkaline Phosphatase Formation in Escherichia coli K-12: I. Substitution of Triazolealanine for Histidine. J. Biol. Chem. 1967, 242 (14), 3369– 3372, DOI: 10.1016/S0021-9258(18)95919-3Google ScholarThere is no corresponding record for this reference.
- 229Fried, S. D.; Bagchi, S.; Boxer, S. G. Extreme Electric Fields Power Catalysis in the Active Site of Ketosteroid Isomerase. Science 2014, 346 (6216), 1510– 1514, DOI: 10.1126/science.1259802Google Scholar229Extreme electric fields power catalysis in the active site of ketosteroid isomeraseFried, Stephen D.; Bagchi, Sayan; Boxer, Steven G.Science (Washington, DC, United States) (2014), 346 (6216), 1510-1514CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Enzymes use protein architecture to impose specific electrostatic fields onto their bound substrates, but the magnitude and catalytic effect of these elec. fields have proven difficult to quantify with std. exptl. approaches. Here, using vibrational Stark effect spectroscopy, the authors found that the active site of ketosteroid isomerase (KSI) exerted an extremely large elec. field onto the C:O chem. bond that undergoes a charge rearrangement in the KSI rate-detg. step. Moreover, the authors found that the magnitude of the elec. field exerted by the active site strongly correlated with the enzyme's catalytic rate enhancement, enabling them to quantify the fraction of the catalytic effect that was electrostatic in origin. The measurements described here may help explain the role of electrostatics in many other enzymes and biomol. systems.
- 230Ortmayer, M.; Hardy, F. J.; Quesne, M. G.; Fisher, K.; Levy, C.; Heyes, D. J.; Catlow, C. R. A.; de Visser, S. P.; Rigby, S. E. J.; Hay, S.; Green, A. P. A Noncanonical Tryptophan Analogue Reveals an Active Site Hydrogen Bond Controlling Ferryl Reactivity in a Heme Peroxidase. JACS Au 2021, 1 (7), 913– 918, DOI: 10.1021/jacsau.1c00145Google ScholarThere is no corresponding record for this reference.
- 231Kang, G.; Taguchi, A. T.; Stubbe, J.; Drennan, C. L. Structure of a Trapped Radical Transfer Pathway within a Ribonucleotide Reductase Holocomplex. Science 2020, 368 (6489), 424– 427, DOI: 10.1126/science.aba6794Google Scholar231Structure of a trapped radical transfer pathway within a ribonucleotide reductase holocomplexKang, Gyunghoon; Taguchi, Alexander T.; Stubbe, JoAnne; Drennan, Catherine L.Science (Washington, DC, United States) (2020), 368 (6489), 424-427CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Ribonucleotide reductases (RNRs) are a diverse family of enzymes that are alone capable of generating 2'-deoxynucleotides de novo and are thus crit. in DNA biosynthesis and repair. The nucleotide redn. reaction in all RNRs requires the generation of a transient active site thiyl radical, and in class I RNRs, this process involves a long-range radical transfer between two subunits, α and β. Because of the transient subunit assocn., an at. resoln. structure of an active α2β2 RNR complex has been elusive. We used a doubly substituted β2, E52Q/(2,3,5)-trifluorotyrosine122-β2, to trap wild-type α2 in a long-lived α2β2 complex. We report the structure of this complex by means of cryo-electron microscopy to 3.6-angstrom resoln., allowing for structural visualization of a 32-angstrom-long radical transfer pathway that affords RNR activity.
- 232Beiboer, S. H. W.; Berg, B. v. d.; Dekker, N.; Cox, R. C.; Verheij, H. M. Incorporation of an Unnatural Amino Acid in the Active Site of Porcine Pancreatic Phospholipase A2. Substitution of Histidine by l,2,4-Triazole-3-Alanine Yields an Enzyme with High Activity at Acidic pH. Protein Eng. Des. Sel. 1996, 9 (4), 345– 352, DOI: 10.1093/protein/9.4.345Google ScholarThere is no corresponding record for this reference.
- 233Soumillion, P.; Fastrez, J. Incorporation of 1,2,4-Triazole-3-Alanine into a Mutant of Phage Lambda Lysozyme Containing a Single Histidine. Protein Eng. Des. Sel. 1998, 11 (3), 213– 217, DOI: 10.1093/protein/11.3.213Google ScholarThere is no corresponding record for this reference.
- 234Blatter, N.; Prokup, A.; Deiters, A.; Marx, A. Modulating the pKa of a Tyrosine in KlenTaq DNA Polymerase that Is Crucial for Abasic Site Bypass by in Vivo Incorporation of a Non-canonical Amino Acid. Chem Bio Chem 2014, 15, 1735– 1737, DOI: 10.1002/cbic.201400051Google ScholarThere is no corresponding record for this reference.
- 235Obeid, S.; Blatter, N.; Kranaster, R.; Schnur, A.; Diederichs, K.; Welte, W.; Marx, A. Replication Through an Abasic DNA Lesion: Structural Basis for Adenine Selectivity. EMBO J. 2010, 29 (10), 1738, DOI: 10.1038/emboj.2010.64Google ScholarThere is no corresponding record for this reference.
- 236Greene, B. L.; Kang, G.; Cui, C.; Bennati, M.; Nocera, D. G.; Drennan, C. L.; Stubbe, J. Ribonucleotide Reductases: Structure, Chemistry, and Metabolism Suggest New Therapeutic Targets. Annu. Rev. Biochem. 2020, 89 (1), 45– 75, DOI: 10.1146/annurev-biochem-013118-111843Google Scholar236Ribonucleotide Reductases: Structure, Chemistry, and Metabolism Suggest New Therapeutic TargetsGreene, Brandon L.; Kang, Gyunghoon; Cui, Chang; Bennati, Marina; Nocera, Daniel G.; Drennan, Catherine L.; Stubbe, JoAnneAnnual Review of Biochemistry (2020), 89 (), 45-75CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)A review. Ribonucleotide reductases (RNRs) catalyze the de novo conversion of nucleotides to deoxynucleotides in all organisms, controlling their relative ratios and abundance. In doing so, they play an important role in fidelity of DNA replication and repair. RNRs' central role in nucleic acid metab. has resulted in five therapeutics that inhibit human RNRs. In this review, we discuss the structural, dynamic, and mechanistic aspects of RNR activity and regulation, primarily for the human and Escherichia coli class Ia enzymes. The unusual radical-based org. chem. of nucleotide redn., the inorg. chem. of the essential metallo-cofactor biosynthesis/maintenance, the transport of a radical over a long distance, and the dynamics of subunit interactions all present distinct entry points toward RNR inhibition that are relevant for drug discovery. We describe the current mechanistic understanding of small mols. that target different elements of RNR function, including downstream pathways that lead to cell cytotoxicity. We conclude by summarizing novel and emergent RNR targeting motifs for cancer and antibiotic therapeutics.
- 237Uhlin, U.; Eklund, H. Structure of Ribonucleotide Reductase Protein R1. Nature 1994, 370 (6490), 533– 539, DOI: 10.1038/370533a0Google Scholar237Structure of ribonucleotide reductase protein R1Uhlin, Ulla; Eklund, HansNature (London, United Kingdom) (1994), 370 (6490), 533-9CODEN: NATUAS; ISSN:0028-0836.The crystal structure ribonucleotide reductase (I) subunit R1 (in complex with subunit R2) at 2.5 Å is reported. The 3-dimensional structure of the R2 subunit was previously reported and refined at 2.2 Å. The R2 tyrosyl radical-based I reaction involves 5 cysteines. Two redox-active R1 cysteines (Cys-225 and Cys-462) are located at adjacent antiparallel strands in a new type of 10-stranded α/β-barrel, and 2 others (Cys-754 and Cys-759) at the C-terminal end in a flexible arm. The 5th cysteine (Cys-439), in a loop in the center of the barrel, is positioned to initiate the radical reaction.
- 238Reece, S. Y.; Seyedsayamdost, M. R.; Stubbe, J.; Nocera, D. G. Electron Transfer Reactions of Fluorotyrosyl Radicals. J. Am. Chem. Soc. 2006, 128 (42), 13654– 13655, DOI: 10.1021/ja0636688Google Scholar238Electron transfer reactions of fluorotyrosyl radicalsReece, Steven Y.; Seyedsayamdost, Mohammad R.; Stubbe, JoAnne; Nocera, Daniel G.Journal of the American Chemical Society (2006), 128 (42), 13654-13655CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The complex Re(bpy)(CO)3CN is an excited state oxidant of tyrosine upon deprotonation of the tyrosyl phenol. Re(bpy-FnY)(CO)3CN complexes ([Re]-FnY: [Re]-Y, [Re]-3-FY, [Re]-3,5-F2Y, [Re]-2,3-F2Y, [Re]-2,3,5-F3Y, [Re]-2,3,6-F3Y, and [Re]-F4Y) (FnY = Me tyrosinate and fluorotyrosinates) were prepd. so as to vary the FnY·/FnY- redn. potential and thus the driving force for electron transfer (ET) in this system. Time-resolved emission and nanosecond absorption spectroscopies were used to measure the rates for charge sepn. (CS), and charge recombination, CR, for each complex. A driving force anal. reveals that CS is well described by Marcus' theory for ET, is strongly driving force dependent (activated), and occurs in the normal region for ET. CR, however, is weakly driving force dependent (near activation-less) and occurs in the inverted region for ET. Fluorotyrosines will be powerful probes for unraveling charge transport mechanisms in enzymes that use tyrosyl radicals. An x-ray crystal structure detn. of Re(bpy)(CO)3CN·MeOH is also presented.
- 239Seyedsayamdost, M. R.; Yee, C. S.; Reece, S. Y.; Nocera, D. G.; Stubbe, J. pH Rate Profiles of FnY356-R2s (n = 2, 3, 4) in Escherichia coli Ribonucleotide Reductase: Evidence that Y356 Is a Redox-Active Amino Acid along the Radical Propagation Pathway. J. Am. Chem. Soc. 2006, 128 (5), 1562– 1568, DOI: 10.1021/ja055927jGoogle Scholar239pH Rate Profiles of FnY356-R2s (n = 2, 3, 4) in Escherichia coli Ribonucleotide Reductase: Evidence that Y356 Is a Redox-Active Amino Acid along the Radical Propagation PathwaySeyedsayamdost, Mohammad R.; Yee, Cyril S.; Reece, Steven Y.; Nocera, Daniel G.; Stubbe, JoAnneJournal of the American Chemical Society (2006), 128 (5), 1562-1568CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The Escherichia coli ribonucleotide reductase (RNR), composed of two subunits (R1 and R2), catalyzes the conversion of nucleotides to deoxynucleotides. Substrate redn. requires that a tyrosyl radical (Y122•) in R2 generate a transient cysteinyl radical (C439•) in R1 through a pathway thought to involve amino acid radical intermediates [Y122• → W48 → Y356 within R2 to Y731 → Y730 → C439 within R1]. To study this radical propagation process, the authors have synthesized R2 semisynthetically using intein technol. and replaced Y356 with a variety of fluorinated tyrosine analogs (2,3-F2Y, 3,5-F2Y, 2,3,5-F3Y, 2,3,6-F3Y, and F4Y) that have been described and characterized in the accompanying paper. These fluorinated tyrosine derivs. have potentials that vary from -50 to +270 mV relative to tyrosine over the accessible pH range for RNR and pKas that range from 5.6 to 7.8. The pH rate profiles of deoxynucleotide prodn. by these FnY356-R2s are reported. The results suggest that the rate-detg. step can be changed from a phys. step to the radical propagation step by altering the redn. potential of Y356• using these analogs. As the difference in potential of the FnY• relative to Y• becomes >80 mV, the activity of RNR becomes inhibited, and by 200 mV, RNR activity is no longer detectable. These studies support the model that Y356 is a redox-active amino acid on the radical-propagation pathway. On the basis of the authors' previous studies with 3-NO2Y356-R2, the authors assume that 2,3,5-F3Y356, 2,3,6-F3Y356, and F4Y356-R2s are all deprotonated at pH >7.5. The authors show that they all efficiently initiate nucleotide redn. If this assumption is correct, then a hydrogen-bonding pathway between W48 and Y356 of R2 and Y731 of R1 does not play a central role in triggering radical initiation nor is hydrogen-atom transfer between these residues obligatory for radical propagation.
- 240Seyedsayamdost, M. R.; Xie, J.; Chan, C. T. Y.; Schultz, P. G.; Stubbe, J. Site-Specific Insertion of 3-Aminotyrosine into Subunit α2 of E. coli Ribonucleotide Reductase: Direct Evidence for Involvement of Y730 and Y731 in Radical Propagation. J. Am. Chem. Soc. 2007, 129 (48), 15060– 15071, DOI: 10.1021/ja076043yGoogle Scholar240Site-Specific Insertion of 3-Aminotyrosine into Subunit α2 of E. coli Ribonucleotide Reductase: Direct Evidence for Involvement of Y730 and Y731 in Radical PropagationSeyedsayamdost, Mohammad R.; Xie, Jianming; Chan, Clement T. Y.; Schultz, Peter G.; Stubbe, JoAnneJournal of the American Chemical Society (2007), 129 (48), 15060-15071CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)E. coli ribonucleotide reductase (RNR) catalyzes the prodn. of deoxynucleotides using complex radical chem. Active RNR is composed of a 1:1 complex of two subunits: α2 and β2. α2 Binds nucleoside diphosphate substrates and deoxynucleotide/ATP allosteric effectors and is the site of nucleotide redn. β2 Contains the stable diiron tyrosyl radical (Y122·) cofactor that initiates deoxynucleotide formation. This process is proposed to involve reversible radical transfer over >35 Å between the Y122 in β2 and C439 in the active site of α2. A docking model of α2β2, based on structures of the individual subunits, suggests that radical initiation involves a pathway of transient, arom. amino acid radical intermediates, including Y730 and Y731 in α2. In this study the function of residues Y730 and Y731 is investigated by their site-specific replacement with 3-aminotyrosine (NH2Y). Using the in vivo suppressor tRNA/aminoacyl-tRNA synthetase method, Y730NH2Y-α2 and Y731NH2Y-α2 have been generated with high fidelity in yields of 4-6 mg/g of cell paste. These mutants have been examd. by stopped flow UV-vis and EPR spectroscopies in the presence of β2, CDP, and ATP. The results reveal formation of an NH2Y radical (NH2Y730· or NH2Y731·) in a kinetically competent fashion. Activity assays demonstrate that both NH2Y-α2s make deoxynucleotides. These results show that the NH2Y· can oxidize C439 suggesting a hydrogen atom transfer mechanism for the radical propagation pathway within α2. The obsd. NH2Y· may constitute the first detection of an amino acid radical intermediate in the proposed radical propagation pathway during turnover.
- 241Seyedsayamdost, M. R.; Chan, C. T. Y.; Mugnaini, V.; Stubbe, J.; Bennati, M. PELDOR Spectroscopy with DOPA-β2 and NH2Y-α2s: Distance Measurements between Residues Involved in the Radical Propagation Pathway of E. coli Ribonucleotide Reductase. J. Am. Chem. Soc. 2007, 129 (51), 15748– 15749, DOI: 10.1021/ja076459bGoogle Scholar241PELDOR Spectroscopy with DOPA-β2 and NH2Y-α2s: Distance Measurements between Residues Involved in the Radical Propagation Pathway of E. coli Ribonucleotide ReductaseSeyedsayamdost, Mohammad R.; Chan, Clement T. Y.; Mugnaini, Veronica; Stubbe, JoAnne; Bennati, MarinaJournal of the American Chemical Society (2007), 129 (51), 15748-15749CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Escherichia coli ribonucleotide reductase (RNR) catalyzes the redn. of nucleotides to 2'-deoxynucleotides. The active enzyme is a 1:1 complex of two homodimeric subunits, α2 and β2. The α2 is the site of nucleotide redn., and β2 harbors a diferric tyrosyl radical (Y122•) cofactor. Turnover requires formation of a cysteinyl radical (C439•) in the active site of α2 at the expense of the Y122• in β2. A docking model for the α2β2 interaction and a pathway for radical transfer from β2 to α2 have been proposed. This pathway contains three Ys: Y356 in β2 and Y731/Y730 in α2. We have previously incorporated 3-hydroxytyrosine and 3-aminotyrosine into these residues and showed that they act as radical traps. In this study, we use these α2/β2 variants and PELDOR spectroscopy to measure the distance between the Y122• in one αβ pair and the newly formed radical in the second αβ pair. The results yield distances that are similar to those predicted by the docking model for radical transfer. Further, they support a long-range radical initiation process for C439• generation and provide a structural constraint for residue Y356, which is thermally labile in all β2 structures solved to date.
- 242Seyedsayamdost, M. R.; Stubbe, J. Site-Specific Replacement of Y356 with 3,4-Dihydroxyphenylalanine in the β2 Subunit of E. coli Ribonucleotide Reductase. J. Am. Chem. Soc. 2006, 128 (8), 2522– 2523, DOI: 10.1021/ja057776qGoogle Scholar242Site-Specific Replacement of Y356 with 3,4-Dihydroxyphenylalanine in the β2 Subunit of E. coli Ribonucleotide ReductaseSeyedsayamdost, Mohammad R.; Stubbe, JoAnneJournal of the American Chemical Society (2006), 128 (8), 2522-2523CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)E. coli ribonucleotide reductase (RNR), composed of the homodimeric subunits α2 and β2, catalyzes the conversion of nucleotides to deoxynucleotides via complex radical chem. The radical initiation process involves a putative proton-coupled electron transfer (PCET) pathway over 35 Å between α2 and β2. Y356 in β2 has been proposed to lie on this pathway. To test this model, intein technol. has been used to make β2 semi-synthetically in which Y356 is replaced with a DOPA-amino acid. Anal. of this mutant with α2 and various combinations of substrate and effector by SF UV-vis spectroscopy and EPR methods demonstrates formation of a DOPA radical concomitant with disappearance of the tyrosyl radical, which initiates the reaction. The results reveal that Y356 lies on the PCET pathway and demonstrate the first kinetically competent conformational changes prior to ET. They further show that substrate binding brings about rapid conformational changes which place the complex into its active form(s) and suggest that the RNR complex is asym.
- 243Seyedsayamdost, M. R.; Stubbe, J. Forward and Reverse Electron Transfer with the Y356DOPA-β2 Heterodimer of E. coli Ribonucleotide Reductase. J. Am. Chem. Soc. 2007, 129 (8), 2226– 2227, DOI: 10.1021/ja0685607Google Scholar243Forward and Reverse Electron Transfer with the Y356DOPA-β2 Heterodimer of E. coli Ribonucleotide ReductaseSeyedsayamdost, Mohammad R.; Stubbe, JoAnneJournal of the American Chemical Society (2007), 129 (8), 2226-2227CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)E. coli ribonucleotide reductase catalyzes the conversion of nucleotides to deoxynucleotides, and consists of two subunits, α2 and β2. β2 Contains a stable diiron tyrosyl radical (Y122•) that is essential for catalysis. α2 harbors the active site, where nucleotide redn. occurs, as well as effector and activity sites which control substrate specificity and turnover rates. In this study, we have used intein methodol. to generate a heterodimer of β2 contg. the unnatural amino acid 3,4-dihydroxyphenylalanine (DOPA) at residue 356 (DOPA-ββ'). In this heterodimer, the β-monomer is full-length (residues 1-375), whereas the β'-monomer is truncated and only contains residues 1-353. DOPA-ββ', upon addn. of α2, CDP, and ATP effector, generates a DOPA• concomitant with loss of the Y122•. Anal. of DOPA• stability by EPR reveal that DOPA•-ββ' can reoxidize Y122 thereby regenerating the Y122•. These results, for the first time, directly demonstrate back electron transfer from residue 356 to Y122.
- 244Yokoyama, K.; Smith, A. A.; Corzilius, B.; Griffin, R. G.; Stubbe, J. Equilibration of Tyrosyl Radicals (Y356•, Y731•, Y730•) in the Radical Propagation Pathway of the Escherichia coli Class Ia Ribonucleotide Reductase. J. Am. Chem. Soc. 2011, 133 (45), 18420– 18432, DOI: 10.1021/ja207455kGoogle Scholar244Equilibration of Tyrosyl Radicals (Y356·, Y731·, Y730·) in the Radical Propagation Pathway of the Escherichia coli Class Ia Ribonucleotide ReductaseYokoyama, Kenichi; Smith, Albert A.; Corzilius, Bjorn; Griffin, Robert G.; Stubbe, JoAnneJournal of the American Chemical Society (2011), 133 (45), 18420-18432CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Escherichia coli ribonucleotide reductase (RNR) is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides using a diferric tyrosyl radical (Y122·) cofactor in β2 to initiate catalysis in α2. Each turnover requires reversible long-range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y122· ↹ [W48] ↹ Y356 within β to Y731 ↹ Y730 ↹ C439 within α). Previously, we reported that a β2 mutant with 3-nitrotyrosyl radical (NO2Y·; 1.2 radicals/β2) in place of Y122· in the presence of α2, CDP, and ATP catalyzes formation of 0.6 equiv of dCDP and accumulates 0.6 equiv of a new Y· proposed to be located on Y356 in β2. We now report three independent methods that establish that Y356 is the predominant location (85-90%) of the radical, with the remaining 10-15% delocalized onto Y731 and Y730 in α2. Pulsed electron-electron double-resonance spectroscopy on samples prepd. by rapid freeze quench (RFQ) methods identified three distances: 30 ± 0.4 Å (88% ± 3%) and 33 ± 0.4 and 38 ± 0.5 Å (12% ± 3%) indicative of NO2Y122·-Y356·, NO2Y122·-NO2Y122·, and NO2Y122·-Y731(730)·, resp. Radical distribution in α2 was supported by RFQ ESR (EPR) studies using Y731(3,5-F2Y) or Y730(3,5-F2Y)-α2, which revealed F2Y·, studies using globally incorporated [β-2H2]Y-α2, and anal. using parameters obtained from 140 GHz EPR spectroscopy. The amt. of Y· delocalized in α2 from these two studies varied from 6% to 15%. The studies together give the first insight into the relative redox potentials of the three transient Y· radicals in the PCET pathway and their conformations.
- 245Lin, C.-Y.; Muñoz Hernández, A. L.; Laremore, T. N.; Silakov, A.; Krebs, C.; Boal, A. K.; Bollinger, J. M., Jr Use of Noncanonical Tyrosine Analogues to Probe Control of Radical Intermediates during Endoperoxide Installation by Verruculogen Synthase (FtmOx1). ACS Catal. 2022, 12 (12), 6968– 6979, DOI: 10.1021/acscatal.2c01037Google ScholarThere is no corresponding record for this reference.
- 246Taylor, A.; Heyes, D. J.; Scrutton, N. S. Catalysis by Nature’s photoenzymes. Curr. Opin. Struct. Biol. 2022, 77, 102491, DOI: 10.1016/j.sbi.2022.102491Google Scholar246Catalysis by Nature's photoenzymesTaylor, Aoife; Heyes, Derren J.; Scrutton, Nigel S.Current Opinion in Structural Biology (2022), 77 (), 102491CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. Photoenzymes use light to initiate biochem. reactions. Although rarely found in nature, their study has advanced understanding of how light energy can be harnessed to facilitate enzyme catalysis, which is also of importance to the design and engineering of man-made photocatalysts. Natural photoenzymes can be assigned to one of two families, based broadly on the nature of the light-sensing chromophores used, those being chlorophyll-like tetrapyrroles or flavins. In all cases, light absorption leads to excited state electron transfer, which in turn initiates photocatalysis. Reviewed here are recent findings relating to the structures and mechanisms of known photoenzymes. We highlight recent advances that have deepened understanding of mechanisms in biol. photocatalysis.
- 247Taylor, A.; Zhang, S.; Johannissen, L. O.; Sakuma, M.; Phillips, R. S.; Green, A. P.; Hay, S.; Heyes, D. J.; Scrutton, N. S. Mechanistic Implications of the Ternary Complex Structural Models for the Photoenzyme Protochlorophyllide Oxidoreductase. FEBS J. 2024, 291, 1404, DOI: 10.1111/febs.17025Google ScholarThere is no corresponding record for this reference.
- 248Horowitz, S.; Adhikari, U.; Dirk, L. M. A.; Del Rizzo, P. A.; Mehl, R. A.; Houtz, R. L.; Al-Hashimi, H. M.; Scheiner, S.; Trievel, R. C. Manipulating Unconventional CH-Based Hydrogen Bonding in a Methyltransferase via Noncanonical Amino Acid Mutagenesis. ACS Chem. Biol. 2014, 9 (8), 1692– 1697, DOI: 10.1021/cb5001185Google Scholar248Manipulating Unconventional CH-Based Hydrogen Bonding in a Methyltransferase via Noncanonical Amino Acid MutagenesisHorowitz, Scott; Adhikari, Upendra; Dirk, Lynnette M. A.; Del Rizzo, Paul A.; Mehl, Ryan A.; Houtz, Robert L.; Al-Hashimi, Hashim M.; Scheiner, Steve; Trievel, Raymond C.ACS Chemical Biology (2014), 9 (8), 1692-1697CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Recent studies have demonstrated that the active sites of S-adenosylmethionine (AdoMet)-dependent methyltransferases form strong carbon-oxygen (CH···O) hydrogen bonds with the substrate's sulfonium group that are important in AdoMet binding and catalysis. To probe these interactions, we substituted the noncanonical amino acid p-aminophenylalanine (pAF) for the active site tyrosine in the lysine methyltransferase SET7/9, which forms multiple CH···O hydrogen bonds to AdoMet and is invariant in SET domain enzymes. Using quantum chem. calcns. to predict the mutation's effects, coupled with biochem. and structural studies, we obsd. that pAF forms a strong CH···N hydrogen bond to AdoMet that is offset by an energetically unfavorable amine group rotamer within the SET7/9 active site that hinders AdoMet binding and activity. Together, these results illustrate that the invariant tyrosine in SET domain methyltransferases functions as an essential hydrogen bonding hub and cannot be readily substituted by residues bearing other hydrogen bond acceptors.
- 249Kirsh, J. M.; Weaver, J. B.; Boxer, S. G.; Kozuch, J. Comprehensive Analysis of Nitrile Probe IR Shifts and Intensities in Proteins: Experiment and Critical Evaluation of Simulations. ChemRxiv 2024, DOI: 10.26434/chemrxiv-2023-j935v-v2Google ScholarThere is no corresponding record for this reference.
- 250Weaver, J. B.; Kozuch, J.; Kirsh, J. M.; Boxer, S. G. Nitrile Infrared Intensities Characterize Electric Fields and Hydrogen Bonding in Protic, Aprotic, and Protein Environments. J. Am. Chem. Soc. 2022, 144 (17), 7562– 7567, DOI: 10.1021/jacs.2c00675Google Scholar250Nitrile Infrared Intensities Characterize Electric Fields and Hydrogen Bonding in Protic, Aprotic, and Protein EnvironmentsWeaver, Jared Bryce; Kozuch, Jacek; Kirsh, Jacob M.; Boxer, Steven G.Journal of the American Chemical Society (2022), 144 (17), 7562-7567CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Nitriles are widely used vibrational probes; however, the interpretation of their IR frequencies is complicated by hydrogen bonding (H-bonding) in protic environments. We report a new vibrational Stark effect (VSE) that correlates the elec. field projected on the -C≡N bond to the transition dipole moment and, by extension, the nitrile peak area or integrated intensity. This linear VSE applies to both H-bonding and non-H-bonding interactions. It can therefore be generally applied to det. elec. fields in all environments. Addnl., it allows for semiempirical extn. of the H-bonding contribution to the blueshift of the nitrile frequency. Nitriles were incorporated at H-bonding and non-H-bonding protein sites using amber suppression, and each nitrile variant was structurally characterized at high resoln. We exploited the combined information available from variations in frequency and integrated intensity and demonstrate that nitriles are a generally useful probe for elec. fields.
- 251Zheng, C.; Mao, Y.; Kozuch, J.; Atsango, A. O.; Ji, Z.; Markland, T. E.; Boxer, S. G. A Two-Directional Vibrational Probe Reveals Different Electric Field Orientations in Solution and an Enzyme Active Site. Nat. Chem. 2022, 14 (8), 891– 897, DOI: 10.1038/s41557-022-00937-wGoogle Scholar251A two-directional vibrational probe reveals different electric field orientations in solution and an enzyme active siteZheng, Chu; Mao, Yuezhi; Kozuch, Jacek; Atsango, Austin O.; Ji, Zhe; Markland, Thomas E.; Boxer, Steven G.Nature Chemistry (2022), 14 (8), 891-897CODEN: NCAHBB; ISSN:1755-4330. (Nature Portfolio)The catalytic power of an elec. field depends on its magnitude and orientation with respect to the reactive chem. species. Understanding and designing new catalysts for electrostatic catalysis thus requires methods to measure the elec. field orientation and magnitude at the mol. scale. Elec. field orientations can be extd. using a two-directional vibrational probe by exploiting the vibrational Stark effect of both the C:O and C-D stretches of a deuterated aldehyde. Combining spectroscopy with mol. dynamics and electronic structure partitioning methods, despite distinct polarities, solvents act similarly in their preference for electrostatically stabilizing large bond dipoles at the expense of destabilizing small ones. In contrast, for an active-site aldehyde inhibitor of liver alc. dehydrogenase, the elec. field orientation deviates markedly from that found in solvents, which provides direct evidence for the fundamental difference between the electrostatic environment of solvents and of a preorganized enzyme active site.
- 252Kedzierski, P.; Zaczkowska, M.; Sokalski, W. A. Extreme Catalytic Power of Ketosteroid Isomerase Related to the Reversal of Proton Dislocations in Hydrogen-Bond Network. J. Phys. Chem. B 2020, 124 (18), 3661– 3666, DOI: 10.1021/acs.jpcb.0c01489Google Scholar252Extreme catalytic power of ketosteroid isomerase related to the reversal of proton dislocations in hydrogen-bond networkKedzierski, Pawel; Zaczkowska, Maria; Sokalski, W. AndrzejJournal of Physical Chemistry B (2020), 124 (18), 3661-3666CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)Dynamic electrostatic catalytic field (DECF) vectors derived from transition state and reactant wavefunctions for the two-step reaction occurring within ketosteroid isomerase (KSI) have been calcd. using MP2/aug-cc-pVTZ and lower theory levels to det. the magnitude of the catalytic effect and the optimal directions of proton transfers in the KSI hydrogen-bond network. The most surprising and meaningful finding is that the KSI catalytic activity is enhanced by proton dislocations proceeding in opposite directions for each of the two consecutive reaction steps in the same hydrogen network. Such a mechanism allows an ultrafast switching of the catalytic proton wire environment, possibly related to the exceptionally high KSI catalytic power.
- 253Pollack, R. M. Enzymatic Mechanisms for Catalysis of Enolization: Ketosteroid Isomerase. Bioorg. Chem. 2004, 32 (5), 341– 353, DOI: 10.1016/j.bioorg.2004.06.005Google Scholar253Enzymatic mechanisms for catalysis of enolization: ketosteroid isomerasePollack, Ralph M.Bioorganic Chemistry (2004), 32 (5), 341-353CODEN: BOCMBM; ISSN:0045-2068. (Elsevier)A review. Breaking a C-H bond adjacent to a carbonyl group is a slow step in a large no. of chem. reactions. However, many enzymes are capable of catalyzing this reaction with great efficiency. One of the most proficient of these enzymes is 3-oxo-Δ5-steroid isomerase (KSI), which catalyzes the isomerization of a wide variety of 3-oxo-Δ5-steroids to their Δ4-conjugated isomers. Here, the reaction mechanism of KSI is discussed, with particular emphasis on energetic considerations. Both exptl. and theor. approaches are considered to explain the mechanistic details of the reaction.
- 254Wu, Y.; Boxer, S. G. A Critical Test of the Electrostatic Contribution to Catalysis with Noncanonical Amino Acids in Ketosteroid Isomerase. J. Am. Chem. Soc. 2016, 138 (36), 11890– 11895, DOI: 10.1021/jacs.6b06843Google Scholar254A Critical Test of the Electrostatic Contribution to Catalysis with Noncanonical Amino Acids in Ketosteroid IsomeraseWu, Yufan; Boxer, Steven G.Journal of the American Chemical Society (2016), 138 (36), 11890-11895CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The vibrational Stark effect (VSE) has been used to measure the elec. field in the active site of ketosteroid isomerase (KSI). These measured fields correlate with ΔG‡ in a series of conventional mutants yielding an est. for the electrostatic contribution to catalysis (Fried et al. Science, 2014, 346, 1510-1513). In this work we test this result with much more conservative variants in which individual Tyr residues in the active site are replaced by 3-chlorotyrosine via amber suppression. The elec. fields sensed at the position of the carbonyl bond involved in charge displacement during catalysis were characterized using the VSE, where the field sensitivity has been calibrated by vibrational Stark spectroscopy, solvatochromism, and MD simulations. A linear relationship is obsd. between the elec. field and ΔG‡ that interpolates between wild-type and more drastic conventional mutations, reinforcing the evaluation of the electrostatic contribution to catalysis in KSI. A simplified model and calcn. are developed to est. changes in the elec. field accompanying changes in the extended hydrogen-bond network in the active site. The results are consistent with a model in which the O-H group of a key active site tyrosine functions by imposing a static electrostatic potential onto the carbonyl bond. The model suggests that the contribution to catalysis from the active site hydrogen bonds is of similar wt. to the distal interactions from the rest of the protein. A similar linear correlation was also obsd. between the proton affinity of KSI's active site and the catalytic rate, suggesting a direct connection between the strength of the H-bond and the elec. field it exerts.
- 255Faraldos, J. A.; Antonczak, A. K.; González, V.; Fullerton, R.; Tippmann, E. M.; Allemann, R. K. Probing Eudesmane Cation-π Interactions in Catalysis by Aristolochene Synthase with Non-canonical Amino Acids. J. Am. Chem. Soc. 2011, 133 (35), 13906– 13909, DOI: 10.1021/ja205927uGoogle Scholar255Probing eudesmane cation-π interactions in catalysis by aristolochene synthase with non-canonical amino acidsFaraldos, Juan A.; Antonczak, Alicja K.; Gonzalez, Veronica; Fullerton, Rebecca; Tippmann, Eric M.; Allemann, Rudolf K.Journal of the American Chemical Society (2011), 133 (35), 13906-13909CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Stabilization of the reaction intermediate, eudesmane cation (I), through interaction with Trp-334 during catalysis by aristolochene synthase of Penicillium roqueforti was investigated by site-directed incorporation of proteinogenic and non-canonical arom. amino acids. The amt. of germacrene A (II) generated by the mutant enzymes served as a measure of the stabilization of I. II is a neutral intermediate, from which I is formed during PR-AS catalysis by protonation of the C6,C7 double bond. The replacement of Trp-334 with para-substituted Phe residues of increasing electron-withdrawing properties led to a progressive accumulation of II that showed a good correlation with the interaction energies of simple cations such as Na+ with substituted benzenes. These results provided compelling evidence for the stabilizing role played by Trp-334 in aristolochene synthase catalysis for the energetically demanding transformation of II to I.
- 256Morikubo, N.; Fukuda, Y.; Ohtake, K.; Shinya, N.; Kiga, D.; Sakamoto, K.; Asanuma, M.; Hirota, H.; Yokoyama, S.; Hoshino, T. Cation-π Interaction in the Polyolefin Cyclization Cascade Uncovered by Incorporating Unnatural Amino Acids into the Catalytic Sites of Squalene Cyclase. J. Am. Chem. Soc. 2006, 128 (40), 13184– 13194, DOI: 10.1021/ja063358pGoogle Scholar256Cation-π Interaction in the Polyolefin Cyclization Cascade Uncovered by Incorporating Unnatural Amino Acids into the Catalytic Sites of Squalene CyclaseMorikubo, Noriko; Fukuda, Yoriyuki; Ohtake, Kazumasa; Shinya, Naoko; Kiga, Daisuke; Sakamoto, Kensaku; Asanuma, Miwako; Hirota, Hiroshi; Yokoyama, Shigeyuki; Hoshino, TsutomuJournal of the American Chemical Society (2006), 128 (40), 13184-13194CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)It has been assumed that the π-electrons of arom. residues in the catalytic sites of triterpene cyclases stabilize the cationic intermediates formed during the polycyclization cascade of squalene or oxidosqualene, but no definitive exptl. evidence has been given. To validate this cation-π interaction, natural and unnatural arom. amino acids were site-specifically incorporated into squalene-hopene cyclase (SHC) from Alicyclobacillus acidocaldarius and the kinetic data of the mutants were compared with that of the wild-type SHC. The catalytic sites of Phe-365 and Phe-605 were substituted with O-methyltyrosine, tyrosine, and tryptophan, which have higher cation-π binding energies than phenylalanine. These replacements actually increased the SHC activity at low temp., but decreased the activity at high temp., as compared with the wild-type SHC. This decreased activity is due to the disorganization of the protein architecture caused by the introduction of the amino acids more bulky than phenylalanine. Then, mono-, di-, and trifluorophenylalanines were incorporated at positions 365 and 605; these amino acids reduce cation-π binding energies but have van der Waals radii similar to that of phenylalanine. The activities of the SHC variants with fluorophenylalanines were found to be inversely proportional to the no. of the fluorine atoms on the arom. ring and clearly correlated with the cation-π binding energies of the ring moiety. No serious structural alteration was obsd. for these variants even at high temp. These results unambiguously show that the π-electron d. of residues 365 and 605 has a crucial role for the efficient polycyclization reaction by SHC. This is the first report to demonstrate exptl. the involvement of cation-π interaction in triterpene biosynthesis.
- 257Herbst, R. W.; Guce, A.; Bryngelson, P. A.; Higgins, K. A.; Ryan, K. C.; Cabelli, D. E.; Garman, S. C.; Maroney, M. J. Role of Conserved Tyrosine Residues in NiSOD Catalysis: A Case of Convergent Evolution. Biochemistry 2009, 48 (15), 3354– 3369, DOI: 10.1021/bi802029tGoogle Scholar257Role of Conserved Tyrosine Residues in NiSOD Catalysis: A Case of Convergent EvolutionHerbst, Robert W.; Guce, Abigail; Bryngelson, Peter A.; Higgins, Khadine A.; Ryan, Kelly C.; Cabelli, Diane E.; Garman, Scott C.; Maroney, Michael J.Biochemistry (2009), 48 (15), 3354-3369CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Superoxide dismutases rely on protein structural elements to adjust the redox potential of the metallocenter to an optimum value near 300 mV (vs. NHE), to provide a source of protons for catalysis, and to control the access of anions to the active site. These aspects of the catalytic mechanism are examd. herein for recombinant prepns. of the nickel-dependent SOD (NiSOD) from Streptomyces coelicolor and for a series of mutants that affect a key tyrosine residue, Tyr9 (Y9F-, Y62F-, Y9F/Y62F-, and D3A-NiSOD). Structural aspects of the nickel sites are examd. by a combination of EPR and X-ray absorption spectroscopies, and by single-crystal X-ray diffraction at ∼1.9 Å resoln. in the case of Y9F- and D3A-NiSODs. The functional effects of the mutations are examd. by kinetic studies employing pulse radiolytic generation of O2- and by redox titrns. These studies reveal that although the structure of the nickel center in NiSOD is unique, the ligand environment is designed to optimize the redox potential at 290 mV and results in the oxidn. of 50% of the nickel centers in the oxidized hexamer. Kinetic investigations show that all of the mutant proteins have considerable activity. In the case of Y9F-NiSOD, the enzyme exhibits satn. behavior that is not obsd. in wild-type (WT) NiSOD and suggests that release of peroxide is inhibited. The crystal structure of Y9F-NiSOD reveals an anion binding site that is occupied by either Cl- or Br- and is located close to but not within bonding distance of the nickel center. The structure of D3A-NiSOD reveals that in addn. to affecting the interaction between subunits, this mutation repositions Tyr9 and leads to altered chem. with peroxide. Comparisons with Mn(SOD) and Fe(SOD) reveal that although different strategies for adjusting the redox potential and supply of protons are employed, NiSOD has evolved a similar strategy for controlling the access of anions to the active site.
- 258Campeciño, J. O.; Dudycz, L. W.; Tumelty, D.; Berg, V.; Cabelli, D. E.; Maroney, M. J. A Semisynthetic Strategy Leads to Alteration of the Backbone Amidate Ligand in the NiSOD Active Site. J. Am. Chem. Soc. 2015, 137 (28), 9044– 9052, DOI: 10.1021/jacs.5b03629Google Scholar258A Semisynthetic Strategy Leads to Alteration of the Backbone Amidate Ligand in the NiSOD Active SiteCampecino, Julius O.; Dudycz, Lech W.; Tumelty, David; Berg, Volker; Cabelli, Diane E.; Maroney, Michael J.Journal of the American Chemical Society (2015), 137 (28), 9044-9052CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Computational investigations have implicated the amidate ligand in nickel superoxide dismutase (NiSOD) in stabilizing Ni-centered redox catalysis and in preventing cysteine thiolate ligand oxidn. To test these predictions, we have used an exptl. approach utilizing a semisynthetic scheme that employs native chem. ligation of a pentapeptide (HCDLP) to recombinant S. coelicolor NiSOD lacking these N-terminal residues, NΔ5-NiSOD. Wild-type enzyme produced in this manner exhibits the characteristic spectral properties of recombinant WT-NiSOD and is as catalytically active. The semisynthetic scheme was also employed to construct a variant where the amidate ligand was converted to a secondary amine, H1*-NiSOD, a novel strategy that retains a backbone N-donor atom. The H1*-NiSOD variant was found to have only ∼1% of the catalytic activity of the recombinant wild-type enzyme, and had altered spectroscopic properties. X-ray absorption spectroscopy reveals a four-coordinate planar site with N2S2-donor ligands, consistent with electronic absorption spectroscopic results indicating that the Ni center in H1*-NiSOD is mostly reduced in the as-isolated sample, as opposed to 50:50 Ni(II)/Ni(III) mixt. that is typical for the recombinant wild-type enzyme. The EPR spectrum of as-isolated H1*-NiSOD accounts for ∼11% of the Ni in the sample and is similar to WT-NiSOD, but more axial, with gz < gx,y. 14N-hyperfine is obsd. on gz, confirming the addn. of the apical histidine ligand in the Ni(III) complex. The altered electronic properties and implications for redox catalysis are discussed in light of predictions based on synthetic and computational models.
- 259Evans, R. M.; Krahn, N.; Murphy, B. J.; Lee, H.; Armstrong, F. A.; Söll, D. Selective Cysteine-To-Selenocysteine Changes in a [NiFe]-Hydrogenase Confirm a Special Position for Catalysis and Oxygen Tolerance. Proc. Natl. Acad. Sci. U.S.A. 2021, 118 (13), e2100921118 DOI: 10.1073/pnas.2100921118Google ScholarThere is no corresponding record for this reference.
- 260Mukai, T.; Sevostyanova, A.; Suzuki, T.; Fu, X.; Söll, D. A Facile Method for Producing Selenocysteine-Containing Proteins. Angew. Chem. Int. Ed. 2018, 57 (24), 7215– 7219, DOI: 10.1002/anie.201713215Google Scholar260A Facile Method for Producing Selenocysteine-Containing ProteinsMukai, Takahito; Sevostyanova, Anastasia; Suzuki, Tateki; Fu, Xian; Soell, DieterAngewandte Chemie, International Edition (2018), 57 (24), 7215-7219CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Selenocysteine (Sec, U) confers new chem. properties on proteins. Improved tools are thus required that enable Sec insertion into any desired position of a protein. We report a facile method for synthesizing selenoproteins with multiple Sec residues by expanding the genetic code of Escherichia coli. We recently discovered allo-tRNAs, tRNA species with unusual structure, that are as efficient serine acceptors as E. coli tRNASer. Ser-allo-tRNA was converted into Sec-allo-tRNA by Aeromonas salmonicida selenocysteine synthase (SelA). Sec-allo-tRNA variants were able to read through five UAG codons in the fdhF mRNA coding for E. coli formate dehydrogenase H, and produced active FDHH with five Sec residues in E. coli. Engineering of the E. coli selenium metab. along with mutational changes in allo-tRNA and SelA improved the yield and purity of recombinant human glutathione peroxidase 1 (to over 80 %). Thus, our allo-tRNAUTu system offers a new selenoprotein engineering platform.
- 261Snider, G. W.; Ruggles, E.; Khan, N.; Hondal, R. J. Selenocysteine Confers Resistance to Inactivation by Oxidation in Thioredoxin Reductase: Comparison of Selenium and Sulfur Enzymes. Biochemistry 2013, 52 (32), 5472– 5481, DOI: 10.1021/bi400462jGoogle Scholar261Selenocysteine Confers Resistance to Inactivation by Oxidation in Thioredoxin Reductase: Comparison of Selenium and Sulfur EnzymesSnider, Gregg W.; Ruggles, Erik; Khan, Nadeem; Hondal, Robert J.Biochemistry (2013), 52 (32), 5472-5481CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Mammalian thioredoxin reductase (TR) is a selenocysteine (Sec)-contg. homodimeric pyridine nucleotide oxidoreductase which catalyzes the redn. of oxidized thioredoxin. We have previously demonstrated the full-length mitochondrial mammalian TR (mTR3) enzyme to be resistant to inactivation from exposure to 50 mM H2O2. Because a Sec residue oxidizes more rapidly than a cysteine (Cys) residue, it has been previously thought that Sec-contg. enzymes are "sensitive to oxidn." compared to Cys-orthologues. Here we show for the first time a direct comparison of the abilities of Sec-contg. mTR3 and the Cys-orthologue from D. melanogaster (DmTR) to resist inactivation by oxidn. from a variety of oxidants including H2O2, hydroxyl radical, peroxynitrite, hypochlorous acid, hypobromous acid, and hypothiocyanous acid. The results show that the Sec-contg. TR is far superior to the Cys-orthologue TR in resisting inactivation by oxidn. To further test our hypothesis that the use of Sec confers strong resistance to inactivation by oxidn., we constructed a chimeric enzyme in which we replaced the active site Cys nucleophile of DmTR with a Sec residue using semisynthesis. The chimeric Sec-contg. enzyme has similar ability to resist inactivation by oxidn. as the wild type Sec-contg. TR from mouse mitochondria. The use of Sec in the chimeric enzyme "rescued" the enzyme from oxidant-induced inactivation for all of the oxidants tested in this study, in direct contrast to previous understanding. We discuss two possibilities for this rescue effect from inactivation under identical conditions of oxidative stress: (i) Sec resists overoxidn. and inactivation, whereas a Cys residue can be permanently overoxidized to the sulfinic acid form, and (ii) Sec protects the body of the enzyme from harmful oxidn. by allowing the enzyme to metabolize (turnover) various oxidants much better than a Cys-contg. TR.
- 262Wu, Z. P.; Hilvert, D. Selenosubtilisin as a Glutathione Peroxidase Mimic. J. Am. Chem. Soc. 1990, 112 (14), 5647– 5648, DOI: 10.1021/ja00170a043Google Scholar262Selenosubtilisin as a glutathione peroxidase mimicWu, Zhen Ping; Hilvert, DonaldJournal of the American Chemical Society (1990), 112 (14), 5647-8CODEN: JACSAT; ISSN:0002-7863.An artificial Se-contg. protein, selenolsubtilisin, mimics the redox properties of the naturally-occurring selenoenzyme glutathione peroxidase. It efficiently catalyzes the redn. of alkyl hydroperoxides by aryl thiols under mild aq. conditions. Kinetic studies suggest that the enzymic reaction proceeds via a ping pong mechanism with a covalent selenenyl sulfide deriv. as a key reaction intermediate. Comparison of the initial rates for the redn. of tert-Bu hydroperoxide by 3-carboxy-4-nitrobenzenethiol catalyzed by the selenoprotein and by di-Ph diselenide indicates that the protein binding site enhances the reaction rate ≥70,000-fold. Artificial peroxidases may be useful both as models for understanding the mechanism of action of the analogous natural enzymes and as antioxidant drugs in medicine or as practical catalysts in chem. synthesis.
- 263Hardy, F. J.; Ortmayer, M.; Green, A. P.; Noble, C. E. M.; Anderson, J. L. R. Recent Advances in Understanding, Enhancing and Creating Heme Peroxidases. In Reference Module in Chemistry, Molecular Sciences and Chemical Engineering; Elsevier, 2020. DOI: 10.1016/B978-0-08-102688-5.00021-0Google ScholarThere is no corresponding record for this reference.
- 264Poulos, T. L. Heme Enzyme Structure and Function. Chem. Rev. 2014, 114, 3919– 3962, DOI: 10.1021/cr400415kGoogle Scholar264Heme Enzyme Structure and FunctionPoulos, Thomas L.Chemical Reviews (Washington, DC, United States) (2014), 114 (7), 3919-3962CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Metalloporphyrins are employed in various capacities throughout the biosphere, and of these, heme (iron protoporphyrin IX) is one of the most abundant and widely used. Heme is well-known for its roles in shuttling electrons between proteins as seen in mitochondrial respiration and in O2 storage as is the case with globins, but it also serves as a cofactor in multiple enzyme-mediated processes. Although heme enzymes can catalyze both reductive and oxidative reactions, the present review focuses primarily on those that catalyze oxidn. reactions, and esp. those for which crystal structures are available.
- 265Behan, R. K.; Green, M. T. On the Status of Ferryl Protonation. J. Inorg. Biochem. 2006, 100 (4), 448– 459, DOI: 10.1016/j.jinorgbio.2005.12.019Google Scholar265On the status of ferryl protonationBehan, Rachel K.; Green, Michael T.Journal of Inorganic Biochemistry (2006), 100 (4), 448-459CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier B.V.)A review. The authors examine the issue of ferryl protonation in heme proteins. An anal. of the results obtained from x-ray crystallog., resonance Raman spectroscopy, and extended x-ray absorption spectroscopy (EXAFS) is presented. Fe-O bond distances obtained from all three techniques are compared using Badger's rule. The long Fe-O bond lengths found in the ferryl crystal structures of myoglobin, cytochrome c peroxidase, horseradish peroxidase, and catalase deviate substantially from the values predict by Badger's rule, while the oxo-like distances obtained from EXAFS measurements are in good agreement with the empirical formula. D. functional calcns., which suggest that Moessbauer spectroscopy can be used to det. ferryl protonation states, are presented. The authors' calcns. indicate that the quadrupole splitting (ΔEq) changes significantly upon ferryl protonation. New resonance Raman data for horse-heart myoglobin compd. II (Mb-II, pH 4.5) are also presented. An Fe-O stretching frequency of 790 cm-1 (shifting to 754 cm-1 with 18O substitution) was obtained. This frequency provides a Badger distance of rFe-O = 1.66 Å. This distance is in agreement with the 1.69 Å Fe-O bond distance obtained from EXAFS measurements but is significantly shorter than the 1.93 Å bond found in the crystal structure of Mb-II (pH 5.2). In light of the available evidence, the authors conclude that the ferryl forms of myoglobin (pKa ≤ 4), horseradish peroxidase (pKa ≤ 4), cytochrome c peroxidase (pKa ≤ 4), and catalase (pKa ≤ 7) are not basic. They are authentic FeIV oxos with Fe-O bonds on the order of 1.65 Å.
- 266Sivaramakrishnan, S.; Ouellet, H.; Du, J.; McLean, K. J.; Medzihradszky, K. F.; Dawson, J. H.; Munro, A. W.; Ortiz de Montellano, P. R. A Novel Intermediate in the Reaction of Seleno CYP119 with m-Chloroperbenzoic Acid. Biochemistry 2011, 50 (14), 3014– 3024, DOI: 10.1021/bi101728yGoogle Scholar266A novel intermediate in the reaction of seleno CYP119 with m-chloroperbenzoic acidSivaramakrishnan, Santhosh; Ouellet, Hugues; Du, Jing; McLean, Kirsty J.; Medzihradszky, Katalin F.; Dawson, John H.; Munro, Andrew W.; Ortiz de Montellano, Paul R.Biochemistry (2011), 50 (14), 3014-3024CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Cytochrome P 450-mediated monooxygenation generally proceeds via a reactive ferryl intermediate coupled to a ligand radical [Fe(IV)≡O]+· termed Compd. I (Cpd I). The proximal cysteine thiolate ligand is a crit. determinant of the spectral and catalytic properties of P 450 enzymes. To explore the effect of an increased level of donation of electrons by the proximal ligand in the P 450 catalytic cycle, we recently reported successful incorporation of SeCys into the active site of CYP119, a thermophilic cytochrome P 450. Here we report relevant phys. properties of SeCYP119 and a detailed anal. of the reaction of SeCYP119 with m-chloroperbenzoic acid (mCPBA). Our results indicate that the selenolate anion reduces rather than stabilizes Cpd I and also protects the heme from oxidative destruction, leading to the generation of a new stable species with an absorbance max. at 406 nm. This stable intermediate can be returned to the normal ferric state by reducing agents and thiols, in agreement with oxidative modification of the selenolate ligand itself. Thus, in the seleno protein, the oxidative damage shifts from the heme to the proximal ligand, presumably because (a) an increased level of donation of electrons more efficiently quenches reactive species such as Cpd I and (b) the protection of the thiolate ligand provided by the protein active site structure is insufficient to shield the more oxidizable selenolate ligand.
- 267Aldag, C.; Gromov, I. A.; García-Rubio, I.; Von Koenig, K.; Schlichting, I.; Jaun, B.; Hilvert, D. Probing the Role of the Proximal Heme Ligand in Cytochrome P450cam by Recombinant Incorporation of Selenocysteine. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (14), 5481– 5486, DOI: 10.1073/pnas.0810503106Google ScholarThere is no corresponding record for this reference.
- 268Jiang, Y.; Sivaramakrishnan, S.; Hayashi, T.; Cohen, S.; Moënne-Loccoz, P.; Shaik, S.; Ortiz de Montellano, P. R. Calculated and Experimental Spin State of Seleno Cytochrome P450. Angew. Chem. Int. Ed. 2009, 48 (39), 7193– 7195, DOI: 10.1002/anie.200901485Google Scholar268Calculated and Experimental Spin State of Seleno Cytochrome P450Jiang, Yongying; Sivaramakrishnan, Santhosh; Hayashi, Takahiro; Cohen, Shimrit; Moenne-Loccoz, Pierre; Shaik, Sason; Ortiz de Montellano, Paul R.Angewandte Chemie, International Edition (2009), 48 (39), 7193-7195, S7193/1-S7193/19CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The cysteine thiolate ligand coordinated to the heme iron atom in cytochrome P 450 is thought to be responsible for the unique spectroscopic and catalytic properties of these enzymes. To explore the role of the proximal ligand in these proteins, the cysteine has been replaced by a variety of other ligands by site-specific mutagenesis. Here, the expression and characterization of a seleno cytochrome P 450 in which the cysteine thiolate iron ligand is replaced by a selenocysteine is reported. CYP119 was used for this substitution because the proximal ligand is the only cysteine in its sequence. The seleno protein was expressed in a cysteine auxotroph BL21(DE3)CysE strain of Escherichia coli that cannot synthesize cysteine owing to a mutation in the CysE gene. A pCWori vector contg. the CYP119 gene encoding a 6-His tag at the C-terminus was transformed into the auxotrophic BL21(DE3)Cys cells, and the seleno protein was expressed in minimal media contg. L-selenocystine. The protein yield was 2.6 mg L-1 of culture after affinity purifn., which is approx. 8-10 times lower than that of the normal thiolate-ligated protein. This approach results in over 70 % replacement of the cysteine by a selenocysteine as judged by the relative peak intensities of the Cys and SeCys proteins by LC/ESI-MS. In conclusion, the first selenium-incorporated P 450 encoding was expressed and characterized. The preliminary results reveal that the spectral characteristics of this novel CYP119 are comparable to those of the corresponding WT protein, indicating the presence of a six-coordinate low-spin heme iron with water as a distal ligand. More importantly, the catalytic activity of the seleno-mutant is comparable to that of the WT enzyme. Furthermore, computational calcns. clearly support the exptl. assigned spin state. Future studies will focus on both examg. how this substitution affects the stability of the putative compd. I species and the development of novel catalysts.
- 269Onderko, E. L.; Silakov, A.; Yosca, T. H.; Green, M. T. Characterization of a Selenocysteine-Ligated P450 Compound I Reveals Direct Link Between Electron Donation and Reactivity. Nat. Chem. 2017, 9 (7), 623– 628, DOI: 10.1038/nchem.2781Google Scholar269Characterization of a selenocysteine-ligated P450 compound I reveals direct link between electron donation and reactivityOnderko, Elizabeth L.; Silakov, Alexey; Yosca, Timothy H.; Green, Michael T.Nature Chemistry (2017), 9 (7), 623-628CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)Strong electron-donation from the axial thiolate ligand of cytochrome P 450 has been proposed to increase the reactivity of compd. I with respect to C-H bond activation. However, it has proven difficult to test this hypothesis, and a direct link between reactivity and electron donation has yet to be established. To make this connection, we prepd. a selenolate-ligated cytochrome P 450 compd. I intermediate. This isoelectronic perturbation allowed for direct comparisons with the wild-type enzyme. Selenium incorporation was achieved using a cysteine auxotrophic Escherichia coli strain. The intermediate was prepd. with m-chloroperbenzoic acid and characterized by UV-visible, Moessbauer, and ESR spectroscopies. Measurements revealed increased asymmetry around the ferryl moiety, consistent with increased electron donation from the axial selenolate ligand. In line with this observation, we found that the selenolate-ligated compd. I cleaved C-H bonds more rapidly than the wild-type intermediate.
- 270Sivaramakrishnan, S.; Ouellet, H.; Matsumura, H.; Guan, S.; Moënne-Loccoz, P.; Burlingame, A. L.; Ortiz De Montellano, P. R. Proximal Ligand Electron Donation and Reactivity of the Cytochrome P450 Ferric-Peroxo Anion. J. Am. Chem. Soc. 2012, 134 (15), 6673– 6684, DOI: 10.1021/ja211499qGoogle Scholar270Proximal Ligand Electron Donation and Reactivity of the Cytochrome P450 Ferric-Peroxo AnionSivaramakrishnan, Santhosh; Ouellet, Hugues; Matsumura, Hirotoshi; Guan, Shenheng; Moenne-Loccoz, Pierre; Burlingame, Alma L.; Ortiz de Montellano, Paul R.Journal of the American Chemical Society (2012), 134 (15), 6673-6684CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)CYP125 from Mycobacterium tuberculosis catalyzes sequential oxidn. of the cholesterol side-chain terminal Me group to the alc., aldehyde, and finally acid. Here, we demonstrate that CYP125 simultaneously catalyzes the formation of five other products, all of which result from deformylation of the sterol side chain. The aldehyde intermediate is shown to be the precursor of both the conventional acid metabolite and the five deformylation products. The acid arises by protonation of the ferric-peroxo anion species and formation of the ferryl-oxene species, also known as Compd. I, followed by hydrogen abstraction and oxygen transfer. The deformylation products arise by addn. of the same ferric-peroxo anion to the aldehyde intermediate to give a peroxyhemiacetal that leads to C-C bond cleavage. This bifurcation of the catalytic sequence has allowed us to examine the effect of electron donation by the proximal ligand on the properties of the ferric-peroxo anion. Replacement of the cysteine thiolate iron ligand by a selenocysteine results in UV-vis, EPR, and resonance Raman spectral changes indicative of an increased electron donation from the proximal selenolate ligand to the iron. Anal. of the product distribution in the reaction of the selenocysteine substituted enzyme reveals a gain in the formation of the acid (Compd. I pathway) at the expense of deformylation products. These observations are consistent with an increase in the pKa of the ferric-peroxo anion, which favors its protonation and, therefore, Compd. I formation.
- 271Ortmayer, M.; Fisher, K.; Basran, J.; Wolde-Michael, E. M.; Heyes, D. J.; Levy, C.; Lovelock, S. L.; Anderson, J. L. R.; Raven, E. L.; Hay, S. Rewiring the “ Push-Pull ” Catalytic Machinery of a Heme Enzyme Using an Expanded Genetic Code. ACS Catal. 2020, 10, 2735– 2746, DOI: 10.1021/acscatal.9b05129Google Scholar271Rewiring the "Push-Pull" Catalytic Machinery of a Heme Enzyme Using an Expanded Genetic CodeOrtmayer, Mary; Fisher, Karl; Basran, Jaswir; Wolde-Michael, Emmanuel M.; Heyes, Derren J.; Levy, Colin; Lovelock, Sarah L.; Anderson, J. L. Ross; Raven, Emma L.; Hay, Sam; Rigby, Stephen E. J.; Green, Anthony P.ACS Catalysis (2020), 10 (4), 2735-2746CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Nature employs a limited no. of genetically encoded axial ligands to control diverse heme enzyme activities. Deciphering the functional significance of these ligands requires a quant. understanding of how their electron-donating capabilities modulate the structures and reactivities of the iconic ferryl intermediates compds. I and II. However, probing these relationships exptl. has proven to be challenging as ligand substitutions accessible via conventional mutagenesis do not allow fine tuning of electron donation and typically abolish catalytic function. Here, we exploit engineered translation components to replace the histidine ligand of cytochrome c peroxidase (CcP) by a less electron-donating Nδ-Me histidine (Me-His) with little effect on the enzyme structure. The rate of formation (k1) and the reactivity (k2) of compd. I are unaffected by ligand substitution. In contrast, proton-coupled electron transfer to compd. II (k3) is 10-fold slower in CcP Me-His, providing a direct link between electron donation and compd. II reactivity, which can be explained by weaker electron donation from the Me-His ligand ("the push") affording an electron-deficient ferryl oxygen with reduced proton affinity ("the pull"). The deleterious effects of the Me-His ligand can be fully compensated by introducing a W51F mutation designed to increase "the pull" by removing a hydrogen bond to the ferryl oxygen. Analogous substitutions in ascorbate peroxidase lead to similar activity trends to those obsd. in CcP, suggesting that a common mechanistic strategy is employed by enzymes using distinct electron transfer pathways. Our study highlights how noncanonical active site substitutions can be used to directly probe and deconstruct highly evolved bioinorg. mechanisms.
- 272Xiao, H.; Peters, F. B.; Yang, P.-Y.; Reed, S.; Chittuluru, J. R.; Schultz, P. G. Genetic Incorporation of Histidine Derivatives Using an Engineered Pyrrolysyl-tRNA Synthetase. ACS Chem. Biol. 2014, 9 (5), 1092– 1096, DOI: 10.1021/cb500032cGoogle Scholar272Genetic Incorporation of Histidine Derivatives Using an Engineered Pyrrolysyl-tRNA SynthetaseXiao, Han; Peters, Francis B.; Yang, Peng-Yu; Reed, Sean; Chittuluru, Johnathan R.; Schultz, Peter G.ACS Chemical Biology (2014), 9 (5), 1092-1096CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)A polyspecific amber suppressor aminoacyl-tRNA synthetase/tRNA pair was evolved that genetically encodes a series of histidine analogs in both Escherichia coli and mammalian cells. In combination with tRNACUAPyl, a pyrrolysyl-tRNA synthetase (PylRS) mutant was able to site-specifically incorporate 3-methylhistidine, 3-pyridylalanine, 2-furylalanine, and 3-(2-thienyl)alanine into proteins in response to an amber codon. Substitution of His66 in the blue fluorescent protein (BFP) with these histidine analogs created mutant proteins with distinct spectral properties. This work further expands the structural and chem. diversity of unnatural amino acids (UAAs) that can be genetically encoded in prokaryotic and eukaryotic organisms and affords new probes of protein structure and function.
- 273Martin, C.; Zhang, Y. The Diverse Functions of Histone Lysine Methylation. Nat. Rev. Mol. Cell Biol. 2005, 6 (11), 838– 849, DOI: 10.1038/nrm1761Google Scholar273The diverse functions of histone lysine methylationMartin, Cyrus; Zhang, YiNature Reviews Molecular Cell Biology (2005), 6 (11), 838-849CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. Covalent modifications of histone tails play fundamental roles in chromatin structure and function. One such modification, lysine methylation, has important functions in many biol. processes that include heterochromatin formation, X-chromosome inactivation, and transcriptional regulation. Here, the authors summarize recent advances in the understanding of how lysine methylation functions in these diverse biol. processes, and raise questions that need to be addressed in the future.
- 274Niu, W.; Guo, J. Co-translational Installation of Posttranslational Modifications by Non-canonical Amino Acid Mutagenesis. ChemBioChem 2023, 24 (9), e202300039 DOI: 10.1002/cbic.202300039Google ScholarThere is no corresponding record for this reference.
- 275Rust, H. L.; Subramanian, V.; West, G. M.; Young, D. D.; Schultz, P. G.; Thompson, P. R. Using Unnatural Amino Acid Mutagenesis To Probe the Regulation of PRMT1. ACS Chem. Biol. 2014, 9 (3), 649– 655, DOI: 10.1021/cb400859zGoogle Scholar275Using Unnatural Amino Acid Mutagenesis To Probe the Regulation of PRMT1Rust, Heather L.; Subramanian, Venkataraman; West, Graham M.; Young, Douglas D.; Schultz, Peter G.; Thompson, Paul R.ACS Chemical Biology (2014), 9 (3), 649-655CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Protein arginine methyltransferase 1 (PRMT1)-dependent methylation contributes to the onset and progression of numerous diseases (e.g., cancer, heart disease, ALS); however, the regulatory mechanisms that control PRMT1 activity are relatively unexplored. We therefore set out to decipher how phosphorylation regulates PRMT1 activity. Curated mass spectrometry data identified Tyr291, a residue adjacent to the conserved THW loop, as being phosphorylated. Natural and unnatural amino acid mutagenesis, including the incorporation of p-carboxymethyl-L-phenylalanine (pCmF) as a phosphotyrosine mimic, were used to show that Tyr291 phosphorylation alters the substrate specificity of PRMT1. Addnl., p-benzoyl-L-phenylalanine (pBpF) was incorporated at the Tyr291 position, and crosslinking expts. with K562 cell exts. identified several proteins (e.g., hnRNP A1 and hnRNP H3) that bind specifically to this site. Moreover, we also demonstrate that Tyr291 phosphorylation impairs PRMT1's ability to bind and methylate both proteins. In total, these studies demonstrate that Tyr291 phosphorylation alters both PRMT1 substrate specificity and protein-protein interactions.
- 276Neumann, H.; Neumann-Staubitz, P.; Witte, A.; Summerer, D. Epigenetic Chromatin Modification by Amber Suppression Technology. Curr. Opin. Chem. Biol. 2018, 45, 1– 9, DOI: 10.1016/j.cbpa.2018.01.017Google Scholar276Epigenetic chromatin modification by amber suppression technologyNeumann, Heinz; Neumann-Staubitz, Petra; Witte, Anna; Summerer, DanielCurrent Opinion in Chemical Biology (2018), 45 (), 1-9CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)The genetic incorporation of unnatural amino acids (UAAs) into proteins by amber suppression technol. provides unique avenues to study protein structure, function and interactions both in vitro and in living cells and organisms. This approach has been particularly useful for studying mechanisms of epigenetic chromatin regulation, since these extensively involve dynamic changes in structure, complex formation and posttranslational modifications that are difficult to access by traditional approaches. Here, we review recent achievements in this field, emphasizing UAAs that help to unravel protein-protein interactions in cells by photo-crosslinking or that allow the biosynthesis of proteins with defined posttranslational modifications for studying their function and turnover in vitro and in cells.
- 277Wang, Z. A.; Cole, P. A. The Chemical Biology of Reversible Lysine Post-translational Modifications. Cell Chem. Biol. 2020, 27 (8), 953– 969, DOI: 10.1016/j.chembiol.2020.07.002Google Scholar277The Chemical Biology of Reversible Lysine Post-translational ModificationsWang, Zhipeng A.; Cole, Philip A.Cell Chemical Biology (2020), 27 (8), 953-969CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)A review. Lysine (Lys) residues in proteins undergo a wide range of reversible post-translational modifications (PTMs), which can regulate enzyme activities, chromatin structure, protein-protein interactions, protein stability, and cellular localization. Here we discuss the "writers," "erasers," and "readers" of some of the common protein Lys PTMs and summarize examples of their major biol. impacts. We also review chem. biol. approaches, from small-mol. probes to protein chem. technologies, that have helped to delineate Lys PTM functions and show promise for a diverse set of biomedical applications.
- 278Wang, T.; Zhou, Q.; Li, F.; Yu, Y.; Yin, X.; Wang, J. Genetic Incorporation of Nε-Formyllysine, a New Histone Post-translational Modification. ChemBioChem 2015, 16 (10), 1440– 1442, DOI: 10.1002/cbic.201500170Google ScholarThere is no corresponding record for this reference.
- 279Cao, L.; Liu, J.; Ghelichkhani, F.; Rozovsky, S.; Wang, L. Genetic Incorporation of ε-N-Benzoyllysine by Engineering Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase. ChemBioChem 2021, 22 (15), 2530– 2534, DOI: 10.1002/cbic.202100218Google ScholarThere is no corresponding record for this reference.
- 280Ren, C.; Wu, Q.; Xiao, R.; Ji, Y.; Yang, X.; Zhang, Z.; Qin, H.; Ma, J.-A.; Xuan, W. Expanding the Scope of Genetically Encoded Lysine Post-Translational Modifications with Lactylation, β-Hydroxybutyrylation and Lipoylation. ChemBioChem 2022, 23 (18), e202200302 DOI: 10.1002/cbic.202200302Google ScholarThere is no corresponding record for this reference.
- 281Nguyen, D. P.; Garcia Alai, M. M.; Kapadnis, P. B.; Neumann, H.; Chin, J. W. Genetically Encoding Nϵ-Methyl-l-lysine in Recombinant Histones. J. Am. Chem. Soc. 2009, 131 (40), 14194– 14195, DOI: 10.1021/ja906603sGoogle Scholar281Genetically encoding Nε-methyl-L-lysine in recombinant histonesNguyen, Duy P.; Garcia Alai, Maria M.; Kapadnis, Prashant B.; Neumann, Heinz; Chin, Jason W.Journal of the American Chemical Society (2009), 131 (40), 14194-14195CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Lysine methylation is an important post-translational modification of histone proteins that defines epigenetic status and controls heterochromatin formation, X-chromosome inactivation, genome imprinting, DNA repair, and transcriptional regulation. Despite considerable efforts by chem. biologists to synthesize modified histones for use in deciphering the mol. role of methylation in these phenomena, no general method exists to synthesize proteins bearing quant. site-specific methylation. Here we demonstrate a general method for the quant. installation of Nε-methyl-L-lysine at defined positions in recombinant histones and demonstrate the use of this method for investigating the methylation dependent binding of HP1 to full length histone H3 monomethylated on K9 (H3K9me1). This strategy will find wide application in defining the mol. mechanisms by which histone methylation orchestrates cellular phenomena.
- 282Wang, Z. A.; Liu, W. R. Proteins with Site-Specific Lysine Methylation. Chem. Eur. J. 2017, 23 (49), 11732– 11737, DOI: 10.1002/chem.201701655Google ScholarThere is no corresponding record for this reference.
- 283Wang, Y.-S.; Wu, B.; Wang, Z.; Huang, Y.; Wan, W.; Russell, W. K.; Pai, P.-J.; Moe, Y. N.; Russell, D. H.; Liu, W. R. A Genetically Encoded Photocaged Nε-Methyl-L-Lysine. Mol. BioSyst. 2010, 6 (9), 1557– 1560, DOI: 10.1039/c002155eGoogle ScholarThere is no corresponding record for this reference.
- 284Neumann, H.; Peak-Chew, S. Y.; Chin, J. W. Genetically Encoding Nε-Acetyllysine in Recombinant Proteins. Nat. Chem. Biol. 2008, 4 (4), 232– 234, DOI: 10.1038/nchembio.73Google Scholar284Genetically encoding Nε-acetyllysine in recombinant proteinsNeumann, Heinz; Peak-Chew, Sew Y.; Chin, Jason W.Nature Chemical Biology (2008), 4 (4), 232-234CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Nε-acetylation of lysine is a reversible post-translational modification with a regulatory role that rivals that of phosphorylation in eukaryotes. No general methods exist to synthesize proteins contg. Nε-acetyllysine at defined sites. The site-specific incorporation of Nε-acetyllysine in recombinant proteins produced in Escherichia coli was achieved via the evolution of an orthogonal Nε-acetyllysyl-tRNA synthetase/tRNACUA pair. This strategy should find wide applications in defining the cellular role of this modification.
- 285Gattner, M. J.; Vrabel, M.; Carell, T. Synthesis of ε-N-Propionyl-, ε-N-Butyryl-, and ε-N-Crotonyl-Lysine Containing Histone H3 Using the Pyrrolysine System. Chem. Commun. 2013, 49 (4), 379– 381, DOI: 10.1039/C2CC37836AGoogle ScholarThere is no corresponding record for this reference.
- 286Wilkins, B. J.; Hahn, L. E.; Heitmüller, S.; Frauendorf, H.; Valerius, O.; Braus, G. H.; Neumann, H. Genetically Encoding Lysine Modifications on Histone H4. ACS Chem. Biol. 2015, 10 (4), 939– 944, DOI: 10.1021/cb501011vGoogle Scholar286Genetically encoding lysine modifications on histone H4Wilkins, Bryan J.; Hahn, Liljan E.; Heitmueller, Svenja; Frauendorf, Holm; Valerius, Oliver; Braus, Gerhard H.; Neumann, HeinzACS Chemical Biology (2015), 10 (4), 939-944CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Post-translational modifications of proteins are important modulators of protein function. In order to identify the specific consequences of individual modifications, general methods are required for homogeneous prodn. of modified proteins. The direct installation of modified amino acids by genetic code expansion facilitates the prodn. of such proteins independent of the knowledge and availability of the enzymes naturally responsible for the modification. The prodn. of recombinant histone H4 with genetically encoded modifications has proven notoriously difficult in the past. Here, we present a general strategy to produce histone H4 with acetylation, propionylation, butyrylation, and crotonylation on lysine residues. We produce homogeneous histone H4 contg. up to four simultaneous acetylations to analyze the impact of the modifications on chromatin array compaction. Furthermore, we explore the ability of antibodies to discriminate between alternative lysine acylations by incorporating these modifications in recombinant histone H4.
- 287Xiao, H.; Xuan, W.; Shao, S.; Liu, T.; Schultz, P. G. Genetic Incorporation of ε-N-2-Hydroxyisobutyryl-Lysine into Recombinant Histones. ACS Chem. Biol. 2015, 10 (7), 1599– 1603, DOI: 10.1021/cb501055hGoogle ScholarThere is no corresponding record for this reference.
- 288Kim, C. H.; Kang, M.; Kim, H. J.; Chatterjee, A.; Schultz, P. G. Site-Specific Incorporation of ε-N-Crotonyllysine into Histones. Angew. Chem. Int. Ed. 2012, 51 (29), 7246– 7249, DOI: 10.1002/anie.201203349Google ScholarThere is no corresponding record for this reference.
- 289Tian, H.; Yang, J.; Guo, A.-D.; Ran, Y.; Yang, Y.-Z.; Yang, B.; Huang, R.; Liu, H.; Chen, X.-H. Genetically Encoded Benzoyllysines Serve as Versatile Probes for Interrogating Histone Benzoylation and Interactions in Living Cells. ACS Chem. Biol. 2021, 16 (11), 2560– 2569, DOI: 10.1021/acschembio.1c00614Google ScholarThere is no corresponding record for this reference.
- 290Fatema, N.; Fan, C. Studying Lysine Acetylation of Citric Acid Cycle Enzymes by Genetic Code Expansion. Mol. Microbiol. 2023, 119 (5), 551– 559, DOI: 10.1111/mmi.15052Google ScholarThere is no corresponding record for this reference.
- 291Araujo, J.; Ottinger, S.; Venkat, S.; Gan, Q.; Fan, C. Studying Acetylation of Aconitase Isozymes by Genetic Code Expansion. Front. Chem. 2022, 10, 1, DOI: 10.3389/fchem.2022.862483Google ScholarThere is no corresponding record for this reference.
- 292Venkat, S.; Chen, H.; Stahman, A.; Hudson, D.; McGuire, P.; Gan, Q.; Fan, C. Characterizing Lysine Acetylation of Isocitrate Dehydrogenase in Escherichia coli. J. Mol. Biol. 2018, 430 (13), 1901– 1911, DOI: 10.1016/j.jmb.2018.04.031Google ScholarThere is no corresponding record for this reference.
- 293Venkat, S.; Gregory, C.; Sturges, J.; Gan, Q.; Fan, C. Studying the Lysine Acetylation of Malate Dehydrogenase. J. Mol. Biol. 2017, 429 (9), 1396– 1405, DOI: 10.1016/j.jmb.2017.03.027Google ScholarThere is no corresponding record for this reference.
- 294Venkat, S.; Chen, H.; McGuire, P.; Stahman, A.; Gan, Q.; Fan, C. Characterizing Lysine Acetylation of Escherichia coli Type II Citrate Synthase. FEBS J. 2019, 286 (14), 2799– 2808, DOI: 10.1111/febs.14845Google ScholarThere is no corresponding record for this reference.
- 295Wright, D. E.; Altaany, Z.; Bi, Y.; Alperstein, Z.; O’Donoghue, P. Acetylation Regulates Thioredoxin Reductase Oligomerization and Activity. Antioxid. Redox Signal. 2018, 29 (4), 377– 388, DOI: 10.1089/ars.2017.7082Google ScholarThere is no corresponding record for this reference.
- 296Rogerson, D. T.; Sachdeva, A.; Wang, K.; Haq, T.; Kazlauskaite, A.; Hancock, S. M.; Huguenin-Dezot, N.; Muqit, M. M. K.; Fry, A. M.; Bayliss, R. Efficient Genetic Encoding of Phosphoserine and its Nonhydrolyzable Analog. Nat. Chem. Biol. 2015, 11 (7), 496– 503, DOI: 10.1038/nchembio.1823Google Scholar296Efficient genetic encoding of phosphoserine and its nonhydrolyzable analogRogerson, Daniel T.; Sachdeva, Amit; Wang, Kaihang; Haq, Tamanna; Kazlauskaite, Agne; Hancock, Susan M.; Huguenin-Dezot, Nicolas; Muqit, Miratul M. K.; Fry, Andrew M.; Bayliss, Richard; Chin, Jason W.Nature Chemical Biology (2015), 11 (7), 496-503CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Serine phosphorylation is a key post-translational modification that regulates diverse biol. processes. Powerful anal. methods have identified thousands of phosphorylation sites, but many of their functions remain to be deciphered. A key to understanding the function of protein phosphorylation is access to phosphorylated proteins, but this is often challenging or impossible. Here we evolve an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair that directs the efficient incorporation of phosphoserine (pSer (1)) into recombinant proteins in Escherichia coli. Moreover, combining the orthogonal pair with a metabolically engineered E. coli enables the site-specific incorporation of a nonhydrolyzable analog of pSer. Our approach enables quant. decoding of the amber stop codon as pSer, and we purify, with yields of several milligrams per L of culture, proteins bearing biol. relevant phosphorylations that were previously challenging or impossible to access-including phosphorylated ubiquitin and the kinase Nek7, which is synthetically activated by a genetically encoded phosphorylation in its activation loop.
- 297Venkat, S.; Sturges, J.; Stahman, A.; Gregory, C.; Gan, Q.; Fan, C. Genetically Incorporating Two Distinct Post-translational Modifications into One Protein Simultaneously. ACS Synth. Biol. 2018, 7 (2), 689– 695, DOI: 10.1021/acssynbio.7b00408Google Scholar297Genetically Incorporating Two Distinct Post-translational Modifications into One Protein SimultaneouslyVenkat, Sumana; Sturges, Jourdan; Stahman, Alleigh; Gregory, Caroline; Gan, Qinglei; Fan, ChenguangACS Synthetic Biology (2018), 7 (2), 689-695CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Post-translational modifications (PTMs) play important roles in regulating a variety of biol. processes. To facilitate PTM studies, the genetic code expansion strategy has been used to cotranslationally incorporate individual PTMs such as acetylation and phosphorylation into proteins at specific sites. However, recent studies demonstrated that PTMs actually work together to regulate protein functions and structures. Thus, simultaneous incorporation of multiple distinct PTMs into one protein is highly desirable. The authors used the genetic incorporation systems of phosphoserine and acetyllysine to install both phosphorylation and acetylation into target proteins simultaneously in Escherichia coli. And this system was used to study the effect of coexisting acetylation and phosphorylation on malate dehydrogenase, demonstrating a practical application of this system in biochem. studies. Furthermore, the authors tested the mutual orthogonality of three widely used genetic incorporation systems, indicating the possibility of incorporating three distinct PTMs into one protein simultaneously.
- 298Zang, J.; Chen, Y.; Liu, C.; Lin, S. Probing the Role of Aurora Kinase A Threonylation with Site-Specific Lysine Threonylation. ACS Chem. Biol. 2023, 18 (4), 674– 678, DOI: 10.1021/acschembio.1c00682Google ScholarThere is no corresponding record for this reference.
- 299Wan, N.; Wang, N.; Yu, S.; Zhang, H.; Tang, S.; Wang, D.; Lu, W.; Li, H.; Delafield, D. G.; Kong, Y. Cyclic Immonium Ion of Lactyllysine Reveals Widespread Lactylation in the Human Proteome. Nat. Methods 2022, 19 (7), 854– 864, DOI: 10.1038/s41592-022-01523-1Google ScholarThere is no corresponding record for this reference.
- 300Whittaker, J. W. Free Radical Catalysis by Galactose Oxidase. Chem. Rev. 2003, 103 (6), 2347– 2364, DOI: 10.1021/cr020425zGoogle Scholar300Free Radical Catalysis by Galactose OxidaseWhittaker, James W.Chemical Reviews (Washington, DC, United States) (2003), 103 (6), 2347-2363CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. The unusual two-electron reactivity of the mononuclear copper active site in galactose oxidase has been explained in terms of the direct participation of the protein in the redox chem. of the active site, forming a stable free radical-copper complex in the active enzyme. The copper-free apoprotein is readily oxidized under mild conditions, forming a stable free radical, with distinctive optical absorption and ESR (EPR) spectra. A single free radical species is obsd., implying a unique reactive site in the protein. At X-band (9 GHz) the EPR spectrum exhibits an av. g-value of 2.005 and a complex pattern of fine structure splittings. Isotopic labeling demonstrates that the free radical site in the apoprotein is derived from a tyrosine residue, and it allows the major splittings to be assigned to hyperfine interactions between the unpaired electron and the β hydrogens in the side chain of a perturbed tyrosine residue. ENDOR spectroscopy (also at X-band) yields more refined ests. of the hyperfine coupling consts. and provides evidence for hydrogen bonding to the phenoxy oxygen. This review will focus on research aimed at understanding the nature of the free radical site, the reactivity of the unique metalloradical complex, and the mechanism of free radical catalysis by galactose oxidase.
- 301Polyakov, K. M.; Boyko, K. M.; Tikhonova, T. V.; Slutsky, A.; Antipov, A. N.; Zvyagilskaya, R. A.; Popov, A. N.; Bourenkov, G. P.; Lamzin, V. S.; Popov, V. O. High-Resolution Structural Analysis of a Novel Octaheme Cytochrome c Nitrite Reductase from the Haloalkaliphilic Bacterium Thioalkalivibrio nitratireducens. J. Mol. Biol. 2009, 389 (5), 846– 862, DOI: 10.1016/j.jmb.2009.04.037Google ScholarThere is no corresponding record for this reference.
- 302Ye, S.; Wu, X. a.; Wei, L.; Tang, D.; Sun, P.; Bartlam, M.; Rao, Z. An Insight into the Mechanism of Human Cysteine Dioxygenase: key roles of the thioether-bonded tyrosine-cysteine cofactor. J. Biol. Chem. 2007, 282 (5), 3391– 3402, DOI: 10.1074/jbc.M609337200Google Scholar302An Insight into the Mechanism of Human Cysteine Dioxygenase. Key Roles of the Thioether-Bonded Tyrosine-Cysteine CofactorYe, Sheng; Wu, Xiao'ai; Wei, Lei; Tang, Danming; Sun, Ping; Bartlam, Mark; Rao, ZiheJournal of Biological Chemistry (2007), 282 (5), 3391-3402CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Cysteine dioxygenase (CDO) is a non-heme mononuclear iron metalloenzyme that catalyzes the oxidn. of cysteine to cysteine sulfinic acid with addn. of mol. dioxygen. This irreversible oxidative catabolism of cysteine initiates several important metabolic pathways related to diverse sulfurate compds. Cysteine dioxygenase is therefore very important for maintaining the proper hepatic concn. of intracellular free cysteine. Mechanisms for mouse and rat cysteine dioxygenases have recently been reported based on their crystal structures in the absence of substrates, although there is still a lack of direct evidence. Here we report the first crystal structure of human cysteine dioxygenase in complex with its substrate L-cysteine to 2.7Å, together with enzymic activity and metal content assays of several single point mutants. Our results provide an insight into a new mechanism of cysteine thiol dioxygenation catalyzed by cysteine dioxygenase, which is tightly assocd. with a thioether-bonded tyrosine-cysteine cofactor involving Tyr-157 and Cys-93. This cross-linked protein-derived cofactor plays several key roles different from those in galactose oxidase. This report provides a new potential target for therapy of diseases related to human cysteine dioxygenase, including neurodegenerative and autoimmune diseases.
- 303Zhou, Q.; Hu, M.; Zhang, W.; Jiang, L.; Perrett, S.; Zhou, J.; Wang, J. Probing the Function of the Tyr-Cys Cross-Link in Metalloenzymes by the Genetic Incorporation of 3-Methylthiotyrosine. Angew. Chem. Int. Ed. 2013, 52 (4), 1203– 1207, DOI: 10.1002/anie.201207229Google ScholarThere is no corresponding record for this reference.
- 304Dominy, J. E.; Hwang, J.; Guo, S.; Hirschberger, L. L.; Zhang, S.; Stipanuk, M. H. Synthesis of Amino Acid Cofactor in Cysteine Dioxygenase Is Regulated by Substrate and Represents a Novel Post-translational Regulation of Activity. J. Biol. Chem. 2008, 283 (18), 12188– 12201, DOI: 10.1074/jbc.M800044200Google Scholar304Synthesis of Amino Acid Cofactor in Cysteine Dioxygenase Is Regulated by Substrate and Represents a Novel Post-translational Regulation of ActivityDominy, John E., Jr.; Hwang, Jesse; Guo, Stephanie; Hirschberger, Lawrence L.; Zhang, Sheng; Stipanuk, Martha H.Journal of Biological Chemistry (2008), 283 (18), 12188-12201CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Cysteine dioxygenase (CDO) catalyzes the conversion of cysteine to cysteinesulfinic acid and is important in the regulation of intracellular cysteine levels in mammals and in the provision of oxidized cysteine metabolites such as sulfate and taurine. Several crystal structure studies of mammalian CDO have shown that there is a cross-linked cofactor present in the active site of the enzyme. The cofactor consists of a thioether bond between the γ-sulfur of residue cysteine 93 and the arom. side chain of residue tyrosine 157. The exact requirements for cofactor synthesis and the contribution of the cofactor to the catalytic activity of the enzyme have yet to be fully described. In this study, therefore, we explored the factors necessary for cofactor biogenesis in vitro and in vivo and examd. what effect cofactor formation had on activity in vitro. Like other cross-linked cofactor-contg. enzymes, formation of the Cys-Tyr cofactor in CDO required a transition metal cofactor (Fe2+) and O2. Unlike other enzymes, however, biogenesis was also strictly dependent upon the presence of substrate. Cofactor formation was also appreciably slower than the rates reported for other enzymes and, indeed, took hundreds of catalytic turnover cycles to occur. In the absence of the Cys-Tyr cofactor, CDO possessed appreciable catalytic activity, suggesting that the cofactor was not essential for catalysis. Nevertheless, at physiol. relevant cysteine concns., cofactor formation increased CDO catalytic efficiency by ∼10-fold. Overall, the regulation of Cys-Tyr cofactor formation in CDO by ambient cysteine levels represents an unusual form of substrate-mediated feed-forward activation of enzyme activity with important physiol. consequences.
- 305Li, J.; Griffith, W. P.; Davis, I.; Shin, I.; Wang, J.; Li, F.; Wang, Y.; Wherritt, D. J.; Liu, A. Cleavage of a Carbon-Fluorine Bond by an Engineered Cysteine Dioxygenase. Nat. Chem. Biol. 2018, 14 (9), 853– 860, DOI: 10.1038/s41589-018-0085-5Google ScholarThere is no corresponding record for this reference.
- 306Li, J.; Koto, T.; Davis, I.; Liu, A. Probing the Cys-Tyr Cofactor Biogenesis in Cysteine Dioxygenase by the Genetic Incorporation of Fluorotyrosine. Biochemistry 2019, 58 (17), 2218– 2227, DOI: 10.1021/acs.biochem.9b00006Google Scholar306Probing the Cys-Tyr Cofactor Biogenesis in Cysteine Dioxygenase by the Genetic Incorporation of FluorotyrosineLi, Jiasong; Koto, Teruaki; Davis, Ian; Liu, AiminBiochemistry (2019), 58 (17), 2218-2227CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Cysteine dioxygenase (CDO) is a nonheme iron enzyme that adds two oxygen atoms from dioxygen to the sulfur atom of L-cysteine. Adjacent to the iron site of mammalian CDO, there is a post-translationally generated Cys-Tyr cofactor, whose presence substantially enhances the oxygenase activity. The formation of the Cys-Tyr cofactor in CDO is an autocatalytic process, and it is challenging to study by traditional techniques because the crosslinking reaction is a side, uncoupled, single-turnover oxidn. buried among multiple turnovers of L-cysteine oxygenation. Here, we take advantage of our recent success in obtaining a purely uncross-linked human CDO due to site-specific incorporation of 3,5-difluoro-L-tyrosine (F2-Tyr) at the crosslinking site through the genetic code expansion strategy. Using EPR spectroscopy, we show that nitric oxide (•NO), an oxygen surrogate, similarly binds to uncross-linked F2-Tyr157 CDO as in wild-type human CDO. We detd. X-ray crystal structures of uncross-linked F2-Tyr157 CDO and mature wild-type CDO in complex with both L-cysteine and •NO. These structural data reveal that the active site cysteine (Cys93 in the human enzyme), rather than the generally expected tyrosine (i.e., Tyr157), is well-aligned to be oxidized should the normal oxidn. reaction uncouple. This structure-based understanding is further supported by a computational study with models built on the uncross-linked ternary complex structure. Together, these results strongly suggest that the first target to oxidize during the iron-assisted Cys-Tyr cofactor biogenesis is Cys93. Based on these data, a plausible reaction mechanism implementing a cysteine radical involved in the crosslink formation is proposed.
- 307Chen, L.; Naowarojna, N.; Song, H.; Wang, S.; Wang, J.; Deng, Z.; Zhao, C.; Liu, P. Use of a Tyrosine Analogue To Modulate the Two Activities of a Nonheme Iron Enzyme OvoA in Ovothiol Biosynthesis, Cysteine Oxidation versus Oxidative C-S Bond Formation. J. Am. Chem. Soc. 2018, 140 (13), 4604– 4612, DOI: 10.1021/jacs.7b13628Google Scholar307Use of a Tyrosine Analogue To Modulate the Two Activities of a Nonheme Iron Enzyme OvoA in Ovothiol Biosynthesis, Cysteine Oxidation versus Oxidative C-S Bond FormationChen, Li; Naowarojna, Nathchar; Song, Heng; Wang, Shu; Wang, Jiangyun; Deng, Zixin; Zhao, Changming; Liu, PinghuaJournal of the American Chemical Society (2018), 140 (13), 4604-4612CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Ovothiol is a histidine thiol deriv. The biosynthesis of ovothiol involves an extremely efficient trans-sulfuration strategy. The nonheme iron enzyme OvoA catalyzed oxidative coupling between cysteine and histidine is one of the key steps. Besides catalyzing the oxidative coupling between cysteine and histidine, OvoA also catalyzes the oxidn. of cysteine to cysteine sulfinic acid (cysteine dioxygenase activity). Thus far, very little mechanistic information is available for OvoA-catalysis. In this report, we measured the kinetic isotope effect (KIE) in OvoA-catalysis using the isotopically sensitive branching method. In addn., by replacing an active site tyrosine (Tyr417) with 2-amino-3-(4-hydroxy-3-(methylthio)phenyl)propanoic acid (MtTyr) through the amber suppressor mediated unnatural amino acid incorporation method, the two OvoA activities (oxidative coupling between cysteine and histidine, and cysteine dioxygenase activity) can be modulated. These results suggest that the two OvoA activities branch out from a common intermediate and that the active site tyrosine residue plays some key roles in controlling the partitioning between these two pathways.
- 308Trumpower, B. L.; Gennis, R. B. Energy Transduction By Cytochrome Complexes In Mitochondrial And Bacterial Respiration: The Enzymology of Coupling Electron Transfer Reactions to Transmembrane Proton Translocation. Annu. Rev. Biochem. 1994, 63 (1), 675– 716, DOI: 10.1146/annurev.bi.63.070194.003331Google ScholarThere is no corresponding record for this reference.
- 309Kim, E.; Chufán, E. E.; Kamaraj, K.; Karlin, K. D. Synthetic Models for Heme-Copper Oxidases. Chem. Rev. 2004, 104 (2), 1077– 1134, DOI: 10.1021/cr0206162Google Scholar309Synthetic Models for Heme-Copper OxidasesKim, Eunsuk; Chufan, Eduardo E.; Kamaraj, Kaliappan; Karlin, Kenneth D.Chemical Reviews (Washington, DC, United States) (2004), 104 (2), 1077-1133CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. The newest directions in heme-copper oxidase modeling have come from examn. of compds. with reduced heme and copper ion complex 1:1 mixts. or heterobinuclear constructs. Thus, generation and characterization of carbon monoxide adducts of heme and/or copper provide preliminary insights into the binding of this O2 surrogate and allow probing of the heme-copper environment. However, it is dioxygen reactivity that has really led to exciting developments, including biomimetic functional modeling studies using electrochem. approaches and O2 reactivity studies leading to discrete superoxoheme (with copper present) and heme-peroxocopper assemblies. The latter may be directly relevant to an enzyme transient intermediate or may be a precursor to such (i.e., by protonation giving a heme hydroperoxo FeIII-OOH···CuII moiety). It was demonstrated that peroxospectroscopic signatures (and perhaps structures) can be influenced by binucleating ligand superstructure and copper-ligand denticity.
- 310Miner, K. D.; Mukherjee, A.; Gao, Y.-G.; Null, E. L.; Petrik, I. D.; Zhao, X.; Yeung, N.; Robinson, H.; Lu, Y. A Designed Functional Metalloenzyme that Reduces O2 to H2O with Over One Thousand Turnovers. Angew. Chem. Int. Ed. 2012, 51 (23), 5589– 5592, DOI: 10.1002/anie.201201981Google ScholarThere is no corresponding record for this reference.
- 311Liu, X.; Yu, Y.; Hu, C.; Zhang, W.; Lu, Y.; Wang, J. Significant Increase of Oxidase Activity through the Genetic Incorporation of a Tyrosine-Histidine Cross-Link in a Myoglobin Model of Heme-Copper Oxidase. Angew. Chem. Int. Ed. 2012, 51 (18), 4312– 4316, DOI: 10.1002/anie.201108756Google Scholar311Significant Increase of Oxidase Activity through the Genetic Incorporation of a Tyrosine-Histidine Cross-Link in a Myoglobin Model of Heme-Copper OxidaseLiu, Xiaohong; Yu, Yang; Hu, Cheng; Zhang, Wei; Lu, Yi; Wang, JiangyunAngewandte Chemie, International Edition (2012), 51 (18), 4312-4316, S4312/1-S4312/6CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)By directly incorporating the unnatural amino acid imiTyr (1) into myoglobins in E. coli in response to the amber codon TAG, we have successfully designed a functional heme copper oxidase (HCO) model imiTyrCuBMb that catalyzes selective and efficient oxygen redn. to water. The HCO model imiTyrCuBMb bearing the Tyr-His cross-link is eightfold more selective with threefold more turnovers than F33YCuBMb, which does not contain the cross-link but harbors His and Tyr residues at the same positions in the same protein. Since the synthesis of imiTyr contains only two steps with 50% overall yield, and mutant proteins bearing imiTyr (1) at any site can be easily obtained and purified in milligram quantities through site-directed mutagenesis and recombinant protein expression, further systematic investigation of the function of the Tyr-His cross-link is now possible. While imiTyrCuBMb exhibits lower enzymic activity (2 02/min) in comparison to native heme-copper oxidase (ca. 300 02/s), it is possible to rapidly improve our HCO model to achieve higher activity through directed evolution and incorporation of unnatural amino acids. Our designed enzyme harbors the unnatural amino acid imiTyr, which is highly analogous to the post-translationally modified tyrosine-histidine ligand found in the CuB site of HCO; this designed enzyme serves as an ideal model for a more detailed understanding of HCOs and allows for potential applications in synthetic biol. and alternative energy.
- 312Yu, Y.; Lv, X.; Li, J.; Zhou, Q.; Cui, C.; Hosseinzadeh, P.; Mukherjee, A.; Nilges, M. J.; Wang, J.; Lu, Y. Defining the Role of Tyrosine and Rational Tuning of Oxidase Activity by Genetic Incorporation of Unnatural Tyrosine Analogs. J. Am. Chem. Soc. 2015, 137 (14), 4594– 4597, DOI: 10.1021/ja5109936Google Scholar312Defining the Role of Tyrosine and Rational Tuning of Oxidase Activity by Genetic Incorporation of Unnatural Tyrosine AnalogsYu, Yang; Lv, Xiaoxuan; Li, Jiasong; Zhou, Qing; Cui, Chang; Hosseinzadeh, Parisa; Mukherjee, Arnab; Nilges, Mark J.; Wang, Jiangyun; Lu, YiJournal of the American Chemical Society (2015), 137 (14), 4594-4597CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)While a conserved tyrosine (Tyr) is found in oxidases, the roles of phenol ring pKa and redn. potential in O2 redn. have not been defined despite many years of research on numerous oxidases and their models. These issues represent major challenges in our understanding of O2 redn. mechanism in bioenergetics. Through genetic incorporation of unnatural amino acid analogs of Tyr, with progressively decreasing pKa of the phenol ring and increasing redn. potential, in the active site of a functional model of oxidase in myoglobin, a linear dependence of both the O2 redn. activity and the fraction of H2O formation with the pKa of the phenol ring has been established. By using these unnatural amino acids as spectroscopic probe, we have provided conclusive evidence for the location of a Tyr radical generated during reaction with H2O2, by the distinctive hyperfine splitting patterns of the halogenated tyrosines and one of its deuterated derivs. incorporated at the 33 position of the protein. These results demonstrate for the first time that enhancing the proton donation ability of the Tyr enhances the oxidase activity, allowing the Tyr analogs to augment enzymic activity beyond that of natural Tyr. 3-Chlorotyrosine (ClTyr), 3,5-difluorotyrosine (F2Tyr), and 2,3,5-trifluorotyrosine.
- 313Yu, Y.; Zhou, Q.; Wang, L.; Liu, X.; Zhang, W.; Hu, M.; Dong, J.; Li, J.; Lv, X.; Ouyang, H. Significant Improvement of Oxidase Activity Through the Genetic Incorporation of a Redox-Active Unnatural Amino Acid. Chem. Sci. 2015, 6 (7), 3881– 3885, DOI: 10.1039/C5SC01126DGoogle Scholar313Significant improvement of oxidase activity through the genetic incorporation of a redox-active unnatural amino acidYu, Yang; Zhou, Qing; Wang, Li; Liu, Xiaohong; Zhang, Wei; Hu, Meirong; Dong, Jianshu; Li, Jiasong; Lv, Xiaoxuan; Ouyang, Hanlin; Li, Han; Gao, Feng; Gong, Weimin; Lu, Yi; Wang, JiangyunChemical Science (2015), 6 (7), 3881-3885CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)While Nature employs various covalent and noncovalent strategies to modulate tyrosine redox potential and pKa in order to optimize enzyme activities, such approaches have not been systematically applied for the design of functional metalloproteins. Here, through the genetic incorporation of 3-methoxytyrosine (I) into myoglobin, the authors replicated important features of cytochrome c oxidase (CcO) in this small sol. protein, which exhibited selective O2 redn. activity while generating a small amt. of reactive O species (ROS). These results demonstrated that the electron-donating ability of a Tyr residue in the active site is important for CcO function. Moreover, the authors elucidated the structural basis for the genetic incorporation of I into proteins by solving the x-ray structure of I-specific aminoacyl-tRNA synthetase complexed with I.
- 314Rigoldi, F.; Donini, S.; Redaelli, A.; Parisini, E.; Gautieri, A. Review: Engineering of Thermostable Enzymes for Industrial Applications. APL Bioengineering 2018, 2 (1), 011501, DOI: 10.1063/1.4997367Google ScholarThere is no corresponding record for this reference.
- 315Sheldon, R. A.; Basso, A.; Brady, D. New Frontiers in Enzyme Immobilisation: Robust Biocatalysts for a Circular Bio-Based Economy. Chem. Soc. Rev. 2021, 50 (10), 5850– 5862, DOI: 10.1039/D1CS00015BGoogle Scholar315New frontiers in enzyme immobilization: robust biocatalysts for a circular bio-based economySheldon, Roger A.; Basso, Alessandra; Brady, DeanChemical Society Reviews (2021), 50 (10), 5850-5862CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. This tutorial review focuses on recent advances in technologies for enzyme immobilization, enabling their cost-effective use in the bio-based economy and continuous processing in general. The application of enzymes, particularly in aq. media, is generally on a single use, throw-away basis which is neither cost-effective nor compatible with a circular economy concept. This shortcoming can be overcome by immobilizing the enzyme as an insol. recyclable solid, that is as a heterogeneous catalyst.
- 316Baker, P. J.; Montclare, J. K. Enhanced Refoldability and Thermoactivity of Fluorinated Phosphotriesterase. ChemBioChem 2011, 12 (12), 1845– 1848, DOI: 10.1002/cbic.201100221Google ScholarThere is no corresponding record for this reference.
- 317Deepankumar, K.; Shon, M.; Nadarajan, S. P.; Shin, G.; Mathew, S.; Ayyadurai, N.; Kim, B.-G.; Choi, S.-H.; Lee, S.-H.; Yun, H. Enhancing Thermostability and Organic Solvent Tolerance of ω-Transaminase through Global Incorporation of Fluorotyrosine. Adv. Synth. Catal. 2014, 356 (5), 993– 998, DOI: 10.1002/adsc.201300706Google ScholarThere is no corresponding record for this reference.
- 318Ohtake, K.; Mukai, T.; Iraha, F.; Takahashi, M.; Haruna, K.-i.; Date, M.; Yokoyama, K.; Sakamoto, K. Engineering an Automaturing Transglutaminase with Enhanced Thermostability by Genetic Code Expansion with Two Codon Reassignments. ACS Synth. Biol. 2018, 7 (9), 2170– 2176, DOI: 10.1021/acssynbio.8b00157Google Scholar318Engineering an Automaturing Transglutaminase with Enhanced Thermostability by Genetic Code Expansion with Two Codon ReassignmentsOhtake, Kazumasa; Mukai, Takahito; Iraha, Fumie; Takahashi, Mihoko; Haruna, Ken-ichi; Date, Masayo; Yokoyama, Keiichi; Sakamoto, KensakuACS Synthetic Biology (2018), 7 (9), 2170-2176CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)In the present study, we simultaneously incorporated 2 types of synthetic components into microbial transglutaminase (MTG) from Streptoverticillium mobaraense, to enhance the utility of this industrial enzyme. The 1st amino acid, 3-chloro-L-tyrosine, was incorporated into MTG in response to in-frame UAG codons, to substitute for the 15 Tyr residues sep. Two substitutions at positions 20 and 62 were found to each increase the thermostability of the enzyme, while 7 substitutions (at positions 24, 34, 75, 146, 171, 217, and 310) exhibited neutral effects. Then, these 2 stabilizing chlorinations were combined with one of the neutral ones, and the most stabilized variant was found to contain 3-chlorotyrosines at positions 20, 62, and 171, exhibiting a 5.1-fold longer half-life than the wild-type enzyme at 60°. Next, this MTG variant was further modified by incorporating the α-hydroxy acid analog of Nε-allyloxycarbonyl-L-lysine (AlocKOH), specified by the AGG codon, at the end of the N-terminal inhibitory peptide. We used an Escherichia coli strain previously engineered to have a synthetic genetic code with 2 codon reassignments, for synthesizing MTG variants contg. both 3-chlorotyrosine and AlocKOH. The ester bond, thus incorporated into the main chain, efficiently self-cleaved under alk. conditions (pH 11.0), achieving the autonomous maturation of the thermostabilized MTG. The results suggested that synthetic genetic codes with multiple codon reassignments would be useful for developing the novel designs of enzymes.
- 319Politzer, P.; Murray, J. S. Halogen Bonding: An Interim Discussion. ChemPhysChem 2013, 14 (2), 278– 294, DOI: 10.1002/cphc.201200799Google Scholar319Halogen Bonding: An Interim DiscussionPolitzer, Peter; Murray, Jane S.ChemPhysChem (2013), 14 (2), 278-294CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Halogen bonding is a noncovalent interaction that is receiving rapidly increasing attention because of its significance in biol. systems and its importance in the design of new materials in a variety of areas, for example, electronics, nonlinear optical activity, and pharmaceuticals. The interactions can be understood in terms of electrostatics/polarization and dispersion; they involve a region of pos. electrostatic potential on a covalently bonded halogen and a neg. site, such as the lone pair of a Lewis base. The pos. potential, labeled a σ hole, is on the extension of the covalent bond to the halogen, which accounts for the characteristic near-linearity of halogen bonding. In many instances, the lateral sides of the halogen have neg. electrostatic potentials, allowing it to also interact favorably with pos. sites. In this discussion, after looking at some of the exptl. observations of halogen bonding, we address the origins of σ holes, the factors that govern the magnitudes of their electrostatic potentials, and the properties of the resulting complexes with neg. sites. The relationship of halogen and hydrogen bonding is examd. We also point out that σ-hole interactions are not limited to halogens, but can also involve covalently bonded atoms of Groups IV-VI. Examples of applications in biol./medicinal chem. and in crystal engineering are mentioned, taking note that halogen bonding can be "tuned" to fit various requirements, i.e., strength of interaction, steric factors, and so forth.
- 320Scholfield, M. R.; Ford, M. C.; Carlsson, A.-C. C.; Butta, H.; Mehl, R. A.; Ho, P. S. Structure-Energy Relationships of Halogen Bonds in Proteins. Biochemistry 2017, 56 (22), 2794– 2802, DOI: 10.1021/acs.biochem.7b00022Google Scholar320Structure-Energy Relationships of Halogen Bonds in ProteinsScholfield, Matthew R.; Ford, Melissa Coates; Carlsson, Anna-Carin C.; Butta, Hawera; Mehl, Ryan A.; Ho, P. ShingBiochemistry (2017), 56 (22), 2794-2802CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The structures and stabilities of proteins are defined by a series of weak non-covalent electrostatic, van der Waals, and H-bond (HB) interactions. Here, we designed and engineered halogen bonds (XBs) site-specifically in order to study their structure-energy relations in a model protein, phage T4 lysozyme. The evidence for XBs is the displacement of the arom. side-chain toward an oxygen acceptor, at distances that are at or less than the sums of their resp. van der Waals radii, when the hydroxyl substituent of the wild-type Tyr residue is replaced by iodine. In addn., thermal melting studies showed that the iodine XB rescued the stabilization energy from an otherwise destabilizing substitution (at an equiv. non-interacting site), indicating that the interaction is also present in soln. Quantum chem. calcns. showed that the XB complements an HB at this site and that solvent structure must also be considered in trying to design mol. interactions such as XBs into biol. systems. A Br substitution also showed displacement of the side-chain, but the distances and geometries did not indicate formation of an XB. Thus, we have dissected the contributions from various noncovalent interactions of halogens introduced into proteins, to drive the application of XBs, particularly in biomol. design.
- 321Acevedo-Rocha, C. G.; Hoesl, M. G.; Nehring, S.; Royter, M.; Wolschner, C.; Wiltschi, B.; Antranikian, G.; Budisa, N. Non-Canonical Amino Acids as a Useful Synthetic Biological Tool for Lipase-Catalysed Reactions in Hostile Environments. Catal. Sci. Technol. 2013, 3 (5), 1198– 1201, DOI: 10.1039/c3cy20712aGoogle ScholarThere is no corresponding record for this reference.
- 322Mendel, D.; Ellman, J. A.; Chang, Z.; Veenstra, D. L.; Kollman, P. A.; Schultz, P. G. Probing Protein Stability with Unnatural Amino Acids. Science 1992, 256 (5065), 1798– 1802, DOI: 10.1126/science.1615324Google Scholar322Probing protein stability with unnatural amino acidsMendel, David; Ellman, Jonathan A.; Chang, Zhiyuh; Veenstra, David L.; Kollman, Peter A.; Schultz, Peter G.Science (Washington, DC, United States) (1992), 256 (5065), 1798-802CODEN: SCIEAS; ISSN:0036-8075.Unnatural amino acid mutagenesis, in combination with mol. modeling and simulation techniques, was used to probe the effect of side chain structure on protein stability. Specific replacements at position 133 in T4 lysozyme included (i) leucine (wt), norvaline, ethyl-glycine, and alanine to measure the cost of stepwise removal of Me groups from the hydrophobic core, (ii) norvaline and O-Me serine to evaluate the effects of side chain solvation, and (iii) leucine, S,S-2-amino-4-methylhexanoic acid, and S-2-amino-3-cyclopentylpropanoic acid to measure the influence of packing d. and side chain conformational entropy on protein stability. All of these factors (hydrophobicity, packing, conformational entropy, and cavity formation) significantly influence protein stability and must be considered when analyzing any structural change to proteins.
- 323Ismail, A. R.; Kashtoh, H.; Baek, K.-H. Temperature-Resistant and Solvent-Tolerant Lipases as Industrial Biocatalysts: Biotechnological Approaches and Applications. Int. J. Biol. Macromol. 2021, 187, 127– 142, DOI: 10.1016/j.ijbiomac.2021.07.101Google Scholar323Temperature-resistant and solvent-tolerant lipases as industrial biocatalysts: Biotechnological approaches and applicationsIsmail, Abdallah R.; Kashtoh, Hamdy; Baek, Kwang-HyunInternational Journal of Biological Macromolecules (2021), 187 (), 127-142CODEN: IJBMDR; ISSN:0141-8130. (Elsevier B.V.)The development of new biocatalytic systems to replace the chem. catalysts, with suitable characteristics in terms of efficiency, stability under high temp. reactions and in the presence of org. solvents, reusability, and eco-friendliness is considered a very important step to move towards the green processes. From this basis, the use of lipase as a catalyst is highly desired for many industrial applications because it offers the reactions in which could be used, stability in harsh conditions, reusability and a greener process. Therefore, the introduction of temp.-resistant and solvent-tolerant lipases have become essential and ideal for industrial applications. Temp.-resistant and solvent-tolerant lipases have been involved in many large-scale applications including biodiesel, detergent, food, pharmaceutical, org. synthesis, biosensing, pulp and paper, textile, animal feed, cosmetics, and leather industry. So, the present review provides a comprehensive overview of the industrial use of lipase. Moreover, special interest in biotechnol. and biochem. techniques for enhancing temp.-resistance and solvent-tolerance of lipases to be suitable for the industrial uses.
- 324Vivek, K.; Sandhia, G. S.; Subramaniyan, S. Extremophilic Lipases for Industrial Applications: A General Review. Biotechnol. Adv. 2022, 60, 108002, DOI: 10.1016/j.biotechadv.2022.108002Google Scholar324Extremophilic lipases for industrial applications: A general reviewVivek, K.; Sandhia, G. S.; Subramaniyan, S.Biotechnology Advances (2022), 60 (), 108002CODEN: BIADDD; ISSN:0734-9750. (Elsevier Inc.)A Review on. With industrialization and development in modern science enzymes and their applications increased widely. There is always a hunt for new proficient enzymes with novel properties to meet specific needs of various industrial sectors. Along with the high efficiency, the green and eco-friendly side of enzymes attracts human attention, as they form a true answer to counter the hazardous and toxic conventional industrial catalyst. Lipases have always earned industrial attention due to the broad range of hydrolytic and synthetic reactions they catalyze. When these catalytic properties get accompanied by features like temp. stability, pH stability, and solvent stability lipases becomes an appropriate tool for use in many industrial processes. Extremophilic lipases offer the same, thermostable: hot and cold active thermophilic and psychrophilic lipases, acid and alkali resistant and active acidophilic and alkaliphilic lipases, and salt tolerant halophilic lipases form excellent biocatalyst for detergent formulations, biofuel synthesis, ester synthesis, food processing, pharmaceuticals, leather, and paper industry. An interesting application of these lipases is in the bioremediation of lipid waste in harsh environments. The review gives a brief account on various extremophilic lipases with emphasis on thermophilic, psychrophilic, halophilic, alkaliphilic, and acidophilic lipases, their sources, biochem. properties, and potential applications in recent decades.
- 325Budisa, N.; Wenger, W.; Wiltschi, B. Residue-Specific Global Fluorination of Candida antarctica Lipase B in Pichia pastoris. Mol. BioSyst. 2010, 6 (9), 1630– 1639, DOI: 10.1039/c002256jGoogle Scholar325Residue-specific global fluorination of Candida antarctica lipase B in Pichia pastorisBudisa, Nediljko; Wenger, Waltraud; Wiltschi, BirgitMolecular BioSystems (2010), 6 (9), 1630-1639CODEN: MBOIBW; ISSN:1742-206X. (Royal Society of Chemistry)We report the in vivo fluorination of the tryptophan, tyrosine, and phenylalanine residues in a glycosylation-deficient mutant of Candida antarctica lipase B, CalB N74D, expressed in the methylotrophic yeast Pichia pastoris and subsequently segregated into the growth medium. To achieve this, a P. pastoris strain auxotrophic for all three arom. amino acids was supplemented with 5-fluoro-L-tryptophan, meta-fluoro-(DL)-tyrosine, or para-fluoro-L-phenylalanine during expression of CalB N74D. The residue-specific replacement of the canonical amino acids by their fluorinated analogs was confirmed by mass anal. Although global fluorination induced moderate changes in the secondary structure of CalB N74D, the fluorous variant proteins were still active lipases. However, their catalytic activity was lower than that of the non-fluorinated parent protein while their resistance to proteolytic degrdn. by proteinase K remained unchanged. Importantly, we obsd. that the global fluorination prolonged the shelf life of the lipase activity, which is an esp. useful feature for the storage of, e.g., therapeutic proteins. Our study represents the first step on the road to the prodn. of biotechnol. and pharmacol. relevant fluorous proteins in P. pastoris.
- 326Merkel, L.; Schauer, M.; Antranikian, G.; Budisa, N. Parallel Incorporation of Different Fluorinated Amino Acids: On the Way to “Teflon” Proteins. ChemBioChem 2010, 11 (11), 1505– 1507, DOI: 10.1002/cbic.201000295Google ScholarThere is no corresponding record for this reference.
- 327Hoesl, M. G.; Acevedo-Rocha, C. G.; Nehring, S.; Royter, M.; Wolschner, C.; Wiltschi, B.; Budisa, N.; Antranikian, G. Lipase Congeners Designed by Genetic Code Engineering. ChemCatChem 2011, 3 (1), 213– 221, DOI: 10.1002/cctc.201000253Google ScholarThere is no corresponding record for this reference.
- 328Kelly, S. A.; Pohle, S.; Wharry, S.; Mix, S.; Allen, C. C. R.; Moody, T. S.; Gilmore, B. F. Application of ω-Transaminases in the Pharmaceutical Industry. Chem. Rev. 2018, 118 (1), 349– 367, DOI: 10.1021/acs.chemrev.7b00437Google Scholar328Application of ω-Transaminases in the Pharmaceutical IndustryKelly, Stephen A.; Pohle, Stefan; Wharry, Scott; Mix, Stefan; Allen, Christopher C. R.; Moody, Thomas S.; Gilmore, Brendan F.Chemical Reviews (Washington, DC, United States) (2018), 118 (1), 349-367CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Chiral amines are valuable building blocks for the pharmaceutical industry. ω-TAms have emerged as an exciting option for their synthesis, offering a potential "green alternative" to overcome the drawbacks assocd. with conventional chem. methods. In this review, we explore the application of ω-TAms for pharmaceutical prodn. We discuss the diverse array of reactions available involving ω-TAms and process considerations of their use in both kinetic resoln. and asym. synthesis. With the aid of specific drug intermediates and APIs, we chart the development of ω-TAms using protein engineering and their contribution to elegant one-pot cascades with other enzymes, including carbonyl reductases (CREDs), hydrolases and monoamine oxidases (MAOs), providing a comprehensive overview of their uses, beginning with initial applications through to the present day.
- 329Mathew, S.; Yun, H. ω-Transaminases for the Production of Optically Pure Amines and Unnatural Amino Acids. ACS Catal. 2012, 2 (6), 993– 1001, DOI: 10.1021/cs300116nGoogle Scholar329ω-Transaminases for the Production of Optically Pure Amines and Unnatural Amino AcidsMathew, Sam; Yun, HyungdonACS Catalysis (2012), 2 (6), 993-1001CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)A review. ω-Transaminases have been increasingly used as efficient biocatalysts due to their ability to produce a wide range of optically pure amine compds. Several approaches have been adopted, including screening, engineering, and development of new techniques in reaction systems for different aspects of the enzymes. This review summarizes the various methodologies and approaches adopted to produce enantiomerically pure amines and unnatural amino acids using ω-transaminases.
- 330Won, Y.; Jeon, H.; Pagar, A. D.; Patil, M. D.; Nadarajan, S. P.; Flood, D. T.; Dawson, P. E.; Yun, H. In vivo Biosynthesis of Tyrosine Analogs and their Concurrent Incorporation into a Residue-Specific Manner for Enzyme Engineering. Chem. Commun. 2019, 55 (100), 15133– 15136, DOI: 10.1039/C9CC08503CGoogle ScholarThere is no corresponding record for this reference.
- 331Votchitseva, Y. A.; Efremenko, E. N.; Varfolomeyev, S. D. Insertion of an Unnatural Amino Acid into the Protein Structure: Preparation and Properties of 3-Fluorotyrosine-Containing Organophosphate Hydrolase. Russ. Chem. Bull. 2006, 55 (2), 369– 374, DOI: 10.1007/s11172-006-0262-7Google ScholarThere is no corresponding record for this reference.
- 332Castro, A. A. d.; Prandi, I. G.; Kuca, K.; Ramalho, T. C. Enzimas Degradantes de Organofosforados: Base Molecular e Perspectivas para Biorremediação Enzimática de Agroquímicos. Ciênc. Agrotec. 2017, 41 (5), 471, DOI: 10.1590/1413-70542017415000417Google ScholarThere is no corresponding record for this reference.
- 333Makkar, R. S.; DiNovo, A. A.; Westwater, C.; Schofield, D. A. Enzyme-Mediated Bioremediation of Organophosphates using Stable Yeast Biocatalysts. J. Bioremed. Biodeg. 2013, 4 (182), 2, DOI: 10.4172/2155-6199.1000182Google ScholarThere is no corresponding record for this reference.
- 334Holzberger, B.; Marx, A. Replacing 32 Proline Residues by a Noncanonical Amino Acid Results in a Highly Active DNA Polymerase. J. Am. Chem. Soc. 2010, 132 (44), 15708– 15713, DOI: 10.1021/ja106525yGoogle Scholar334Replacing 32 Proline Residues by a Noncanonical Amino Acid Results in a Highly Active DNA PolymeraseHolzberger, Bastian; Marx, AndreasJournal of the American Chemical Society (2010), 132 (44), 15708-15713CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein engineering may be achieved by rational design, directed evolution-based methods, or computational protein design. Mostly these methods make recourse to the restricted pool of the 20 natural amino acids. With the ability to introduce different new kinds of functionalities into proteins, the use of noncanonical amino acids became a promising new method in protein engineering. Here, we report on the generation of a multifluorinated DNA polymerase. DNA polymerases are highly dynamic enzymes that catalyze DNA synthesis in a template-dependent manner, thereby passing several conformational states during the catalytic cycle. Here, we globally replaced 32 proline residues by the noncanonical imino acid (4R)-fluoroproline in a DNA polymerase of 540 amino acids (KlenTaq DNA polymerase). Interestingly, the substitution level of the proline residues was very efficient (92%). Nonetheless, the introduction of (4R)-fluoroproline into the DNA polymerase resulted in a highly active fluorinated enzyme, which was investigated in primer extension and PCR assays to analyze activity, selectivity, and stability in comparison to the parental enzyme. The DNA polymerase retained fidelity, activity, and sensitivity as the parental wild-type enzyme accompanied by some loss in thermostability. These results demonstrate that a noncanonical amino acid can be used for substitutions of natural counterparts in a highly dynamic enzyme with high mol. wt. without effecting crucial enzyme properties. Furthermore, the employed DNA polymerase represents a promising starting point for directed DNA polymerase evolution with noncanonical amino acids.
- 335Holzberger, B.; Obeid, S.; Welte, W.; Diederichs, K.; Marx, A. Structural Insights into the Potential of 4-Fluoroproline to Modulate Biophysical Properties of Proteins. Chem. Sci. 2012, 3 (10), 2924– 2931, DOI: 10.1039/c2sc20545aGoogle ScholarThere is no corresponding record for this reference.
- 336Panchenko, T.; Zhu, W. W.; Montclare, J. K. Influence of Global Fluorination on Chloramphenicol Acetyltransferase Activity and Stability. Biotechnol. Bioeng. 2006, 94 (5), 921– 930, DOI: 10.1002/bit.20910Google Scholar336Influence of global fluorination on chloramphenicol acetyltransferase activity and stabilityPanchenko, Tatyana; Zhu, Wan Wen; Montclare, Jin KimBiotechnology and Bioengineering (2006), 94 (5), 921-930CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)Varied levels of fluorinated amino acid have been introduced biosynthetically to test the functional limits of global substitution on enzymic activity and stability. Replacement of all the leucine (LEU) residues in the enzyme chloramphenicol acetyltransferase (CAT) with the analog, 5',5',5'-trifluoroleucine (TFL), results in the maintenance of enzymic activity under ambient temps. as well as an enhancement in secondary structure but loss in stability against heat and denaturants or org. co-solvents. Although catalytic activity of the fully substituted CAT is preserved under std. reaction conditions compared to the wild-type enzyme both in vitro and in vivo, as the incorporation levels increase, a concomitant redn. in thermostability and chemostability is obsd. CD studies reveal that although fluorination greatly improves the secondary structure of CAT, a large structural destabilization upon increased levels of TFL incorporation occurs at elevated temps. These data suggest that enhanced secondary structure afforded by TFL incorporation does not necessarily lead to an improvement in stability.
- 337Yang, C.-Y.; Renfrew, P. D.; Olsen, A. J.; Zhang, M.; Yuvienco, C.; Bonneau, R.; Montclare, J. K. Improved Stability and Half-Life of Fluorinated Phosphotriesterase Using Rosetta. ChemBioChem 2014, 15 (12), 1761– 1764, DOI: 10.1002/cbic.201402062Google ScholarThere is no corresponding record for this reference.
- 338Ohtake, K.; Yamaguchi, A.; Mukai, T.; Kashimura, H.; Hirano, N.; Haruki, M.; Kohashi, S.; Yamagishi, K.; Murayama, K.; Tomabechi, Y. Protein Stabilization Utilizing a Redefined Codon. Sci. Rep. 2015, 5 (1), 9762, DOI: 10.1038/srep09762Google ScholarThere is no corresponding record for this reference.
- 339Carlsson, A. C.; Scholfield, M. R.; Rowe, R. K.; Ford, M. C.; Alexander, A. T.; Mehl, R. A.; Ho, P. S. Increasing Enzyme Stability and Activity through Hydrogen Bond-Enhanced Halogen Bonds. Biochemistry 2018, 57 (28), 4135– 4147, DOI: 10.1021/acs.biochem.8b00603Google Scholar339Increasing Enzyme Stability and Activity through Hydrogen Bond-Enhanced Halogen BondsCarlsson, Anna-Carin C.; Scholfield, Matthew R.; Rowe, Rhianon K.; Ford, Melissa Coates; Alexander, Austin T.; Mehl, Ryan A.; Ho, P. ShingBiochemistry (2018), 57 (28), 4135-4147CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The construction of more stable proteins is important in biomol. engineering, particularly in the design of biologics based therapeutics. We show here that replacing the tyrosine at position 18 (Y18) of T4 lysozyme with the unnatural amino acid meta-chlorotyrosine (mClY) increases both the thermal stability (raising the melting temp. by ∼1°C and melting enthalpy by 3 kcal/mol) and enzymic activity at elevated temps. (15% higher than the parent enzyme at 40°C) of this classic enzyme. The chlorine of mClY forms a halogen bond (XB) to the carbonyl oxygen of the peptide bond at glycine 28 (G28) in a tight loop near the active site. In this case, the XB potential of the typically weak XB donor Cl is shown from quantum chem. calcns. to be significantly enhanced by polarization via an intramol. hydrogen bond (HB) from the adjacent hydroxyl substituent of the tyrosyl side-chain, resulting in a distinctive synergistic HB enhanced XB (or HeX-B for short) interaction. The larger halogens (bromine and iodine) are not well accommodated within this same loop and, consequently, do not exhibit the effects on protein stability or function assocd. with the HeX-B interaction. Thus, we have for the first time demonstrated that an XB can be engineered to stabilize and increase the activity of an enzyme, with the increased stabilizing potential of the HeX-B further extending the application of halogenated amino acids in the design of more stable protein therapeutics.
- 340Nicholson, H.; Anderson, D. E.; Dao Pin, S.; Matthews, B. W. Analysis of the Interaction Between Charged Side Chains and the Alpha-Helix Dipole Using Designed Thermostable Mutants of Phage T4 Lysozyme. Biochemistry 1991, 30 (41), 9816– 9828, DOI: 10.1021/bi00105a002Google ScholarThere is no corresponding record for this reference.
- 341Klink, T. A.; Woycechowsky, K. J.; Taylor, K. M.; Raines, R. T. Contribution of Disulfide Bonds to the Conformational Stability and Catalytic Activity Of Ribonuclease A. Eur. J. Biochem. 2000, 267 (2), 566– 572, DOI: 10.1046/j.1432-1327.2000.01037.xGoogle Scholar341Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease AKlink, Tony A.; Woycechowsky, Kenneth J.; Taylor, Kimberly M.; Raines, Ronald T.European Journal of Biochemistry (2000), 267 (2), 566-572CODEN: EJBCAI; ISSN:0014-2956. (Blackwell Science Ltd.)Disulfide bonds between the side-chains of Cys residues are the only common crosslinks in proteins. Bovine pancreatic RNase A is a 124-residue enzyme that contains 4 interweaving disulfide bonds (Cys-26-Cys-84, Cys-40-Cys-95, Cys-58-Cys-110, and Cys-65-Cys-72) and catalyzes the cleavage of RNA. Here, the contribution of each disulfide bond to the conformational stability and catalytic activity of RNase A was detd. by using variants in which each cystine residue was replaced independently with a pair of Ala residues. Thermal unfolding expts. monitored by UV spectroscopy and DSC revealed that wild-type RNase A and each disulfide variant unfolded in a 2-state process and that each disulfide bond contributed substantially to conformational stability. The 2 terminal disulfide bonds in the amino acid sequence (Cys-26-Cys-84 and Cys-58-Cys-110) enhanced the stability more than did the 2 embedded ones (Cys-40-Cys-95 and Cys-65-Cys-72). Removing either one of the terminal disulfide bonds liberated a similar no. of residues and had a similar effect on conformational stability, decreasing the midpoint of the thermal transition by almost 40°. The disulfide variants catalyzed the cleavage of poly(cytidylic acid) with values of kcat/Km that were 2- to 40-fold less than that of wild-type RNase A. The 2 embedded disulfide bonds, which were least important to conformational stability, were most important to catalytic activity. These embedded disulfide bonds likely contribute to the proper alignment of residues (such as Lys-41 and Lys-66) that are necessary for efficient catalysis of RNA cleavage.
- 342Yin, X.; Hu, D.; Li, J.-F.; He, Y.; Zhu, T.-D.; Wu, M.-C. Contribution of Disulfide Bridges to the Thermostability of a Type A Feruloyl Esterase from Aspergillus usamii. PLOS ONE 2015, 10 (5), e0126864 DOI: 10.1371/journal.pone.0126864Google Scholar342Contribution of disulfide bridges to the thermostability of a type A feruloyl esterase from Aspergillus usamiiYin, Xin; Hu, Die; Li, Jian-Fang; He, Yao; Zhu, Tian-Di; Wu, Min-ChenPLoS One (2015), 10 (5), e0126864/1-e0126864/16CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)The contribution of disulfide bridges to the thermostability of a type A feruloyl esterase (AuFaeA) from Aspergillus usamii E001 was studied by introducing an extra disulfide bridge or eliminating a native one from the enzyme. MODIP and DbD, two computational tools that can predict the possible disulfide bridges in proteins for thermostability improvement, and mol. dynamics (MD) simulations were used to design the extra disulfide bridge. One residue pair A126-N152 was chosen, and the resp. amino acid residues were mutated to cysteine. The wild-type AuFaeA and its variants were expressed in Pichia pastoris GS115. The temp. optimum of the recombinant (re-) AuFaeAA126C-N152C was increased by 6°C compared to that of re-AuFaeA. The thermal inactivation half-lives of re-AuFaeAA126C-N152C at 55 and 60°C were 188 and 40 min, which were 12.5- and 10-folds longer than those of re-AuFaeA. The catalytic efficiency (kcat/Km) of re-AuFaeAA126C-N152C was similar to that of re-AuFaeA. Addnl., after elimination of each native disulfide bridge in AuFaeA, a great decrease in expression level and at least 10°C decrease in thermal stability of recombinant AuEaeA variants were also obsd.
- 343Gihaz, S.; Bash, Y.; Rush, I.; Shahar, A.; Pazy, Y.; Fishman, A. Bridges to Stability: Engineering Disulfide Bonds Towards Enhanced Lipase Biodiesel Synthesis. ChemCatChem 2020, 12 (1), 181– 192, DOI: 10.1002/cctc.201901369Google ScholarThere is no corresponding record for this reference.
- 344Zhou, X.; Xu, Z.; Li, Y.; He, J.; Zhu, H. Improvement of the Stability and Activity of an LPMO Through Rational Disulfide Bonds Design. Front. bioeng. biotechnol. 2022, DOI: 10.3389/fbioe.2021.815990Google ScholarThere is no corresponding record for this reference.
- 345Dombkowski, A. A.; Sultana, K. Z.; Craig, D. B. Protein Disulfide Engineering. FEBS Lett. 2014, 588 (2), 206– 212, DOI: 10.1016/j.febslet.2013.11.024Google Scholar345Protein disulfide engineeringDombkowski, Alan A.; Sultana, Kazi Zakia; Craig, Douglas B.FEBS Letters (2014), 588 (2), 206-212CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)A review. Improving the stability of proteins is an important goal in many biomedical and industrial applications. A logical approach is to emulate stabilizing mol. interactions found in nature. Disulfide bonds are covalent interactions that provide substantial stability to many proteins and conform to well-defined geometric conformations, thus making them appealing candidates in protein engineering efforts. Disulfide engineering is the directed design of novel disulfide bonds into target proteins. This important biotechnol. tool has achieved considerable success in a wide range of applications, yet the rules that govern the stabilizing effects of disulfide bonds are not fully characterized. Contrary to expectations, many designed disulfide bonds have resulted in decreased stability of the modified protein. The authors review progress in disulfide engineering, with an emphasis on the issue of stability and computational methods that facilitate engineering efforts.
- 346Liu, T.; Wang, Y.; Luo, X.; Li, J.; Reed, S. A.; Xiao, H.; Young, T. S.; Schultz, P. G. Enhancing Protein Stability with Extended Disulfide Bonds. Proc. Natl. Acad. Sci. U.S.A. 2016, 113 (21), 5910– 5915, DOI: 10.1073/pnas.1605363113Google Scholar346Enhancing protein stability with extended disulfide bondsLiu, Tao; Wang, Yan; Luo, Xiaozhou; Li, Jack; Reed, Sean A.; Xiao, Han; Young, Travis S.; Schultz, Peter G.Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (21), 5910-5915CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Disulfide bonds play an important role in protein folding and stability. However, the crosslinking of sites within proteins by cysteine disulfides has significant distance and dihedral angle constraints. Here we report the genetic encoding of noncanonical amino acids contg. long side-chain thiols that are readily incorporated into both bacterial and mammalian proteins in good yields and with excellent fidelity. These amino acids can pair with cysteines to afford extended disulfide bonds and allow crosslinking of more distant sites and distinct domains of proteins. To demonstrate this notion, we preformed growth-based selection expts. at nonpermissive temps. using a library of random β-lactamase mutants contg. these noncanonical amino acids. A mutant enzyme that is cross-linked by one such extended disulfide bond and is stabilized by ∼9 °C was identified. This result indicates that an expanded set of building blocks beyond the canonical 20 amino acids can lead to proteins with improved properties by unique mechanisms, distinct from those possible through conventional mutagenesis schemes.
- 347Hecky, J.; Müller, K. M. Structural Perturbation and Compensation by Directed Evolution at Physiological Temperature Leads to Thermostabilization of β-Lactamase. Biochemistry 2005, 44 (38), 12640– 12654, DOI: 10.1021/bi0501885Google Scholar347Structural perturbation and compensation by directed evolution at physiological temperature leads to thermostabilization of β-lactamaseHecky, Jochen; Mueller, Kristian M.Biochemistry (2005), 44 (38), 12640-12654CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The choice of protein for use in tech. and medical applications is limited by stability issues, making understanding and engineering of stability key. Here, enzyme destabilization by truncation was combined with directed evolution to create stable variants of TEM-1 β-lactamase (I). I was chosen because of its implication in prodrug activation therapy, pathogen resistance to lactam antibiotics, and reporter enzyme bioassays. Removal of 5 N-terminal residues generated a mutant which did not confer antibiotic resistance at 37°. Accordingly, the half-life time in vitro was only 7 s at 40°. However, 3 cycles comprising random mutagenesis, DNA shuffling, and metabolic selection at 37° yielded mutants providing resistance levels significantly higher than that of wild-type I. These mutants demonstrated increased thermoactivity and thermostability in time-resolved kinetics at various temps. Chem. denaturation revealed improved thermodn. stabilities of a 3-state unfolding pathway exceeding wild-type construct stability. Elongation of one optimized deletion mutant to full length increased its stability even further. Compared to that of wild-type I, the temp. optimum was shifted from 35 to 50°, and the beginning of heat inactivation increased by 20° while full activity at low temps. was maintained. These effects were attributed mainly to 2 independently acting boundary interface residue exchanges (M182T and A224V). Thus, structural perturbation by terminal truncation, evolutionary compensation at physiol. temps., and elongation is an efficient way to analyze and improve thermostability without the need for high-temp. selection, structural information, or homologous proteins.
- 348Brown, N. G.; Pennington, J. M.; Huang, W.; Ayvaz, T.; Palzkill, T. Multiple Global Suppressors of Protein Stability Defects Facilitate the Evolution of Extended-Spectrum TEM β-Lactamases. J. Mol. Biol. 2010, 404 (5), 832– 846, DOI: 10.1016/j.jmb.2010.10.008Google Scholar348Multiple Global Suppressors of Protein Stability Defects Facilitate the Evolution of Extended-Spectrum TEM β-LactamasesBrown, Nicholas G.; Pennington, Jeanine M.; Huang, Wanzhi; Ayvaz, Tulin; Palzkill, TimothyJournal of Molecular Biology (2010), 404 (5), 832-846CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)The introduction of extended-spectrum cephalosporins and β-lactamase inhibitors has driven the evolution of extended-spectrum β-lactamases (ESBLs) that possess the ability to hydrolyze these drugs. The evolved TEM ESBLs from clin. isolates of bacteria often contain substitutions that occur in the active site and alter the catalytic properties of the enzyme to provide an increased hydrolysis of extended-spectrum cephalosporins or an increased resistance to inhibitors. These active-site substitutions often result in a cost in the form of reduced enzyme stability. The evolution of TEM ESBLs is facilitated by mutations that act as global suppressors of protein stability defects in the sense that they allow the enzyme to absorb multiple amino acid changes despite incremental losses in stability assocd. with the substitutions. The best-studied example is the M182T substitution, which corrects protein stability defects and is commonly found in TEM ESBLs or inhibitor-resistant variants from clin. isolates. In this study, a genetic selection for second-site mutations that could partially restore function to a severely destabilized primary mutant enabled the identification of A184V, T265M, R275Q, and N276D, which are known to occur in TEM ESBLs from clin. isolates, as suppressors of TEM-1 protein stability defects. Further characterization demonstrated that these substitutions increased the thermal stability of TEM-1 and were able to correct the stability defects of two different sets of destabilizing mutations. The acquisition of compensatory global suppressors of stability costs assocd. with active-site mutations may be a common mechanism for the evolution of novel protein function.
- 349Moore, E. J.; Zorine, D.; Hansen, W. A.; Khare, S. D.; Fasan, R. Enzyme Stabilization via Computationally Guided Protein Stapling. Proc. Natl. Acad. Sci. U.S.A. 2017, 114 (47), 12472– 12477, DOI: 10.1073/pnas.1708907114Google Scholar349Enzyme stabilization via computationally guided protein staplingMoore, Eric J.; Zorine, Dmitri; Hansen, William A.; Khare, Sagar D.; Fasan, RudiProceedings of the National Academy of Sciences of the United States of America (2017), 114 (47), 12472-12477CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Thermostabilization represents a crit. and often obligatory step toward enhancing the robustness of enzymes for org. synthesis and other applications. While directed evolution methods have provided valuable tools for this purpose, these protocols are laborious and time-consuming and typically require the accumulation of several mutations, potentially at the expense of catalytic function. Here, we report a minimally invasive strategy for enzyme stabilization that relies on the installation of genetically encoded, nonreducible covalent staples in a target protein scaffold using computational design. This methodol. enables the rapid development of myoglobin-based cyclopropanation biocatalysts featuring dramatically enhanced thermostability (ΔTm = +18.0 °C and ΔT50 = +16.0 °C) as well as increased stability against chem. denaturation [ΔCm (GndHCl) = 0.53 M], without altering their catalytic efficiency and stereoselectivity properties. In addn., the stabilized variants offer superior performance and selectivity compared with the parent enzyme in the presence of a high concn. of org. cosolvents, enabling the more efficient cyclopropanation of a water-insol. substrate. This work introduces and validates an approach for protein stabilization which should be applicable to a variety of other proteins and enzymes.
- 350Bi, J.; Jing, X.; Wu, L.; Zhou, X.; Gu, J.; Nie, Y.; Xu, Y. Computational Design of Noncanonical Amino Acid-Based Thioether Staples at N/C-Terminal Domains of Multi-Modular Pullulanase for Thermostabilization in Enzyme Catalysis. Comput. Struct. Biotechnol. J. 2021, 19, 577– 585, DOI: 10.1016/j.csbj.2020.12.043Google Scholar350Computational design of noncanonical amino acid-based thioether staples at N/C-terminal domains of multi-modular pullulanase for thermostabilization in enzyme catalysisBi, Jiahua; Jing, Xiaoran; Wu, Lunjie; Zhou, Xia; Gu, Jie; Nie, Yao; Xu, YanComputational and Structural Biotechnology Journal (2021), 19 (), 577-585CODEN: CSBJAC; ISSN:2001-0370. (Elsevier B.V.)Enzyme thermostabilization is considered a crit. and often obligatory step in biosynthesis, because thermostability is a significant property of enzymes that can be used to evaluate their feasibility for industrial applications. However, conventional strategies for thermostabilizing enzymes generally introduce non-covalent interactions and/or natural covalent bonds caused by natural amino acid substitutions, and the trade-off between the activity and stability of enzymes remains a challenge. Here, we developed a computationally guided strategy for constructing thioether staples by incorporating noncanonical amino acid (ncAA) into the more flexible N/C-terminal domains of the multi-modular pullulanase from Bacillus thermoleovorans (BtPul) to enhance its thermostability. First, potential thioether staples located in the N/C-terminal domains of BtPul were predicted using RosettaMatch. Next, eight variants involving stable thioether staples were precisely predicted using FoldX and Rosetta ddg_monomer. Six pos. variants were obtained, of which T73(O2beY)-171C had a 157% longer half-life at 70 °C and an increase of 7.0 °C in Tm, when compared with the wild-type (WT). T73(O2beY)-171C/T126F/A72R exhibited an even more improved thermostability, with a 211% increase in half-life at 70 °C and a 44% enhancement in enzyme activity compared with the WT, which was attributed to further optimization of the local interaction network. This work introduces and validates an efficient strategy for enhancing the thermostability and activity of multi-modular enzymes.
- 351Iannuzzelli, J. A.; Bacik, J.-P.; Moore, E. J.; Shen, Z.; Irving, E. M.; Vargas, D. A.; Khare, S. D.; Ando, N.; Fasan, R. Tuning Enzyme Thermostability via Computationally Guided Covalent Stapling and Structural Basis of Enhanced Stabilization. Biochemistry 2022, 61 (11), 1041– 1054, DOI: 10.1021/acs.biochem.2c00033Google Scholar351Tuning enzyme thermostability via computationally guided covalent stapling and structural basis of enhanced stabilizationIannuzzelli, Jacob A.; Bacik, John-Paul; Moore, Eric J.; Shen, Zhuofan; Irving, Ellen M.; Vargas, David A.; Khare, Sagar D.; Ando, Nozomi; Fasan, RudiBiochemistry (2022), 61 (11), 1041-1054CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Enhancing the thermostability of enzymes without impacting their catalytic function represents an important yet challenging goal in protein engineering and biocatalysis. We recently introduced a novel method for enzyme thermostabilization that relies on the computationally guided installation of genetically encoded thioether "staples" into a protein via cysteine alkylation with the noncanonical amino acid O-2-bromoethyl tyrosine (O2beY). Here, we demonstrate the functionality of an expanded set of electrophilic amino acids featuring chloroacetamido, acrylamido, and vinylsulfonamido side-chain groups for protein stapling using this strategy. Using a myoglobin-based cyclopropanase as a model enzyme, our studies show that covalent stapling with p-chloroacetamido-phenylalanine (pCaaF) provides higher stapling efficiency and enhanced stability (thermodn. and kinetic) compared to the other stapled variants and the parent protein. Interestingly, mol. simulations of conformational flexibility of the crosslinks show that the pCaaF staple allows fewer energetically feasible conformers than the other staples, and this property may be a broader indicator of stability enhancement. Using this strategy, pCaaF-stapled variants with significantly enhanced stability against thermal denaturation (ΔTm' = +27°C) and temp.-induced heme loss (ΔT50 = +30°C) were obtained while maintaining high levels of catalytic activity and stereoselectivity. Crystallog. analyses of singly and doubly stapled variants provide key insights into the structural basis for stabilization, which includes both direct interactions of the staples with protein residues and indirect interactions through adjacent residues involved in heme binding. This work expands the toolbox of protein stapling strategies available for protein stabilization.
- 352Gur, E.; Biran, D.; Gazit, E.; Ron, E. Z. In vivo Aggregation of a Single Enzyme Limits Growth of Escherichia coli at Elevated Temperatures. Mol. Microbiol. 2002, 46 (5), 1391– 1397, DOI: 10.1046/j.1365-2958.2002.03257.xGoogle Scholar352In vivo aggregation of a single enzyme limits growth of Escherichia coli at elevated temperaturesGur, Eyal; Biran, Dvora; Gazit, Ehud; Ron, Eliora Z.Molecular Microbiology (2002), 46 (5), 1391-1397CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Science Ltd.)The formation of protein aggregates is assocd. with unfolding and denaturation of proteins. Recent studies have indicated that, in Escherichia coli, cellular proteins tend to aggregate when the bacteria are exposed to thermal stress. Here, we show that the aggregation of one single E. coli cytoplasmic protein limits growth at elevated temps. in minimal media. Homoserine trans-succinylase (HTS), the first enzyme in the methionine biosynthetic pathway, aggregates at temps. higher than 44°C in vitro. Above this temp., we can also observe in vivo aggregation that results in the complete disappearance of the enzyme from the sol. fraction. Moreover, reducing the in vivo level of HTS aggregation enables growth at non-permissive temps. This is the first demonstration of the physiol. role of aggregation of a specific protein in the growth of wild-type bacteria.
- 353Li, J. C.; Liu, T.; Wang, Y.; Mehta, A. P.; Schultz, P. G. Enhancing Protein Stability with Genetically Encoded Noncanonical Amino Acids. J. Am. Chem. Soc. 2018, 140 (47), 15997– 16000, DOI: 10.1021/jacs.8b07157Google Scholar353Enhancing Protein Stability with Genetically Encoded Noncanonical Amino AcidsLi, Jack C.; Liu, Tao; Wang, Yan; Mehta, Angad P.; Schultz, Peter G.Journal of the American Chemical Society (2018), 140 (47), 15997-16000CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The ability to add noncanonical amino acids to the genetic code may allow one to evolve proteins with new or enhanced properties using a larger set of building blocks. To this end, we have been able to select mutant proteins with enhanced thermal properties from a library of Escherichia coli homoserine O-succinyltransferase (metA) mutants contg. randomly incorporated noncanonical amino acids. Here, we showed that substitution of Phe-21 with p-benzoyl-L-phenylalanine (pBzF), increased the melting temp. of E. coli metA by 21°. This dramatic increase in thermostability, arising from a single mutation, likely resulted from a covalent adduct between Cys-90 and the keto group of pBzF that stabilized the dimeric form of the enzyme. These expts. show that an expanded genetic code can provide unique solns. to the evolution of proteins with enhanced properties.
- 354Xuan, W.; Li, J.; Luo, X.; Schultz, P. G. Genetic Incorporation of a Reactive Isothiocyanate Group into Proteins. Angew. Chem. Int. Ed. 2016, 55 (34), 10065– 10068, DOI: 10.1002/anie.201604891Google Scholar354Genetic Incorporation of a Reactive Isothiocyanate Group into ProteinsXuan, Weimin; Li, Jack; Luo, Xiaozhou; Schultz, Peter G.Angewandte Chemie, International Edition (2016), 55 (34), 10065-10068CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Methods for the site-specific modification of proteins are useful for introducing biol. probes into proteins and engineering proteins with novel activities. Herein, the authors genetically encode a novel noncanonical amino acid (ncAA) that contains an aryl isothiocyanate group which can form stable thiourea crosslinks with amines under mild conditions. This ncAA (pNCSF) allows the selective conjugation of proteins to amine-contg. mol. probes through formation of a thiourea bridge. PNCSF was also used to replace a native salt bridge in myoglobin with an intramol. crosslink to a proximal Lys residue, leading to increased thermal stability. Finally, pNCSF can form stable intermol. crosslinks between two interacting proteins.
- 355Li, J. C.; Nastertorabi, F.; Xuan, W.; Han, G. W.; Stevens, R. C.; Schultz, P. G. A Single Reactive Noncanonical Amino Acid Is Able to Dramatically Stabilize Protein Structure. ACS Chem. Biol. 2019, 14 (6), 1150– 1153, DOI: 10.1021/acschembio.9b00002Google ScholarThere is no corresponding record for this reference.
- 356Deiters, A.; Cropp, T. A.; Summerer, D.; Mukherji, M.; Schultz, P. G. Site-Specific PEGylation of Proteins Containing Unnatural Amino Acids. Bioorg. Med. Chem. Lett. 2004, 14 (23), 5743– 5745, DOI: 10.1016/j.bmcl.2004.09.059Google ScholarThere is no corresponding record for this reference.
- 357Schoffelen, S.; Lambermon, M. H. L.; Eldijk, M. B. v.; Hest, J. C. M. v. Site-Specific Modification of Candida antarctica Lipase B via Residue-Specific Incorporation of a Non-Canonical Amino Acid. Bioconjug. Chem. 2008, 19 (6), 1127– 1131, DOI: 10.1021/bc800019vGoogle Scholar357Site-Specific Modification of Candida antarctica Lipase B via Residue-Specific Incorporation of a Non-Canonical Amino AcidSchoffelen, Sanne; Lambermon, Mark H. L.; van Eldijk, Mark B.; van Hest, Jan C. M.Bioconjugate Chemistry (2008), 19 (6), 1127-1131CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)In order to modify proteins in a controlled way, new functionalities need to be introduced in a defined manner. One way to accomplish this is by the incorporation of a non-natural amino acid of which the side chain can selectively be reacted to other mols. We have investigated whether the relatively simple method of residue-specific replacement of methionine by azidohomoalanine can be used to achieve monofunctionalization of the model enzyme Candida antarctica lipase B. A protein variant was engineered with one addnl. methionine residue. Due to the high hydrophobicity and low abundance of methionine, this was the only residue out of five that was exposed to the solvent. The use of the CuI-catalyzed [3+2] cycloaddn. under native conditions resulted in a monofunctionalized enzyme which retained hydrolytic activity. The strategy can be considered a convenient tool to modify proteins at a single position as long as one solvent-exposed methionine is available.
- 358van Dongen, S. F. M.; Nallani, M.; Schoffelen, S.; Cornelissen, J. J. L. M.; Nolte, R. J. M.; van Hest, J. C. M. A Block Copolymer for Functionalisation of Polymersome Surfaces. Macromol. Rapid Commun. 2008, 29 (4), 321– 325, DOI: 10.1002/marc.200700765Google Scholar358A block copolymer for functionalisation of polymersome surfacesvan Dongen, Stijn F. M.; Nallani, Madhavan; Schoffelen, Sanne; Cornelissen, Jeroen J. L. M.; Nolte, Roeland J. M.; van Hest, Jan C. M.Macromolecular Rapid Communications (2008), 29 (4), 321-325CODEN: MRCOE3; ISSN:1022-1336. (Wiley-VCH Verlag GmbH & Co. KGaA)A block copolymer was designed to functionalize the surface of polystyrene-based polymersomes via coaggregation. An α,ω-diacetylene-functionalized poly(ethylene glycol) (PEG) was coupled to an azide-terminated polystyrene via a Cu(I)-catalyzed cycloaddn. to produce a PS-b-PEG polymer with an acetylene at its hydrophilic extremity. Incorporation of this 'anchor' compd. in the bilayer of a polymersome places its bio-orthogonal group at the surface of this aggregate. Its accessibility was demonstrated using an azido-functionalized Candida antarctica Lipase B (CalB), which retained its activity while immobilized on the polymersome.
- 359Teeuwen, R. L. M.; van Berkel, S. S.; van Dulmen, T. H. H.; Schoffelen, S.; Meeuwissen, S. A.; Zuilhof, H.; de Wolf, F. A.; van Hest, J. C. M. “Clickable” Elastins: Elastin-Like Polypeptides Functionalized with Azide or Alkyne Groups. Chem. Commun. 2009, (27), 4022– 4024, DOI: 10.1039/b903903aGoogle ScholarThere is no corresponding record for this reference.
- 360Debets, M. F.; van Berkel, S. S.; Schoffelen, S.; Rutjes, F. P. J. T.; van Hest, J. C. M.; van Delft, F. L. Aza-Dibenzocyclooctynes for Fast and Efficient Enzyme PEGylation via Copper-Free (3 + 2) Cycloaddition. Chem. Commun. 2010, 46 (1), 97– 99, DOI: 10.1039/B917797CGoogle Scholar360Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3 + 2) cycloadditionDebets, Marjoke F.; van Berkel, Sander S.; Schoffelen, Sanne; Rutjes, Floris P. J. T.; van Hest, Jan C. M.; van Delft, Floris L.Chemical Communications (Cambridge, United Kingdom) (2010), 46 (1), 97-99CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Didehydrodibenzazocines (azadibenzocyclooctynes) I [R = PhCH2OCO, HO2C(CH2)3CO, Me(OCH2CH2)nCH2CH2NHCO(CH2)3CO] are prepd. as potential reagents for copper-free Huisgen dipolar cycloaddns. with azide-labeled proteins to form triazole-substituted protein conjugates. I [R = HO2C(CH2)3CO] is prepd. in nine steps from 2-iodobenzyl alc., 2-ethynylaniline, and 5-methoxy-5-oxopentanoyl chloride. The kinetics of copper-free Huisgen dipolar cycloaddns. of I [R = PhCH2OCO, HO2C(CH2)3CO] with benzyl azide and with (S)-α-azidopropanoic acid are detd.; I react at comparable or larger rates with benzyl azide than other cyclooctyne reagents. Conjugates of I [R = Me(OCH2CH2)nCH2CH2NHCO(CH2)3CO] with Candida antarctica lipase B contg. five azidohomoalanine residues and an azide-substituted horseradish peroxidase are generated, indicating that I [R = Me(OCH2CH2)nCH2CH2NHCO(CH2)3CO] can be used for the PEGylation of azide-labeled proteins.
- 361Wilding, K. M.; Smith, A. K.; Wilkerson, J. W.; Bush, D. B.; Knotts, T. A. I. V.; Bundy, B. C. The Locational Impact of Site-Specific PEGylation: Streamlined Screening with Cell-Free Protein Expression and Coarse-Grain Simulation. ACS Synth. Biol. 2018, 7 (2), 510– 521, DOI: 10.1021/acssynbio.7b00316Google Scholar361The Locational Impact of Site-Specific PEGylation: Streamlined Screening with Cell-Free Protein Expression and Coarse-Grain SimulationWilding, Kristen M.; Smith, Addison K.; Wilkerson, Joshua W.; Bush, Derek B.; Knotts, Thomas A.; Bundy, Bradley C.ACS Synthetic Biology (2018), 7 (2), 510-521CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Although polyethylene glycol (PEG) is commonly used to improve protein stability and therapeutic efficacy, the optimal location for attaching PEG onto proteins is not well understood. Here, we present a cell-free protein synthesis-based screening platform that facilitates site-specific PEGylation and efficient evaluation of PEG attachment efficiency, thermal stability, and activity for different variants of PEGylated T4 lysozyme, including a di-PEGylated variant. We also report developing a computationally efficient coarse-grain simulation model as a potential tool to narrow exptl. screening candidates. We use this simulation method as a novel tool to evaluate the locational impact of PEGylation. Using this screen, we also evaluated the predictive impact of PEGylation site solvent accessibility, conjugation site structure, PEG size, and double PEGylation. Our findings indicate that PEGylation efficiency, protein stability, and protein activity varied considerably with PEGylation site, variations that were not well predicted by common PEGylation guidelines. Overall our results suggest current guidelines are insufficiently predictive, highlighting the need for exptl. and simulation screening systems such as the one presented here.
- 362Wu, J. C. Y.; Hutchings, C. H.; Lindsay, M. J.; Werner, C. J.; Bundy, B. C. Enhanced Enzyme Stability Through Site-Directed Covalent Immobilization. J. Biotechnol. 2015, 193, 83– 90, DOI: 10.1016/j.jbiotec.2014.10.039Google Scholar362Enhanced enzyme stability through site-directed covalent immobilizationWu, Jeffrey Chun Yu; Hutchings, Christopher Hayden; Lindsay, Mark Jeffrey; Werner, Christopher James; Bundy, Bradley CharlesJournal of Biotechnology (2015), 193 (), 83-90CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)Breakthroughs in enzyme immobilization have enabled increased enzyme recovery and reusability, leading to significant decreases in the cost of enzyme use and fueling biocatalysis growth. However, current enzyme immobilization techniques suffer from leaching, enzyme stability, and recoverability and reusability issues. Moreover, these techniques lack the ability to control the orientation of the immobilized enzymes. To det. the impact of orientation on covalently immobilized enzyme activity and stability, the authors applied their PRECISE (Protein Residue-Explicit Covalent Immobilization for Stability Enhancement) system to a model enzyme, phage T4 lysozyme. The PRECISE system uses non-canonical amino acid incorporation and the Huisgen 1,3-dipolar cycloaddn. "click" reaction to enable directed enzyme immobilization at rationally chosen residues throughout an enzyme. Unlike previous site-specific systems, the PRECISE system is a truly covalent immobilization method. Utilizing this system, enzymes immobilized at proximate and distant locations from the active site were tested for activity and stability under denaturing conditions. The results demonstrated that orientation control of covalently immobilized enzymes could provide activity and stability benefits exceeding that of traditional random covalent immobilization techniques. PRECISE immobilized enzymes were 50 and 73% more active than randomly immobilized enzymes after harsh freeze-thaw and chem. denaturant treatments.
- 363Basso, A.; Serban, S. Industrial Applications of Immobilized Enzymes─A Review. Mol. Catal. 2019, 479, 110607, DOI: 10.1016/j.mcat.2019.110607Google Scholar363Industrial applications of immobilized enzymes-A reviewBasso, Alessandra; Serban, SimonaMolecular Catalysis (2019), 479 (), 110607CODEN: MCOADH ISSN:. (Elsevier B.V.)A review. The use of immobilized enzymes is now a routine process for the manuf. of many industrial products in the pharmaceutical, chem. and food industry. Some enzymes, such as lipases, are naturally robust and efficient, can be used for the prodn. of many different mols. and have a wide range of industrial applications thanks to their broad selectivity. As an example, lipase from Candida antarctica (CalB) has been used by BASF to produce chiral compds., such as the herbicide Dimethenamide-P, which was previously made chem. The use of the immobilized enzyme has provided significant advantages over a chem. process, such as the possibility to use equimolar concn. of substrates, obtain an enantiomeric excess > 99%, use relatively low temps. (< 60 °C) in org. solvent, obtain a single enantiomer instead of the racemate as in the chem. process and use a column configuration that allows dramatic increases in productivity. This process would not have been possible without the use of an immobilized enzyme, since it runs in org. solvent [1]. Some more specific enzymes, like transaminases, have required protein engineering to become suitable for applications in prodn. of APIs (Active Pharmaceutical Ingredients) in conditions which are extreme for a wild type enzyme. The process developed by Merck for sitagliptin manuf. is a good example of challenging enzyme engineering applied to API manuf. The previous process of sitagliptin involved hydrogenation of enamine at high pressure using a rhodium-based chiral catalyst. By developing an engineered transaminase, the enzymic process was able to convert 200 g/l of prositagliptin in the final product, with e.e. >99.5% and using an immobilized enzyme in the presence of DMSO as a cosolvent [2]. For all enzymes, the possibility to be immobilized and used in a heterogeneous form brings important industrial and environmental advantages, such as simplified downstream processing or continuous process operations. Here, we present a series of large-scale applications of immobilized enzymes with benefits for the food, chem., pharmaceutical, cosmetics and medical device industries, some of which have been scarcely reported on previously. In general, all enzymic reactions can benefit from the immobilization, however, the final choice to use them in immobilized form depends on the economic evaluation of costs assocd. with their use vs. benefits obtained in the process. It can be concluded that the benefits are rather significant, since the use of immobilized enzymes in industry is increasing.
- 364Hernandez, K.; Fernandez-Lafuente, R. Control of Protein Immobilization: Coupling Immobilization and Site-Directed Mutagenesis to Improve Biocatalyst or Biosensor Performance. Enzyme Microb. Technol. 2011, 48 (2), 107– 122, DOI: 10.1016/j.enzmictec.2010.10.003Google Scholar364Control of protein immobilization: Coupling immobilization and site-directed mutagenesis to improve biocatalyst or biosensor performanceHernandez, Karel; Fernandez-Lafuente, RobertoEnzyme and Microbial Technology (2011), 48 (2), 107-122CODEN: EMTED2; ISSN:0141-0229. (Elsevier B.V.)A review. Mutagenesis and immobilization are usually considered to be unrelated techniques with potential applications to improve protein properties. However, there are several reports showing that the use of site-directed mutagenesis to improve enzyme properties directly, but also how enzymes are immobilized on a support, can be a powerful tool to improve the properties of immobilized biomols. for use as biosensors or biocatalysts. Std. immobilizations are not fully random processes, but the protein orientation may be difficult to alter. Initially, most efforts using this idea were addressed towards controlling the orientation of the enzyme on the immobilization support, in many cases to facilitate electron transfer from the support to the enzyme in redox biosensors. Usually, Cys residues are used to directly immobilize the protein on a support that contains disulfide groups or that is made from gold. There are also some examples using His in the target areas of the protein and using supports modified with immobilized metal chelates and other tags (e.g., using immobilized antibodies). Furthermore, site-directed mutagenesis to control immobilization is useful for improving the activity, the stability and even the selectivity of the immobilized protein, for example, via site-directed rigidification of selected areas of the protein. Initially, only Cys and disulfide supports were employed, but other supports with higher potential to give multipoint covalent attachment are being employed (e.g., glyoxyl or epoxy-disulfide supports). The advances in support design and the deeper knowledge of the mechanisms of enzyme-support interactions have permitted exploration of the possibilities of the coupled use of site-directed mutagenesis and immobilization in a new way. This paper intends to review some of the advances and possibilities that these coupled strategies permit.
- 365Guan, D.; Kurra, Y.; Liu, W.; Chen, Z. A Click Chemistry Approach to Site-Specific Immobilization of a Small Laccase Enables Efficient Direct Electron Transfer in a Biocathode. Chem. Commun. 2015, 51 (13), 2522– 2525, DOI: 10.1039/C4CC09179EGoogle Scholar365A click chemistry approach to site-specific immobilization of a small laccase enables efficient direct electron transfer in a biocathodeGuan, Dongli; Kurra, Yadagiri; Liu, Wenshe; Chen, ZhileiChemical Communications (Cambridge, United Kingdom) (2015), 51 (13), 2522-2525CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Controlled orientation of a small laccase on a multi-walled carbon nanotube (MWCNT) electrode was achieved via copper-free click chem.-mediated immobilization. Modification of the enzyme was limited to only the tethering site and involved the genetic incorporation of the unnatural amino acid 4-azido-L-phenylalanine (AzF). This approach enabled efficient direct electron transfer (DET).
- 366Lim, S. I.; Mizuta, Y.; Takasu, A.; Kim, Y. H.; Kwon, I. Site-Specific Bioconjugation of a Murine Dihydrofolate Reductase Enzyme by Copper(I)-Catalyzed Azide-Alkyne Cycloaddition with Retained Activity. PLOS ONE 2014, 9 (6), e98403 DOI: 10.1371/journal.pone.0098403Google Scholar366Site-specific bioconjugation of a murine dihydrofolate reductase enzyme by copper(I)-catalyzed azide-alkyne cycloaddition with retained activityLim, Sung In; Mizuta, Yukina; Takasu, Akinori; Kim, Yong Hwan; Kwon, InchanPLoS One (2014), 9 (6), e98403/1-e98403/10, 10 pp.CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Cu(I)-catalyzed azide-alkyne cycloaddn. (CuAAC) is an efficient reaction linking an azido and an alkynyl group in the presence of copper catalyst. Incorporation of a non-natural amino acid (NAA) contg. either an azido or an alkynyl group into a protein allows site-specific bioconjugation in mild conditions via CuAAC. Despite its great potential, bioconjugation of an enzyme has been hampered by several issues including low yield, poor soly. of a ligand, and protein structural/functional perturbation by CuAAC components. In the present study, we incorporated an alkyne-bearing NAA into an enzyme, murine dihydrofolate reductase (mDHFR), in high cell d. cultivation of Escherichia coli, and performed CuAAC conjugation with fluorescent azide dyes to evaluate enzyme compatibility of various CuAAC conditions comprising combination of com. available Cu(I)-chelating ligands and reductants. The condensed culture improves the protein yield 19-fold based on the same amt. of non-natural amino acid, and the enzyme incubation under the optimized reaction condition did not lead to any activity loss but allowed a fast and high-yield bioconjugation. Using the established conditions, a biotin-azide spacer was efficiently conjugated to mDHFR with retained activity leading to the site-specific immobilization of the biotin-conjugated mDHFR on a streptavidin-coated plate. These results demonstrate that the combination of reactive non-natural amino acid incorporation and the optimized CuAAC can be used to bioconjugate enzymes with retained enzymic activity.
- 367Wang, A.; Du, F.; Pei, X.; Chen, C.; Wu, S. G.; Zheng, Y. Rational Immobilization of Lipase by Combining the Structure Analysis and Unnatural Amino Acid Insertion. J. Mol. Catal. B Enzym. 2016, 132, 54– 60, DOI: 10.1016/j.molcatb.2016.06.015Google Scholar367Rational immobilization of lipase by combining the structure analysis and unnatural amino acid insertionWang, Anming; Du, Fangchuan; Pei, Xiaolin; Chen, Canyu; Wu, Stephen Gang; Zheng, YuguoJournal of Molecular Catalysis B: Enzymatic (2016), 132 (), 54-60CODEN: JMCEF8; ISSN:1381-1177. (Elsevier B.V.)Improving the conventional covalent immobilization of enzyme and avoiding random covalent linkage to protect enzyme's active sites from unwanted covalent linkage at the mean time are the fundamental topics for enzyme immobilization. In this study, unnatural amino acid was introduced into a recombinant lipase and applied for the rational and smart covalent enzyme immobilization. In the first step, Tyr50, 137, 243, 274, and 355 of lipase were replaced with AzPhe unnatural amino acid based on the anal. of enzyme structure. Then, these novel recombinant lipases were coupled to support using strain-promoted azide-alkyne cycloaddn. (SPAAC), resp. Subsequently, both the effect of the immobilization site and the thermo-stability of immobilized lipases were also examd. The relative activities of the immobilized AzPhe-Lip243 and AzPhe-Lip274 were enhanced to 121.33% and 137.06%, resp., presenting 6.0 and 6.8 fold higher than those of the lipase traditionally immobilized using glutaraldehyde (IM-Lip-GA). In addn., all the immobilized lipases presented better specific activity except for AzPhe-Lip355, whose immobilization site was close to its active site. The rational immobilized lipases also presented better thermo-stability than those by traditionally immobilization method (glutaraldehyde). To sum up, with the aid of protein structure anal., unnatural amino acid can be rationally inserted into enzyme sequence to inform and direct the covalent enzyme immobilization. This method can be further developed for one-step enzyme purifn. and immobilization and applied to a broad scope of enzymes.
- 368Li, H.; Yin, Y.; Wang, A.; Li, N.; Wang, R.; Zhang, J.; Chen, X.; Pei, X.; Xie, T. Stable Immobilization of Aldehyde Ketone Reductase Mutants Containing Nonstandard Amino Acids on an Epoxy Resin via Strain-Promoted Alkyne-Azide Cycloaddition. RSC Adv. 2020, 10 (5), 2624– 2633, DOI: 10.1039/C9RA09067CGoogle Scholar368Stable immobilization of aldehyde ketone reductase mutants containing nonstandard amino acids on an epoxy resin via strain-promoted alkyne-azide cycloadditionLi, Huimin; Yin, Youcheng; Wang, Anming; Li, Ningning; Wang, Ru; Zhang, Jing; Chen, Xinxin; Pei, Xiaolin; Xie, TianRSC Advances (2020), 10 (5), 2624-2633CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)To avoid random chem. linkage and achieve precisely directed immobilization, mutant enzymes were obtained and immobilized using an incorporated reactive nonstandard amino acid (NSAA). For this purpose, aldehyde ketone reductase (AKR) was used as a model enzyme, and 110Y, 114Y, 143Y, 162Q and 189Q were each replaced with p-azido-L-phenylalanine (pAzF). Then, the mutant AKR was coupled to the functionalized support by strain-promoted alkyne-azide cycloaddn. (SPAAC). The effects of the incorporation no. and site of NSAAs on the loading and thermal stability of the immobilized AKR were examd. The results show that the mutant enzymes presented better specific activity than the wild type, except for AKR-110Y, and AKR-114Y showed 1.16-fold higher activity than the wild type. Moreover, the half-life (t1/2) of the five-point immobilized AKR reached 106 h and 45 h, 13 and 7 times higher than that of the free enzyme at 30 °C and 60 °C, resp. Comparison of these three types of enzymes shows that multi-point immobilization provides improved loading and thermal stability and facilitates one-step purifn. We expect this platform to facilitate a fundamental understanding of precisely oriented and controllable covalent immobilization and enable bio-manufg. paradigms for fine chems. and pharmaceuticals.
- 369Umeda, A.; Thibodeaux, G. N.; Zhu, J.; Lee, Y.; Zhang, Z. J. Site-specific Protein Cross-Linking with Genetically Incorporated 3,4-Dihydroxy-L-Phenylalanine. ChemBioChem 2009, 10 (8), 1302– 1304, DOI: 10.1002/cbic.200900127Google ScholarThere is no corresponding record for this reference.
- 370Deepankumar, K.; Nadarajan, S. P.; Mathew, S.; Lee, S.-G.; Yoo, T. H.; Hong, E. Y.; Kim, B.-G.; Yun, H. Engineering Transaminase for Stability Enhancement and Site-Specific Immobilization through Multiple Noncanonical Amino Acids Incorporation. ChemCatChem 2015, 7 (3), 417– 421, DOI: 10.1002/cctc.201402882Google Scholar370Engineering Transaminase for Stability Enhancement and Site-Specific Immobilization through Multiple Noncanonical Amino Acids IncorporationDeepankumar, Kanagavel; Nadarajan, Saravanan Prabhu; Mathew, Sam; Lee, Sun-Gu; Yoo, Tae Hyeon; Hong, Eun Young; Kim, Byung-Gee; Yun, HyungdonChemCatChem (2015), 7 (3), 417-421CODEN: CHEMK3; ISSN:1867-3880. (Wiley-VCH Verlag GmbH & Co. KGaA)In general, conventional enzyme engineering utilizes 20 canonical amino acids to alter and improve the functional properties of proteins such as stability, and activity. In this study, we utilized the noncanonical amino acid (NCAA) incorporation technique to enhance the functional properties of ω-transaminase (ω-TA). Herein, we enhanced the stability of ω-TA by residue-specific incorporation of (4R)-fluoroproline [(4R)-FP] and successfully immobilized onto chitosan or polystyrene (PS) beads with site-specifically incorporated L-3,4-dihydroxyphenylalanine (DOPA) moiety. The immobilization of ω-TAdopa and ω-TAdp[(4R)-FP] onto PS beads showed excellent reusability for 10 cycles in the kinetic resoln. of chiral amines. Compared to the ω-TAdopa, the ω-TAdp[(4R)-FP] immobilized onto PS beads exerted more stability that can serve as suitable biocatalyst for the asym. synthesis of chiral amines.
- 371Bednar, R. M.; Golbek, T. W.; Kean, K. M.; Brown, W. J.; Jana, S.; Baio, J. E.; Karplus, P. A.; Mehl, R. A. Immobilization of Proteins with Controlled Load and Orientation. ACS Appl. Mater. Interfaces 2019, 11 (40), 36391– 36398, DOI: 10.1021/acsami.9b12746Google Scholar371Immobilization of Proteins with Controlled Load and OrientationBednar, Riley M.; Golbek, Thaddeus W.; Kean, Kelsey M.; Brown, Wesley J.; Jana, Subhashis; Baio, Joe E.; Karplus, P. Andrew; Mehl, Ryan A.ACS Applied Materials & Interfaces (2019), 11 (40), 36391-36398CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)Biomaterials based on immobilized proteins are key elements of many biomedical and industrial technologies. However, applications are limited by an inability to precisely construct materials of high homogeneity and defined content. The authors present here a general "protein-limited immobilization" strategy by combining the rapid, bioorthogonal, and biocompatible properties of a tetrazine-strained-trans-cyclooctene reaction with genetic code expansion to site-specifically place the tetrazine into a protein. For the first time, the authors use this strategy to immobilize defined amts. of oriented proteins onto beads and flat surfaces in under five minutes at sub-micromolar concns. without compromising activity. This approach opens the door to generating and studying diverse protein-based biomaterials that are much more precisely defined and characterized, providing a greater ability to engineer properties across a wide range of applications.
- 372Blizzard, R. J.; Backus, D. R.; Brown, W.; Bazewicz, C. G.; Li, Y.; Mehl, R. A. Ideal Bioorthogonal Reactions Using A Site-Specifically Encoded Tetrazine Amino Acid. J. Am. Chem. Soc. 2015, 137 (32), 10044– 10047, DOI: 10.1021/jacs.5b03275Google Scholar372Ideal Bioorthogonal Reactions Using A Site-Specifically Encoded Tetrazine Amino AcidBlizzard, Robert J.; Backus, Dakota R.; Brown, Wes; Bazewicz, Christopher G.; Li, Yi; Mehl, Ryan A.Journal of the American Chemical Society (2015), 137 (32), 10044-10047CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Bioorthogonal reactions for labeling biomols. in live cells have been limited by slow reaction rates or low component selectivity and stability. Ideal bioorthogonal reactions with high reaction rates, high selectivity, and high stability would allow for stoichiometric labeling of biomols. in minutes and eliminate the need to wash out excess labeling reagent. Currently, no general method exists for controlled stoichiometric or substoichiometric labeling of proteins in live cells. To overcome this limitation, the authors developed a significantly improved tetrazine-contg. amino acid (Tet-v2.0, I) and genetically encoded I with an evolved aminoacyl-tRNA synthetase/tRNA(CUA) pair. The authors demonstrated in cellulo that protein contg. I reacts selectively with cyclopropane-fused trans-cyclooctene (sTCO) with a bimol. rate const. of 72,500 ± 1660 M-1 s-1 without reacting with other cellular components. This bioorthogonal ligation of I-protein reacts in cellulo with substoichiometric amts. of sTCO-label fast enough to remove the labeling reagent from media in minutes, thereby eliminating the need to wash out label. This ideal bioorthogonal reaction will enable the monitoring of a larger window of cellular processes in real time.
- 373Switzer, H. J.; Howard, C. A.; Halonski, J. F.; Peairs, E. M.; Smith, N.; Zamecnik, M. P.; Verma, S.; Young, D. D. Employing Non-Canonical Amino Acids Towards the Immobilization of a Hyperthermophilic Enzyme to Increase Protein Stability. RSC Adv. 2023, 13 (13), 8496– 8501, DOI: 10.1039/D3RA00392BGoogle ScholarThere is no corresponding record for this reference.
- 374Ray, S.; Chand, S.; Zhang, Y.; Nussbaum, S.; Rajeshwar, K.; Perera, R. Implications of Active Site Orientation in Myoglobin for Direct Electron Transfer and Electrocatalysis Based on Monolayer and Multilayer Covalent Immobilization on Gold Electrodes. Electrochim. Acta 2013, 99, 85– 93, DOI: 10.1016/j.electacta.2013.03.080Google Scholar374Implications of active site orientation in myoglobin for direct electron transfer and electrocatalysis based on monolayer and multilayer covalent immobilization on gold electrodesRay, Sriparna; Chand, Subhash; Zhang, Yanbo; Nussbaum, Sherry; Rajeshwar, Krishnan; Perera, RoshanElectrochimica Acta (2013), 99 (), 85-93CODEN: ELCAAV; ISSN:0013-4686. (Elsevier Ltd.)Cyclic voltammetry (CV) and at. force microscopy (AFM) were used to study the importance of active site orientation of an immobilized protein for direct electron transfer (DET) and electrocatalysis. While the bioconjugated wild-type myoglobin (WT Mb) was immobilized on the modified Au electrode surface in a random multilayered fashion, the Ser3 replaced with unnatural amino acid, 3-amino-L-tyrosine, (NH2Tyr) mutant Mb was immobilized via a Diels-Alder reaction specific to NH2Tyr residue to form a homogeneous monolayer. Electrochem. calcns. for the no. of surface exposed redox-active mols. on the electrode surface (Γ) and heterogeneous rate const. for DET were 1.29 × 10-10 mol cm-2; 2.3 s-1 for the WT Mb and 1.54 × 10-10 mol cm-2; 1.3 s-1 for the S3NH2Tyr Mb mutant, resp. Electro-catalytic conversion of thioanisole to sulfoxide products showed similar turnover frequencies (TOF) around 1.9 × 103 s-1 (with 87% conversion) for the WT Mb, and 1.5 × 103 s-1 for the mutant S3NH2Tyr Mb (with 81% conversion). Site-directed single monolayer immobilization affords almost the same no. of surface exposed Mb active sites as the random multilayer immobilization strategy, though the latter contains a greater no. of protein mols. on the electrode surface, as obsd. from the AFM data.
- 375Xia, L.; Han, M.-J.; Zhou, L.; Huang, A.; Yang, Z.; Wang, T.; Li, F.; Yu, L.; Tian, C.; Zang, Z. S-Click Reaction for Isotropic Orientation of Oxidases on Electrodes to Promote Electron Transfer at Low Potentials. Angew. Chem. Int. Ed. 2019, 58 (46), 16480– 16484, DOI: 10.1002/anie.201909203Google ScholarThere is no corresponding record for this reference.
- 376Pan, Y.; Li, G.; Liu, R.; Guo, J.; Liu, Y.; Liu, M.; Zhang, X.; Chi, L.; Xu, K.; Wu, R. Unnatural Activities and Mechanistic Insights of Cytochrome P450 PikC Gained from Site-Specific Mutagenesis by Non-Canonical Amino Acids. Nature Commun. 2023, 14 (1), 1669, DOI: 10.1038/s41467-023-37288-0Google ScholarThere is no corresponding record for this reference.
- 377Kolev, J. N.; Zaengle, J. M.; Ravikumar, R.; Fasan, R. Enhancing the Efficiency and Regioselectivity of P450 Oxidation Catalysts by Unnatural Amino Acid Mutagenesis. ChemBioChem 2014, 15 (7), 1001– 1010, DOI: 10.1002/cbic.201400060Google Scholar377Enhancing the Efficiency and Regioselectivity of P450 Oxidation Catalysts by Unnatural Amino Acid MutagenesisKolev, Joshua N.; Zaengle, Jacqueline M.; Ravikumar, Rajesh; Fasan, RudiChemBioChem (2014), 15 (7), 1001-1010CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)The development of effective strategies for modulating the reactivity and selectivity of cytochrome P 450 enzymes represents a key step toward expediting the use of these biocatalysts for synthetic applications. We have investigated the potential of unnatural amino acid mutagenesis to aid efforts in this direction. Four unnatural amino acids with diverse arom. side chains were incorporated at 11 active-site positions of a substrate-promiscuous CYP102A1 variant. The resulting "uP450s" were then tested for their catalytic activity and regioselectivity in the oxidn. of two representative substrates: a small-mol. drug and a natural product. Large shifts in regioselectivity resulted from these single mutations, and in particular, for para-acetyl-Phe substitutions at positions close to the heme cofactor. Screening this mini library of uP 450s enabled us to identify P 450 catalysts for the selective hydroxylation of four aliph. positions in the target substrates, including a C(Sp3)-H site not oxidized by the parent enzyme. Furthermore, we discovered a general activity-enhancing effect of active-site substitutions involving the unnatural amino acid para-amino-Phe, which resulted in P 450 catalysts capable of supporting the highest total turnover no. reported to date on a complex mol. (34 650). The functional changes induced by the unnatural amino acids could not be reproduced by any of the 20 natural amino acids. This study thus demonstrates that unnatural amino acid mutagenesis constitutes a promising new strategy for improving the catalytic activity and regioselectivity of P 450 oxidn. catalysts.
- 378Ma, H.; Yang, X.; Lu, Z.; Liu, N.; Chen, Y. The “Gate Keeper” Role of Trp222 Determines the Enantiopreference of Diketoreductase toward 2-Chloro-1-Phenylethanone. PLOS ONE 2014, 9 (7), e103792 DOI: 10.1371/journal.pone.0103792Google ScholarThere is no corresponding record for this reference.
- 379Yu, Z.; Yu, H.; Tang, H.; Wang, Z.; Wu, J.; Yang, L.; Xu, G. Site-specifically Incorporated Non-Canonical Amino Acids into Pseudomonas alcaligenes Lipase to Hydrolyze L-menthol Propionate among the Eight Isomers. ChemCatChem 2021, 13 (11), 2691– 2701, DOI: 10.1002/cctc.202100358Google Scholar379Site-specifically Incorporated Non-Canonical Amino Acids into Pseudomonas alcaligenes Lipase to Hydrolyze L-menthol Propionate among the Eight IsomersYu, Zhonglang; Yu, Haoran; Tang, Haibin; Wang, Zhe; Wu, Jianping; Yang, Lirong; Xu, GangChemCatChem (2021), 13 (11), 2691-2701CODEN: CHEMK3; ISSN:1867-3880. (Wiley-VCH Verlag GmbH & Co. KGaA)It remains a challenge to improve the diastereopreference of enzymes when there are multiple chiral centers in the substrate, mainly because the limited understanding of mechanism detg. diastereoselectivity. Compared with natural amino acids, non-canonical amino acids (ncAAs) provide side chains with wider range of functional groups and genetically encoded ncAAs have been applied in probing the complex enzyme mechanisms, improving catalytic activity, and even designing enzymes with new catalytic mechanisms. Here, the ncAAs were site-specifically incorporated into a lipase (PaL) produced by Pseudomonas alcaligenes to explore its diastereopreference mechanism. Menthol propionate has three chiral centers, eight isomers in total. Mol. dynamics (MD) simulations were first applied to analyze the interactions between the active sites of PaL and the target substrate L-menthol propionate. Furthermore, the four ncAAs (o-bromophenylalanine, o-chlorophenylalanine, p-cyanophenylalanine and p-aminophenylalanine) were substituted for 9 amino acids sites that potentially influenced three chiral centers and several variants with significant improvement in the diastereopreference were obtained. The diastereomer selectivity of beat variant at Ala253 was 100% higher than that of the wild-type. A linear relationship was found between vol., flexibility of the active center and diastereoselectivity.
- 380Drienovská, I.; Gajdoš, M.; Kindler, A.; Takhtehchian, M.; Darnhofer, B.; Birner-Gruenberger, R.; Dörr, M.; Bornscheuer, U. T.; Kourist, R. Folding Assessment of Incorporation of Noncanonical Amino Acids Facilitates Expansion of Functional-Group Diversity for Enzyme Engineering. Chem. Eur. J. 2020, 26 (54), 12338– 12342, DOI: 10.1002/chem.202002077Google ScholarThere is no corresponding record for this reference.
- 381Green, A. P.; Hayashi, T.; Mittl, P. R. E.; Hilvert, D. A Chemically Programmed Proximal Ligand Enhances the Catalytic Properties of a Heme Enzyme. J. Am. Chem. Soc. 2016, 138 (35), 11344– 11352, DOI: 10.1021/jacs.6b07029Google Scholar381A chemically programmed proximal ligand enhances the catalytic properties of a heme enzymeGreen, Anthony P.; Hayashi, Takahiro; Mittl, Peer R. E.; Hilvert, DonaldJournal of the American Chemical Society (2016), 138 (35), 11344-11352CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Enzymes rely on complex interactions between precisely positioned active site residues as a mechanism to compensate for the limited functionality contained within the genetic code. Heme enzymes provide a striking example of this complexity, whereby the electronic properties of reactive ferryl intermediates are finely tuned through H-bonding interactions between proximal ligands and neighboring amino acids. Here, the authors show that introduction of a chem. programmed proximal Nδ-methylhistidine (NMH) ligand into an engineered ascorbate peroxidase (APX2) overcomes the reliance on the conserved Asp-His H-bonding interaction, leading to a catalytically modified enzyme (APX2 NMH), which is able to achieve a significantly higher no. of turnovers compared with APX2 without compromising catalytic efficiency. Structural, spectroscopic, and kinetic characterization of APX2 NMH and several active site variants provided valuable insights into the role of the Asp-His-Fe triad of heme peroxidases. More significantly, simplification of catalytic mechanisms through the incorporation of chem. optimized ligands may facilitate efforts to create and evolve new active site heme environments within proteins.
- 382Sharp, K. H.; Mewies, M.; Moody, P. C. E.; Raven, E. L. Crystal Structure of the Ascorbate Peroxidase-Ascorbate Complex. Nat. Struct. Biol. 2003, 10 (4), 303– 307, DOI: 10.1038/nsb913Google ScholarThere is no corresponding record for this reference.
- 383Vojtechovský, J.; Chu, K.; Berendzen, J.; Sweet, R. M.; Schlichting, I. Crystal Structures of Myoglobin-Ligand Complexes at Near-Atomic Resolution. Biophys. J. 1999, 77 (4), 2153– 2174, DOI: 10.1016/S0006-3495(99)77056-6Google ScholarThere is no corresponding record for this reference.
- 384Pott, M.; Hayashi, T.; Mori, T.; Mittl, P. R. E.; Green, A. P.; Hilvert, D. A Noncanonical Proximal Heme Ligand Affords an Efficient Peroxidase in a Globin Fold. J. Am. Chem. Soc. 2018, 140 (4), 1535– 1543, DOI: 10.1021/jacs.7b12621Google Scholar384A Noncanonical Proximal Heme Ligand Affords an Efficient Peroxidase in a Globin FoldPott, Moritz; Hayashi, Takahiro; Mori, Takahiro; Mittl, Peer R. E.; Green, Anthony P.; Hilvert, DonaldJournal of the American Chemical Society (2018), 140 (4), 1535-1543CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Expanding the range of genetically encoded metal coordination environments accessible within tunable protein scaffolds presents excellent opportunities for the creation of metalloenzymes with augmented properties and novel activities. Here, we demonstrate that installation of a noncanonical Nδ-Me histidine (NMH) as the proximal heme ligand in the oxygen binding protein myoglobin (Mb) leads to substantial increases in heme redox potential and promiscuous peroxidase activity. Structural characterization of this catalytically modified myoglobin variant (Mb NMH) revealed significant changes in the proximal pocket, including alterations to hydrogen-bonding interactions involving the prosthetic porphyrin cofactor. Further optimization of Mb NMH via a combination of rational modification and several rounds of lab. evolution afforded efficient peroxidase biocatalysts within a globin fold, with activities comparable to those displayed by nature's peroxidases.
- 385Matsuo, T.; Fukumoto, K.; Watanabe, T.; Hayashi, T. Precise Design of Artificial Cofactors for Enhancing Peroxidase Activity of Myoglobin: Myoglobin Mutant H64D Reconstituted with a ″Single-Winged Cofactor″ Is Equivalent to Native Horseradish Peroxidase in Oxidation Activity. Chem. Asian J. 2011, 6 (9), 2491– 2499, DOI: 10.1002/asia.201100107Google ScholarThere is no corresponding record for this reference.
- 386Hayashi, T.; Tinzl, M.; Mori, T.; Krengel, U.; Proppe, J.; Soetbeer, J.; Klose, D.; Jeschke, G.; Reiher, M.; Hilvert, D. Capture and Characterization of a Reactive Haem-Carbenoid Complex in an Artificial Metalloenzyme. Nat. Catal. 2018, 1 (8), 578– 584, DOI: 10.1038/s41929-018-0105-6Google ScholarThere is no corresponding record for this reference.
- 387Pott, M.; Tinzl, M.; Hayashi, T.; Ota, Y.; Dunkelmann, D. L.; Mittl, P. R. E.; Hilvert, D. Noncanonical Heme Ligands Steer Carbene Transfer Reactivity in an Artificial Metalloenzyme. Angew. Chem. Int. Ed 2021, 60, 15063– 15068, DOI: 10.1002/anie.202103437Google Scholar387Noncanonical Heme Ligands Steer Carbene Transfer Reactivity in an Artificial MetalloenzymePott, Moritz; Tinzl, Matthias; Hayashi, Takahiro; Ota, Yusuke; Dunkelmann, Daniel; Mittl, Peer R. E.; Hilvert, DonaldAngewandte Chemie, International Edition (2021), 60 (27), 15063-15068CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Changing the primary metal coordination sphere is a powerful strategy for tuning metalloprotein properties. Here we used amber stop codon suppression with engineered pyrrolysyl-tRNA synthetases, including two newly evolved enzymes, to replace the proximal histidine in myoglobin with Nδ-methylhistidine, 5-thiazoylalanine, 4-thiazoylalanine and 3-(3-thienyl)alanine. In addn. to tuning the heme redox potential over a >200 mV range, these noncanonical ligands modulate the protein's carbene transfer activity with Et diazoacetate. Variants with increased redn. potential proved superior for cyclopropanation and N-H insertion, whereas variants with reduced Eo values gave higher S-H insertion activity. Given the functional importance of histidine in many enzymes, these genetically encoded analogs could be valuable tools for probing mechanism and enabling new chemistries.
- 388Carminati, D. M.; Fasan, R. Stereoselective Cyclopropanation of Electron-Deficient Olefins with a Cofactor Redesigned Carbene Transferase Featuring Radical Reactivity. ACS Catal. 2019, 9 (10), 9683– 9697, DOI: 10.1021/acscatal.9b02272Google Scholar388Stereoselective Cyclopropanation of Electron-Deficient Olefins with a Cofactor Redesigned Carbene Transferase Featuring Radical ReactivityCarminati, Daniela M.; Fasan, RudiACS Catalysis (2019), 9 (10), 9683-9697CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Engineered myoglobins and other hemoproteins have recently emerged as promising catalysts for asym. olefin cyclopropanation reactions via carbene-transfer chem. Despite this progress, the transformation of electron-poor alkenes has proven to be very challenging using these systems. Here, we describe the design of a myoglobin-based carbene transferase incorporating a non-native iron-porphyrin cofactor and axial ligand, as an efficient catalyst for the asym. cyclopropanation of electron-deficient alkenes. Using this metalloenzyme, a broad range of both electron-rich and electron-deficient alkenes are cyclopropanated with high efficiency and high diastereo- and enantioselectivity (up to >99% de and ee). Mechanistic studies revealed that the expanded reaction scope of this carbene transferase is dependent upon the acquisition of metallocarbene radical reactivity as a result of the reconfigured coordination environment around the metal center. The radical-based reactivity of this system diverges from the electrophilic reactivity of myoglobin and most of the known organometallic carbene-transfer catalysts. This work showcases the value of cofactor redesign toward tuning and expanding the reactivity of metalloproteins in abiol. reactions, and it provides a biocatalytic soln. to the asym. cyclopropanation of electron-deficient alkenes. The metallocarbene radical reactivity exhibited by this biocatalyst is anticipated to prove useful in the context of a variety of other synthetic transformations.
- 389Moore, E. J.; Fasan, R. Effect of Proximal Ligand Substitutions on the Carbene and Nitrene Transferase Activity of Myoglobin. Tetrahedron 2019, 75 (16), 2357– 2363, DOI: 10.1016/j.tet.2019.03.009Google Scholar389Effect of proximal ligand substitutions on the carbene and nitrene transferase activity of myoglobinMoore, Eric J.; Fasan, RudiTetrahedron (2019), 75 (16), 2357-2363CODEN: TETRAB; ISSN:0040-4020. (Elsevier Ltd.)Engineered myoglobins (Mbs) were recently shown to be effective catalysts for abiol. carbene and nitrene transfer reactions. Here, we investigated the impact of substituting the conserved heme-coordinating histidine residue with both proteinogenic (Cys, Ser, Tyr, Asp) and non-proteinogenic Lewis basic amino acids (3-(3'-pyridyl)-alanine, p-aminophenylalanine, and β-(3-thienyl)-alanine), on the reactivity of this metalloprotein toward these abiotic transformations. These studies showed that mutation of the proximal histidine residue with both natural and non-natural amino acids result in stable myoglobin variants that can function as both carbene and nitrene transferases. In addn., substitution of the proximal histidine with an aspartate residue led to a myoglobin-based catalyst capable of promoting stereoselective olefin cyclopropanation under nonreducing conditions. Overall, these studies demonstrate that proximal ligand substitution provides a promising strategy to tune the reactivity of myoglobin-based carbene and nitrene transfer catalysts and provide a first, proof-of-principle demonstration of the viability of pyridine-, thiophene-, and aniline-based unnatural amino acids for metalloprotein engineering.
- 390Gan, F.; Liu, R.; Wang, F.; Schultz, P. G. Functional Replacement of Histidine in Proteins To Generate Noncanonical Amino Acid Dependent Organisms. J. Am. Chem. Soc. 2018, 140 (11), 3829– 3832, DOI: 10.1021/jacs.7b13452Google Scholar390Functional replacement of histidine in proteins to generate noncanonical amino acid dependent organismsGan, Fei; Liu, Renhe; Wang, Feng; Schultz, Peter G.Journal of the American Chemical Society (2018), 140 (11), 3829-3832CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Simple strategies to produce organisms whose growth is strictly dependent on the presence of a noncanonical amino acid are useful for the generation of live vaccines and the biol. containment of recombinant organisms. To this end, we report an approach based on genetically replacing key histidine (His) residues in essential proteins with functional His analogs. We demonstrate that 3-methyl-L-histidine (MeH) functionally substitutes for a key metal binding ligand, H264, in the zinc-contg. metalloenzyme mannose-6-phosphate isomerase (ManA). An evolved variant, Opt5, harboring both N262S and H264MeH substitutions exhibited comparable activities to wild type ManA. An engineered Escherichia coli strain contg. the ManA variant Opt5 was strictly dependent on MeH for growth with an extremely low reversion rate. This straightforward strategy should be applicable to other metallo- or nonmetalloproteins that contain essential His residues.
- 391Chand, S.; Ray, S.; Yadav, P.; Samanta, S.; Pierce, B. S.; Perera, R. Abiological Catalysis by Myoglobin Mutant with a Genetically Incorporated Unnatural Amino Acid. Biochem. J. 2021, 478 (9), 1795– 1808, DOI: 10.1042/BCJ20210091Google Scholar391Abiological catalysis by myoglobin mutant with a genetically incorporated unnatural amino acidChand, Subhash; Ray, Sriparna; Yadav, Poonam; Samanta, Susruta; Pierce, Brad S.; Perera, RoshanBiochemical Journal (2021), 478 (9), 1795-1808CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)To inculcate biocatalytic activity in the oxygen-storage protein myoglobin (Mb), a genetically engineered myoglobin mutant H64DOPA (DOPA = L-3,4-dihydroxyphenylalanine) has been created. Incorporation of unnatural amino acids has already demonstrated their ability to accomplish many non-natural functions in proteins efficiently. Herein, the presence of redox-active DOPA residue in the active site of mutant Mb presumably stabilizes the compd. I in the catalytic oxidn. process by participating in an addnl. hydrogen bonding (H-bonding) as compared to the WT Mb. Specifically, a general acid-base catalytic pathway was achieved due to the availability of the hydroxyl moieties of DOPA. The redn. potential values of WT (E° = -260 mV) and mutant Mb (E° = -300 mV), w.r.t. Ag/AgCl ref. electrode, in the presence of hydrogen peroxide, indicated an addnl. H-bonding in the mutant protein, which is responsible for the peroxidase activity of the mutant Mb. We obsd. that in the presence of 5 mM H2O2, H64DOPA Mb oxidizes thioanisole and benzaldehyde with a 10 and 54 folds higher rate, resp., as opposed to WT Mb. Based on spectroscopic, kinetic, and electrochem. studies, we deduce that DOPA residue, when present within the distal pocket of mutant Mb, alone serves the role of His/Arg-pair of peroxidases.
- 392Jackson, J. C.; Duffy, S. P.; Hess, K. R.; Mehl, R. A. Improving Nature’s Enzyme Active Site with Genetically Encoded Unnatural Amino Acids. J. Am. Chem. Soc. 2006, 128 (34), 11124– 11127, DOI: 10.1021/ja061099yGoogle Scholar392Improving Nature's enzyme active site with genetically encoded unnatural amino acidsJackson, Jennifer C.; Duffy, Sean P.; Hess, Kenneth R.; Mehl, Ryan A.Journal of the American Chemical Society (2006), 128 (34), 11124-11127CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The ability to site-specifically incorporate a diverse set of unnatural amino acids (>30) into proteins and quickly add new structures of interest has recently changed our approach to protein use and study. One important question yet unaddressed with unnatural amino acids (UAAs) is whether they can improve the activity of an enzyme beyond that available from the natural 20 amino acids. Herein, we report the >30-fold improvement of prodrug activator nitroreductase activity with an UAA over that of the native active site and a >2.3-fold improvement over the best possible natural amino acid. Because immense structural and electrostatic diversity at a single location can be sampled very quickly, UAAs can be implemented to improve enzyme active sites and tune a site to multiple substrates.
- 393Grove, J. I.; Lovering, A. L.; Guise, C.; Race, P. R.; Wrighton, C. J.; White, S. A.; Hyde, E. I.; Searle, P. F. Generation of Escherichia coli Nitroreductase Mutants Conferring Improved Cell Sensitization to the Prodrug CB19541. Cancer Res. 2003, 63 (17), 5532– 5537Google ScholarThere is no corresponding record for this reference.
- 394Ugwumba, I. N.; Ozawa, K.; Xu, Z.-Q.; Ely, F.; Foo, J.-L.; Herlt, A. J.; Coppin, C.; Brown, S.; Taylor, M. C.; Ollis, D. L. Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino Acids. J. Am. Chem. Soc. 2011, 133 (2), 326– 333, DOI: 10.1021/ja106416gGoogle Scholar394Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino AcidsUgwumba, Isaac N.; Ozawa, Kiyoshi; Xu, Zhi-Qiang; Ely, Fernanda; Foo, Jee-Loon; Herlt, Anthony J.; Coppin, Chris; Brown, Sue; Taylor, Matthew C.; Ollis, David L.; Mander, Lewis N.; Schenk, Gerhard; Dixon, Nicholas E.; Otting, Gottfried; Oakeshott, John G.; Jackson, Colin J.Journal of the American Chemical Society (2011), 133 (2), 326-333CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and to probe the role of peripheral active site residues in nonchem. steps of the catalytic cycle (substrate binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with unnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino acids, as well as leucine, phenylalanine, and tryptophan. Kinetic anal. suggests that the 7-hydroxyl group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product release step of substrate turnover as a result of its electrostatic repulsion of the neg. charged 4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to the difficulty in improving this native activity through screening hundreds of thousands of mutants with natural amino acids. These results demonstrate that designer amino acids provide easy access to new and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.
- 395Pagar, A. D.; Jeon, H.; Khobragade, T. P.; Sarak, S.; Giri, P.; Lim, S.; Yoo, T. H.; Ko, B. J.; Yun, H. Non-Canonical Amino Acid-Based Engineering of (R)-Amine Transaminase. Front. Chem. 2022, DOI: 10.3389/fchem.2022.839636Google ScholarThere is no corresponding record for this reference.
- 396Savile, C. K.; Janey, J. M.; Mundorff, E. C.; Moore, J. C.; Tam, S.; Jarvis, W. R.; Colbeck, J. C.; Krebber, A.; Fleitz, F. J.; Brands, J. Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin Manufacture. Science 2010, 329 (5989), 305– 309, DOI: 10.1126/science.1188934Google Scholar396Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin ManufactureSavile, Christopher K.; Janey, Jacob M.; Mundorff, Emily C.; Moore, Jeffrey C.; Tam, Sarena; Jarvis, William R.; Colbeck, Jeffrey C.; Krebber, Anke; Fleitz, Fred J.; Brands, Jos; Devine, Paul N.; Huisman, Gjalt W.; Hughes, Gregory J.Science (Washington, DC, United States) (2010), 329 (5989), 305-309CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Pharmaceutical synthesis can benefit greatly from the selectivity gains assocd. with enzymic catalysis. Here, we report an efficient biocatalytic process to replace a recently implemented rhodium-catalyzed asym. enamine hydrogenation for the large-scale manuf. of the antidiabetic compd. sitagliptin. Starting from an enzyme that had the catalytic machinery to perform the desired chem. but lacked any activity toward the prositagliptin ketone, we applied a substrate walking, modeling, and mutation approach to create a transaminase with marginal activity for the synthesis of the chiral amine; this variant was then further engineered via directed evolution for practical application in a manufg. setting. The resultant biocatalysts showed broad applicability toward the synthesis of chiral amines that previously were accessible only via resoln. This work underscores the maturation of biocatalysis to enable efficient, economical, and environmentally benign processes for the manuf. of pharmaceuticals.
- 397Wilkinson, H. C.; Dalby, P. A. Fine-Tuning the Activity and Stability of an Evolved Enzyme Active-Site Through Noncanonical Amino-Acids. FEBS J. 2021, 288 (6), 1935– 1955, DOI: 10.1111/febs.15560Google ScholarThere is no corresponding record for this reference.
- 398Parsons, J. F.; Xiao, G.; Gilliland, G. L.; Armstrong, R. N. Enzymes Harboring Unnatural Amino Acids: Mechanistic and Structural Analysis of the Enhanced Catalytic Activity of a Glutathione Transferase Containing 5-Fluorotryptophan. Biochemistry 1998, 37 (18), 6286– 6294, DOI: 10.1021/bi980219eGoogle Scholar398Enzymes Harboring Unnatural Amino Acids: Mechanistic and Structural Analysis of the Enhanced Catalytic Activity of a Glutathione Transferase Containing 5-FluorotryptophanParsons, James F.; Xiao, Gaoyi; Gilliland, Gary L.; Armstrong, Richard N.Biochemistry (1998), 37 (18), 6286-6294CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The catalytic characteristics and structure of the M1-1 isoenzyme of rat glutathione (GSH) transferase in which all four tryptophan residues in each monomer are replaced with 5-fluorotryptophan are described. The fluorine-for-hydrogen substitution does not change the interaction of the enzyme with GSH even though two tryptophan residues (Trp7 and Trp45) are involved in direct hydrogen-bonding interactions with the substrate. The rate consts. for assocn. and dissocn. of the peptide, measured by stopped-flow spectrometry, remain unchanged by the unnatural amino acid. The 5-FTrp-substituted enzyme exhibits a kcat of 73 s-1 as compared to 18 s-1 for the native enzyme toward 1-chloro-2,4-dinitrobenzene. That the increase in the turnover no. is due to an enhanced rate of product release in the mutant is confirmed by the kinetics of the approach to equil. for binding of the product. The crystal structure of the 5-FTrp-contg. enzyme was solved at a resoln. of 2.0 Å by difference Fourier techniques. The structure reveals local conformational changes in the structural elements that define the approach to the active site which are attributed to steric interactions of the fluorine atoms assocd. with 5-FTrp146 and 5-FTrp214 in domain II. These changes appear to result in the enhanced rate of product release. This structure represents the first of a protein substituted with 5-fluorotryptophan.
- 399Dominguez, M. A., Jr; Thornton, K. C.; Melendez, M. G.; Dupureur, C. M. Differential Effects of Isomeric Incorporation of Fluorophenylalanines into PvuII Endonuclease. Proteins 2001, 45 (1), 55– 61, DOI: 10.1002/prot.1123Google ScholarThere is no corresponding record for this reference.
- 400Hoesl, M. G.; Budisa, N. Expanding and Engineering the Genetic Code in a Single Expression Experiment. ChemBioChem 2011, 12, 552– 555, DOI: 10.1002/cbic.201000586Google Scholar400Expanding and Engineering the Genetic Code in a Single Expression ExperimentHoesl, Michael G.; Budisa, NediljkoChemBioChem (2011), 12 (4), 552-555CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)The concept of expanded and engineered genetic code was exptl. verified by simultaneous insertions of p-benzoyl-phenylalanine at UAG stop codons together with the global replacements Met→norleucine in TTL or Pro→cis-4-fluoroproline in EGFP model proteins. In this way, residue-specific, sense codon reassignment ("code engineering") was combined with position-specific stop-codon suppression ("code expansion") in a single in vivo expression expt.
- 401Cirino, P. C.; Tang, Y.; Takahashi, K.; Tirrell, D. A.; Arnold, F. H. Global Incorporation of Norleucine in Place of Methionine in Cytochrome P450 BM-3 Heme Domain Increases Peroxygenase Activity. Biotechnol. Bioeng. 2003, 83 (6), 729– 734, DOI: 10.1002/bit.10718Google ScholarThere is no corresponding record for this reference.
- 402Xiao, H.; Nasertorabi, F.; Choi, S.-h.; Han, G. W.; Reed, S. A.; Stevens, R. C.; Schultz, P. G. Exploring the Potential Impact of an Expanded Genetic Code on Protein Function. Proc. Natl. Acad. Sci. U.S.A. 2015, 112 (22), 6961– 6966, DOI: 10.1073/pnas.1507741112Google ScholarThere is no corresponding record for this reference.
- 403Young, T. S.; Ahmad, I.; Yin, J. A.; Schultz, P. G. An Enhanced System for Unnatural Amino Acid Mutagenesis in E. coli. J. Mol. Biol. 2010, 395, 361– 374, DOI: 10.1016/j.jmb.2009.10.030Google Scholar403An enhanced system for unnatural amino acid mutagenesis in E. coliYoung, Travis S.; Ahmad, Insha; Yin, Jun A.; Schultz, Peter G.Journal of Molecular Biology (2010), 395 (2), 361-374CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)We report a new vector, pEVOL, for the incorporation of unnatural amino acids into proteins in Escherichia coli using evolved Methanocaldococcus jannaschii aminoacyl-tRNA synthetase(s) (aaRS)/suppressor tRNA pairs. This new system affords higher yields of mutant proteins through the use of both constitutive and inducible promoters to drive the transcription of two copies of the M. jannaschii aaRS gene. Yields were further increased by coupling the dual-aaRS promoter system with a newly optimized suppressor tRNACUA opt in a single-vector construct. The optimized suppressor tRNACUA opt afforded increased plasmid stability compared with previously reported vectors for unnatural amino acid mutagenesis. To demonstrate the utility of this new system, we introduced 14 mutant aaRS into pEVOL and compared their ability to insert unnatural amino acids in response to three independent amber nonsense codons in sperm whale myoglobin or green fluorescent protein. When cultured in rich media in shake flasks, pEVOL was capable of producing more than 100 mg/L mutant GroEL protein. The versatility, increased yields, and increased stability of the pEVOL vector will further facilitate the expression of proteins with unnatural amino acids.
- 404Schoffelen, S.; Beekwilder, J.; Debets, M. F.; Bosch, D.; Hest, J. C. M. v. Construction of a Multifunctional Enzyme Complex via the Strain-Promoted Azide-Alkyne Cycloaddition. Bioconjug. Chem. 2013, 24 (6), 987– 996, DOI: 10.1021/bc400021jGoogle Scholar404Construction of a Multifunctional Enzyme Complex via the Strain-Promoted Azide-Alkyne CycloadditionSchoffelen, Sanne; Beekwilder, Jules; Debets, Marjoke F.; Bosch, Dirk; Hest, Jan C. M. vanBioconjugate Chemistry (2013), 24 (6), 987-996CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)Inspired by the multienzyme complexes occurring in nature, enzymes have been brought together in vitro as well. We report a co-localization strategy milder than nonspecific crosslinking, and free of any scaffold and affinity tags. Using non-natural amino acid incorporation, two heterobifunctional linkers, and the strain-promoted azide-alkyne cycloaddn. as conjugation reaction, three metabolic enzymes are linked together in a controlled manner. Conjugate formation was demonstrated by size-exclusion chromatog. and gel electrophoresis. The multienzyme complexes were further characterized by native mass spectrometry. It was shown that the complexes catalyzed the three-step biosynthesis of piceid in vitro with comparable kinetic behavior to the uncoupled enzymes. The approach is envisioned to have high potential for various biotechnol. applications, in which multiple biocatalysts collaborate at low concns., in which diffusion may be limited and/or side-reactions are prone to occur.
- 405Lim, S. I.; Cho, J.; Kwon, I. Double Clicking for Site-Specific Coupling of Multiple Enzymes. Chem. Commun. 2015, 51 (71), 13607– 13610, DOI: 10.1039/C5CC04611DGoogle ScholarThere is no corresponding record for this reference.
- 406Lim, S. I.; Yang, B.; Jung, Y.; Cha, J.; Cho, J.; Choi, E.-S.; Kim, Y. H.; Kwon, I. Controlled Orientation of Active Sites in a Nanostructured Multienzyme Complex. Sci. Rep. 2016, 6 (1), 39587, DOI: 10.1038/srep39587Google Scholar406Controlled Orientation of Active Sites in a Nanostructured Multienzyme ComplexLim, Sung In; Yang, Byungseop; Jung, Younghan; Cha, Jaehyun; Cho, Jinhwan; Choi, Eun-Sil; Kim, Yong Hwan; Kwon, InchanScientific Reports (2016), 6 (), 39587CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)Multistep cascade reactions in nature maximize reaction efficiency by co-assembling related enzymes. Such organization facilitates the processing of intermediates by downstream enzymes. Previously, the studies on multienzyme nanocomplexes assembled on DNA scaffolds demonstrated that closer interenzyme distance enhances the overall reaction efficiency. However, it remains unknown how the active site orientation controlled at nanoscale can have an effect on multienzyme reaction. Here, we show that controlled alignment of active sites promotes the multienzyme reaction efficiency. By genetic incorporation of a non-natural amino acid and two compatible bioorthogonal chemistries, we conjugated mannitol dehydrogenase to formate dehydrogenase with the defined active site arrangement with the residue-level accuracy. The study revealed that the multienzyme complex with the active sites directed towards each other exhibits four-fold higher relative efficiency enhancement in the cascade reaction and produces 60% more D-mannitol than the other complex with active sites directed away from each other.
- 407Ha, J. M.; Jeon, S. T.; Yoon, H. J.; Lee, H. H. Formate Dehydrogenase. PDB 2014, DOI: 10.2210/pdb3WR5/pdbGoogle ScholarThere is no corresponding record for this reference.
- 408Kavanagh, K. L.; Klimacek, M.; Nidetzky, B.; Wilson, D. K. Crystal Structure of Pseudomonas fluorescens Mannitol 2-Dehydrogenase Binary and Ternary Complexes: Specificity And Catalytic Mechanism. J. Biol. Chem. 2002, 277 (45), 43433– 43442, DOI: 10.1074/jbc.M206914200Google ScholarThere is no corresponding record for this reference.
- 409Li, J.; Jia, S.; Chen, P. R. Diels-Alder Reaction-Triggered Bioorthogonal Protein Decaging in Living Cells. Nat. Chem. Biol. 2014, 10 (12), 1003– 1005, DOI: 10.1038/nchembio.1656Google Scholar409Diels-Alder reaction-triggered bioorthogonal protein decaging in living cellsLi, Jie; Jia, Shang; Chen, Peng R.Nature Chemical Biology (2014), 10 (12), 1003-1005CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Small mols. that specifically activate an intracellular protein of interest are highly desirable. A generally applicable strategy, however, remains elusive. Herein we describe a small mol.-triggered bioorthogonal protein decaging technique that relies on the inverse electron-demand Diels-Alder reaction for eliminating a chem. caged protein side chain within living cells. This method permits the efficient activation of a given protein (for example, an enzyme) in its native cellular context within minutes.
- 410Li, J.; Yu, J.; Zhao, J.; Wang, J.; Zheng, S.; Lin, S.; Chen, L.; Yang, M.; Jia, S.; Zhang, X. Palladium-Triggered Deprotection Chemistry for Protein Activation in Living Cells. Nat. Chem. 2014, 6 (4), 352– 361, DOI: 10.1038/nchem.1887Google Scholar410Palladium-triggered deprotection chemistry for protein activation in living cellsLi, Jie; Yu, Juntao; Zhao, Jingyi; Wang, Jie; Zheng, Siqi; Lin, Shixian; Chen, Long; Yang, Maiyun; Jia, Shang; Zhang, Xiaoyu; Chen, Peng R.Nature Chemistry (2014), 6 (4), 352-361CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)Employing small mols. or chem. reagents to modulate the function of an intracellular protein, particularly in a gain-of-function fashion, remains a challenge. In contrast to inhibitor-based loss-of-function approaches, methods based on a gain of function enable specific signalling pathways to be activated inside a cell. Here we report a chem. rescue strategy that uses a palladium-mediated deprotection reaction to activate a protein within living cells. We identify biocompatible and efficient palladium catalysts that cleave the propargyl carbamate group of a protected lysine analog to generate a free lysine. The lysine analog can be genetically and site-specifically incorporated into a protein, which enables control over the reaction site. This deprotection strategy is shown to work with a range of different cell lines and proteins. We further applied this biocompatible protection group/catalyst pair for caging and subsequent release of a crucial lysine residue in a bacterial Type III effector protein within host cells, which reveals details of its virulence mechanism.
- 411Wang, J.; Zheng, S.; Liu, Y.; Zhang, Z.; Lin, Z.; Li, J.; Zhang, G.; Wang, X.; Li, J.; Chen, P. R. Palladium-Triggered Chemical Rescue of Intracellular Proteins via Genetically Encoded Allene-Caged Tyrosine. J. Am. Chem. Soc. 2016, 138 (46), 15118– 15121, DOI: 10.1021/jacs.6b08933Google Scholar411Palladium-Triggered Chemical Rescue of Intracellular Proteins via Genetically Encoded Allene-Caged TyrosineWang, Jie; Zheng, Siqi; Liu, Yanjun; Zhang, Zhaoyue; Lin, Zhi; Li, Jiaofeng; Zhang, Gong; Wang, Xin; Li, Jie; Chen, Peng R.Journal of the American Chemical Society (2016), 138 (46), 15118-15121CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Chem. de-caging has emerged as an attractive strategy for gain-of-function study of proteins via small-mol. reagents. The previously reported chem. de-caging reactions have been largely centered on liberating the side chain of lysine on a given protein. Herein, the authors developed an allene-based caging moiety and the corresponding palladium de-caging reagents for chem. rescue of tyrosine (Tyr) activity on intracellular proteins. This bioorthogonal de-caging pair has been successfully applied to unmask enzymic Tyr sites (e.g., Y671 on Taq polymerase and Y728 on Anthrax lethal factor) as well as the posttranslational Tyr modification site (Y416 on Src kinase) in vitro and in living cells. The strategy provides a general platform for chem. rescue of Tyr-dependent protein activity inside cells.
- 412Georgianna, W. E.; Lusic, H.; McIver, A. L.; Deiters, A. Photocleavable Polyethylene Glycol for the Light-Regulation of Protein Function. Bioconjug. Chem. 2010, 21 (8), 1404– 1407, DOI: 10.1021/bc100084nGoogle Scholar412Photocleavable Polyethylene Glycol for the Light-Regulation of Protein FunctionGeorgianna, Wesleigh E.; Lusic, Hrvoje; McIver, Andrew L.; Deiters, AlexanderBioconjugate Chemistry (2010), 21 (8), 1404-1407CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)PEGylation is commonly employed to enhance the pharmacokinetic properties of proteins, but it can interfere with natural protein function. Protein activity can thus be abrogated through PEGylation, and a controllable means to remove the polyethylene glycol (PEG) group from the protein is desirable. As such, light affords a unique control over biomols. through the application of photosensitive groups. Herein, the authors report the synthesis of a photocleavable PEG reagent (PhotoPEG) and its application to the light-regulation of enzyme activity.
- 413Wu, N.; Deiters, A.; Cropp, T. A.; King, D.; Schultz, P. G. A Genetically Encoded Photocaged Amino Acid. J. Am. Chem. Soc. 2004, 126 (44), 14306– 14307, DOI: 10.1021/ja040175zGoogle Scholar413A Genetically Encoded Photocaged Amino AcidWu, Ning; Deiters, Alexander; Cropp, T. Ashton; King, David; Schultz, Peter G.Journal of the American Chemical Society (2004), 126 (44), 14306-14307CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We have developed a second orthogonal tRNA/synthetase pair for use in yeast based on the Escherichia coli tRNALeu/leucyl tRNA-synthetase pair. Using a novel genetic selection, we have identified a series of synthetase mutants that selectively charge the amber suppressor tRNA with the α-aminocaprylic acid, O-methyltyrosine and o-nitrobenzyl cysteine (photocaged amino acid) allowing them to be incorporated into proteins in yeast in response to the amber nonsense codon, TAG. Biosynthesis and photoactivation of photocaged cysteine-contg. superoxide dismutase and caspase-3 is demonstrated.
- 414Deiters, A.; Groff, D.; Ryu, Y.; Xie, J.; Schultz, P. G. A Genetically Encoded Photocaged Tyrosine. Angew. Chem. Int. Ed. 2006, 45 (17), 2728– 2731, DOI: 10.1002/anie.200600264Google Scholar414A genetically encoded photocaged tyrosineDeiters, Alexander; Groff, Dan; Ryu, Youngha; Xie, Jianming; Schultz, Peter G.Angewandte Chemie, International Edition (2006), 45 (17), 2728-2731CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A photocaged tyrosine was genetically encoded in Escherichia coli in response to the amber codon TAG. Substitution of Tyr503 in the active site of β-galactosidase allowed photoactivation of this enzyme in vitro or directly in bacteria with 360-nm light. This method should allow photoregulation of the activity of a variety of biol. processes including transcription, signal transduction, and cellular trafficking.
- 415Chou, C.; Young, D. D.; Deiters, A. A Light-Activated DNA Polymerase. Angew. Chem. Int. Ed. 2009, 48 (32), 5950– 5953, DOI: 10.1002/anie.200901115Google ScholarThere is no corresponding record for this reference.
- 416Chou, C.; Young, D. D.; Deiters, A. Photocaged T7 RNA Polymerase for the Light Activation of Transcription and Gene Function in Pro- and Eukaryotic Cells. ChemBioChem 2010, 11 (7), 972– 977, DOI: 10.1002/cbic.201000041Google Scholar416Photocaged T7 RNA Polymerase for the Light Activation of Transcription and Gene Function in Pro- and Eukaryotic CellsChou, Chungjung; Young, Douglas D.; Deiters, AlexanderChemBioChem (2010), 11 (7), 972-977CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)A light-activatable bacteriophage T7 RNA polymerase (T7RNAP) has been generated through the site-specific introduction of a photocaged tyrosine residue at the crucial position Tyr639 within the active site of the enzyme. The photocaged tyrosine disrupts polymerase activity by blocking the incoming nucleotide from reaching the active site of the enzyme. However, a brief irradn. with nonphototoxic UV light of 365 nm removes the ortho-nitrobenzyl caging group from Tyr639 and restores the RNA polymerase activity of T7RNAP. The complete orthogonality of T7RNAP to all endogenous RNA polymerases in pro- and eukaryotic systems allowed for the photochem. activation of gene expression in bacterial and mammalian cells. Specifically, E. coli cells were engineered to produce photocaged T7RNAP in the presence of a GFP reporter gene under the control of a T7 promoter. UV irradn. of these cells led to the spatiotemporal activation of GFP expression. In an analogous fashion, caged T7RNAP was transfected into human embryonic kidney (HEK293T) cells. Irradn. with UV light led to the activation of T7RNAP, thereby inducing RNA polymn. and expression of a luciferase reporter gene in tissue culture. The ability to achieve spatiotemporal regulation of orthogonal RNA synthesis enables the precise dissection and manipulation of a wide range of cellular events, including gene function.
- 417Chou, C.; Deiters, A. Light-Activated Gene Editing with a Photocaged Zinc-Finger Nuclease. Angew. Chem. Int. Ed. 2011, 50 (30), 6839, DOI: 10.1002/anie.201101157Google ScholarThere is no corresponding record for this reference.
- 418Gautier, A.; Nguyen, D. P.; Lusic, H.; An, W.; Deiters, A.; Chin, J. W. Genetically Encoded Photocontrol of Protein Localization in Mammalian Cells. J. Am. Chem. Soc. 2010, 132 (12), 4086– 4088, DOI: 10.1021/ja910688sGoogle Scholar418Genetically Encoded Photocontrol of Protein Localization in Mammalian CellsGautier, Arnaud; Nguyen, Duy P.; Lusic, Hrvoje; An, Wenlin; Deiters, Alexander; Chin, Jason W.Journal of the American Chemical Society (2010), 132 (12), 4086-4088CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Precise photochem. control of protein function can be achieved through the site-specific introduction of caging groups. Chem. and enzymic methods, including in vitro translation and chem. ligation, have been used to photocage proteins in vitro. These methods have been extended to allow the introduction of caged proteins into cells by permeabilization or microinjection, but cellular delivery remains challenging. Since lysine residues are key determinants for nuclear localization sequences, the target of key post-translational modifications (including ubiquitination, methylation, and acetylation), and key residues in many important enzyme active sites, we were interested in photocaging lysine to control protein localization, post-translational modification, and enzymic activity. Photochem. control of these important functions mediated by lysine residues in proteins has not previously been demonstrated in living cells. Here we synthesized 1 and evolved a pyrrolysyl-tRNA synthetase/tRNA pair to genetically encode the incorporation of this amino acid in response to an amber codon in mammalian cells. To exemplify the utility of this amino acid, we caged the nuclear localization sequences (NLSs) of nucleoplasmin and the tumor suppressor p53 in human cells, thus mislocalizing the proteins in the cytosol. We triggered protein nuclear import with a pulse of light, allowing us to directly quantify the kinetics of nuclear import.
- 419Gautier, A.; Deiters, A.; Chin, J. W. Light-Activated Kinases Enable Temporal Dissection of Signaling Networks in Living Cells. J. Am. Chem. Soc. 2011, 133 (7), 2124– 2127, DOI: 10.1021/ja1109979Google Scholar419Light-activated kinases enable temporal dissection of signaling networks in living cellsGautier, Arnaud; Deiters, Alexander; Chin, Jason W.Journal of the American Chemical Society (2011), 133 (7), 2124-2127CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We report a general strategy for creating protein kinases in mammalian cells that are poised for very rapid activation by light. By photoactivating a caged version of MEK1, we demonstrate the specific, rapid, and receptor independent activation of an artificial subnetwork within the Raf/MEK/ERK pathway. Time-lapse microscopy allowed us to precisely characterize the kinetics of elementary steps in the signaling cascade and provided insight into adaptive feedback and rate-detg. processes in the pathway.
- 420Hemphill, J.; Chou, C.; Chin, J. W.; Deiters, A. Genetically Encoded Light-Activated Transcription for Spatiotemporal Control of Gene Expression and Gene Silencing in Mammalian Cells. J. Am. Chem. Soc. 2013, 135 (36), 13433– 13439, DOI: 10.1021/ja4051026Google Scholar420Genetically encoded light-activated transcription for spatiotemporal control of gene expression and gene silencing in mammalian cellsHemphill, James; Chou, Chungjung; Chin, Jason W.; Deiters, AlexanderJournal of the American Chemical Society (2013), 135 (36), 13433-13439CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Photocaging provides a method to spatially and temporally control biol. function and gene expression with high resoln. Proteins can be photochem. controlled through the site-specific installation of caging groups on amino acid side chains that are essential for protein function. The photocaging of a synthetic gene network using unnatural amino acid mutagenesis in mammalian cells was demonstrated with an engineered bacteriophage RNA polymerase. A caged T7 RNA polymerase was expressed in cells with an expanded genetic code and used in the photochem. activation of genes under control of an orthogonal T7 promoter, demonstrating tight spatial and temporal control. The synthetic gene expression system was validated with two reporter genes (luciferase and EGFP) and applied to the light-triggered transcription of short hairpin RNA constructs for the induction of RNA interference.
- 421Hemphill, J.; Borchardt, E. K.; Brown, K.; Asokan, A.; Deiters, A. Optical Control of CRISPR/Cas9 Gene Editing. J. Am. Chem. Soc. 2015, 137 (17), 5642– 5645, DOI: 10.1021/ja512664vGoogle Scholar421Optical control of CRISPR/Cas9 gene editingHemphill, James; Borchardt, Erin K.; Brown, Kalyn; Asokan, Aravind; Deiters, AlexanderJournal of the American Chemical Society (2015), 137 (17), 5642-5645CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The CRISPR/Cas9 system has emerged as an important tool in biomedical research for a wide range of applications, with significant potential for genome engineering and gene therapy. In order to achieve conditional control of the CRISPR/Cas9 system, a genetically encoded light-activated Cas9 was engineered through the site-specific installation of a caged lysine amino acid. Several potential lysine residues were identified as viable caging sites that can be modified to optically control Cas9 function, as demonstrated through optical activation and deactivation of both exogenous and endogenous gene function.
- 422Nguyen, D. P.; Mahesh, M.; Elsässer, S. J.; Hancock, S. M.; Uttamapinant, C.; Chin, J. W. Genetic Encoding of Photocaged Cysteine Allows Photoactivation of TEV Protease in Live Mammalian Cells. J. Am. Chem. Soc. 2014, 136 (6), 2240– 2243, DOI: 10.1021/ja412191mGoogle Scholar422Genetic Encoding of Photocaged Cysteine Allows Photoactivation of TEV Protease in Live Mammalian CellsNguyen, Duy P.; Mahesh, Mohan; Elsasser, Simon J.; Hancock, Susan M.; Uttamapinant, Chayasith; Chin, Jason W.Journal of the American Chemical Society (2014), 136 (6), 2240-2243CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The authors demonstrate the evolution of the PylRS/tRNACUA pair for genetically encoding photocaged cysteine (I). By characterizing the incorporation in Escherichia coli and mammalian cells, and the photodeprotection process in vitro and in mammalian cells, the authors establish conditions for rapid efficient photodeprotection to reveal native proteins in live cells. They demonstrate the utility of this approach by rapidly activating TEV protease following illumination of single cells.
- 423Uprety, R.; Luo, J.; Liu, J.; Naro, Y.; Samanta, S.; Deiters, A. Genetic Encoding of Caged Cysteine and Caged Homocysteine in Bacterial and Mammalian Cells. ChemBioChem 2014, 15 (12), 1793– 1799, DOI: 10.1002/cbic.201400073Google ScholarThere is no corresponding record for this reference.
- 424Yang, X.; Zhao, L.; Wang, Y.; Ji, Y.; Su, X. C.; Ma, J. A.; Xuan, W. Constructing Photoactivatable Protein with Genetically Encoded Photocaged Glutamic Acid. Angew. Chem. Int. Ed. 2023, 135 (40), e202308472 DOI: 10.1002/ange.202308472Google ScholarThere is no corresponding record for this reference.
- 425Givens, R. S.; Weber, J. F.; Jung, A. H.; Park, C.-H. New Photoprotecting Groups: Desyl and p-Hydroxyphenacyl Phosphate and Carboxylate Esters. In Methods in enzymology; Elsevier, 1998; Vol. 291, pp 1– 29. DOI: 10.1016/s0076-6879(98)91004-7 .Google ScholarThere is no corresponding record for this reference.
- 426Mangubat-Medina, A. E.; Ball, Z. T. Triggering Biological Processes: Methods and Applications of Photocaged Peptides and Proteins. Chem. Soc. Rev. 2021, 50 (18), 10403– 10421, DOI: 10.1039/D0CS01434FGoogle Scholar426Triggering biological processes: methods and applications of photocaged peptides and proteinsMangubat-Medina, Alicia E.; Ball, Zachary T.Chemical Society Reviews (2021), 50 (18), 10403-10421CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. There has been a significant push in recent years to deploy fundamental knowledge and methods of photochem. toward biol. ends. Photoreactive groups have enabled chemists to activate biol. function using the concept of photocaging. By granting spatiotemporal control over protein activation, these photocaging methods are fundamental in understanding biol. processes. Peptides and proteins are an important group of photocaging targets that present conceptual and tech. challenges, requiring precise chemoselectivity in complex polyfunctional environments. This review focuses on recent advances in photocaging techniques and methodologies, as well as their use in living systems. Photocaging methods include genetic and chem. approaches that require a deep understanding of structure-function relationships based on subtle changes in primary structure. Successful implementation of these ideas can shed light on important spatiotemporal aspects of living systems.
- 427Grasso, K. T.; Singha Roy, S. J.; Osgood, A. O.; Yeo, M. J. R.; Soni, C.; Hillenbrand, C. M.; Ficaretta, E. D.; Chatterjee, A. A Facile Platform to Engineer Escherichia coli Tyrosyl-tRNA Synthetase Adds New Chemistries to the Eukaryotic Genetic Code, Including a Phosphotyrosine Mimic. ACS Cent. Sci. 2022, 8 (4), 483– 492, DOI: 10.1021/acscentsci.1c01465Google ScholarThere is no corresponding record for this reference.
- 428Edwards, W. F.; Young, D. D.; Deiters, A. Light-Activated Cre Recombinase as a Tool for the Spatial and Temporal Control of Gene Function in Mammalian Cells. ACS Chem. Biol. 2009, 4 (6), 441– 445, DOI: 10.1021/cb900041sGoogle Scholar428Light-activated Cre recombinase as a tool for the spatial and temporal control of gene function in mammalian cellsEdwards, Wesleigh F.; Young, Douglas D.; Deiters, AlexanderACS Chemical Biology (2009), 4 (6), 441-445CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Cre recombinase catalyzes DNA exchange between two conserved lox recognition sites. The enzyme has extensive biol. application, from basic cloning to engineering knock-out and knock-in organisms. Widespread use of Cre is due to its simplicity and effectiveness, but the enzyme and the recombination event remain difficult to control with high precision. To obtain such control we report the installation of a light-responsive o-nitrobenzyl caging group directly in the catalytic site of Cre, inhibiting its activity. Prior to irradn., caged Cre is completely inactive, as demonstrated both in vitro and in mammalian cell culture. Exposure to non-damaging UVA light removes the caging group and restores recombinase activity. Tight spatio-temporal control over DNA recombination is thereby achieved.
- 429Le Provost, F.; Lillico, S.; Passet, B.; Young, R.; Whitelaw, B.; Vilotte, J.-L. Zinc Finger Nuclease Technology Heralds a New Era in Mammalian Transgenesis. Trends Biotechnol. 2010, 28 (3), 134– 141, DOI: 10.1016/j.tibtech.2009.11.007Google ScholarThere is no corresponding record for this reference.
- 430Porteus, M. H. Mammalian Gene Targeting with Designed Zinc Finger Nucleases. Molecular Therapy 2006, 13 (2), 438– 446, DOI: 10.1016/j.ymthe.2005.08.003Google ScholarThere is no corresponding record for this reference.
- 431Porteus, M. H.; Carroll, D. Gene Targeting Using Zinc Finger Nucleases. Nat. Biotechnol. 2005, 23 (8), 967– 973, DOI: 10.1038/nbt1125Google ScholarThere is no corresponding record for this reference.
- 432Urnov, F. D.; Rebar, E. J.; Holmes, M. C.; Zhang, H. S.; Gregory, P. D. Genome Editing with Engineered Zinc Finger Nucleases. Nat. Rev. Genet. 2010, 11 (9), 636– 646, DOI: 10.1038/nrg2842Google Scholar432Genome editing with engineered zinc finger nucleasesUrnov, Fyodor D.; Rebar, Edward J.; Holmes, Michael C.; Zhang, H. Steve; Gregory, Philip D.Nature Reviews Genetics (2010), 11 (9), 636-646CODEN: NRGAAM; ISSN:1471-0056. (Nature Publishing Group)A review. Zinc finger nucleases (ZFNs) are versatile tools for making precise modifications to genomes, and their use is now established in a range of model systems. ZFNs are also showing potential in human gene therapy, and several clin. trials are underway. Reverse genetics in model organisms such as Drosophila melanogaster, Arabidopsis thaliana, zebrafish and rats, efficient genome engineering in human embryonic stem and induced pluripotent stem cells, targeted integration in crop plants, and HIV resistance in immune cells - this broad range of outcomes has resulted from the application of the same core technol.: targeted genome cleavage by engineered, sequence-specific zinc finger nucleases followed by gene modification during subsequent repair. Such 'genome editing' is now established in human cells and a no. of model organisms, thus opening the door to a range of new exptl. and therapeutic possibilities.
- 433Rémy, S.; Tesson, L.; Ménoret, S.; Usal, C.; Scharenberg, A. M.; Anegon, I. Zinc-Finger Nucleases: A Powerful Tool for Genetic Engineering of Animals. Transgenic Res. 2010, 19, 363– 371, DOI: 10.1007/s11248-009-9323-7Google ScholarThere is no corresponding record for this reference.
- 434Li, Y.; Korolev, S.; Waksman, G. Crystal Structures of Open and Closed Forms of Binary and Ternary Complexes of the Large Fragment of Thermus aquaticus DNA Polymerase I: Structural Basis for Nucleotide Incorporation. EMBO J. 1998, 17 (24), 7514– 7525, DOI: 10.1093/emboj/17.24.7514Google Scholar434Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporationLi, Ying; Korolev, Sergey; Waksman, GabrielEMBO Journal (1998), 17 (24), 7514-7525CODEN: EMJODG; ISSN:0261-4189. (Oxford University Press)The crystal structures of two ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I (Klentaq1) with a primer/template DNA and dideoxycytidine triphosphate, and that of a binary complex of the same enzyme with a primer/template DNA, were detd. to a resoln. of 2.3, 2.3 and 2.5 Å, resp. One ternary complex structure differs markedly from the two other structures by a large reorientation of the tip of the fingers domain. This structure, designated 'closed', represents the ternary polymerase complex caught in the act of incorporating a nucleotide. In the two other structures, the tip of the fingers domain is rotated outward by 46° ('open') in an orientation similar to that of the apo form of Klentaq1. These structures provide the first direct evidence in DNA polymerase I enzymes of a large conformational change responsible for assembling an active ternary complex.
- 435Suzuki, M.; Baskin, D.; Hood, L.; Loeb, L. A. Random Mutagenesis of Thermus aquaticus DNA Polymerase I: Concordance of Immutable Sites in vivo with the Crystal Structure. Proc. Natl. Acad. Sci. U.S.A. 1996, 93 (18), 9670– 9675, DOI: 10.1073/pnas.93.18.9670Google ScholarThere is no corresponding record for this reference.
- 436Paul, N.; Shum, J.; Le, T. Hot Start PCR. RT-PCR Protocols, Second ed.; 2010; pp 301– 318. DOI: 10.1007/978-1-60761-629-0Google ScholarThere is no corresponding record for this reference.
- 437Cramer, P. Common Structural Features of Nucleic Acid Polymerases. Bioessays 2002, 24 (8), 724– 729, DOI: 10.1002/bies.10127Google ScholarThere is no corresponding record for this reference.
- 438Banghart, M. R.; Mourot, A.; Fortin, D. L.; Yao, J. Z.; Kramer, R. H.; Trauner, D. Photochromic Blockers of Voltage-Gated Potassium Channels. Angew. Chem. Int. Ed. 2009, 48 (48), 9097– 9101, DOI: 10.1002/anie.200904504Google Scholar438Photochromic Blockers of Voltage-Gated Potassium ChannelsBanghart, Matthew R.; Mourot, Alexandre; Fortin, Doris L.; Yao, Jennifer Z.; Kramer, Richard H.; Trauner, DirkAngewandte Chemie, International Edition (2009), 48 (48), 9097-9101, S9097/1-S9097/14CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Photochromic blockers of voltage-gated potassium channels were prepd., with potential to be used as tools in neurobiol. and possibly in therapy of vision disorders.
- 439Beharry, A. A.; Wong, L.; Tropepe, V.; Woolley, G. A. Fluorescence Imaging of Azobenzene Photoswitching in vivo. Angew. Chem. Int. Ed. 2011, 50 (6), 1325– 1327, DOI: 10.1002/anie.201006506Google ScholarThere is no corresponding record for this reference.
- 440Bléger, D.; Schwarz, J.; Brouwer, A. M.; Hecht, S. o-Fluoroazobenzenes as Readily Synthesized Photoswitches Offering Nearly Quantitative Two-Way Isomerization with Visible Light. J. Am. Chem. Soc. 2012, 134 (51), 20597– 20600, DOI: 10.1021/ja310323yGoogle Scholar440o-Fluoroazobenzenes as Readily Synthesized Photoswitches Offering Nearly Quantitative Two-Way Isomerization with Visible LightBleger, David; Schwarz, Jutta; Brouwer, Albert M.; Hecht, StefanJournal of the American Chemical Society (2012), 134 (51), 20597-20600CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Azobenzene functionalized with ortho-fluorine atoms has a lower energy of the n-orbital of the Z-isomer, resulting in a sepn. of the E and Z isomers' n→π* absorption bands. Introducing para-substituents allows for further tuning of the absorption spectra of o-fluoroazobenzenes. In particular, electron-withdrawing ester groups give rise to a 50 nm sepn. of the n→π* transitions. Green and blue light can therefore be used to induce E→Z and Z→E isomerizations, resp. The o-fluoroazobenzene scaffold is readily synthesized and can be inserted into larger structures via its aryl termini. These new azobenzene derivs. can be switched in both ways with high photoconversions, and their Z-isomers display a remarkably long thermal half-life.
- 441Bonardi, F.; London, G.; Nouwen, N.; Feringa, B. L.; Driessen, A. J. Light-Induced Control of Protein Translocation by the SecYEG Complex. Angew. Chem. Int. Ed. 2010, 49 (40), 7234– 7238, DOI: 10.1002/anie.201002243Google Scholar441Light-induced control of protein translocation by the secYEG complexBonardi, Francesco; London, Gabor; Nouwen, Nico; Feringa, Ben L.; Driessen, Arnold J. M.Angewandte Chemie, International Edition (2010), 49 (40), 7234-7238, S7234/1-S7234/13CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)An organochem. photoswitch was introduced into two transmembrane segments that comprise the lateral gate of the bacterial-membrane-embedded protein-conducting pore. Reversible switching of the azobenzene between the trans and cis configurations by irradn. with visible and UV light enforced the opening and closure of the protein-conducting pore.
- 442Gorostiza, P.; Isacoff, E. Y. Nanoengineering Ion Channels for Optical Control. Physiology 2008, 23 (5), 238– 247, DOI: 10.1152/physiol.00018.2008Google Scholar442Nanoengineering ion channels for optical controlGorostiza Pau; Isacoff Ehud YPhysiology (Bethesda, Md.) (2008), 23 (), 238-47 ISSN:1548-9213.Chemical modification with photoisomerizable tethered ligands endows proteins with sensitivity to light. These optically actuated proteins are revolutionizing research in biology by making it possible to manipulate biological processes noninvasively and with unprecedented spatiotemporal resolution.
- 443Knie, C.; Utecht, M.; Zhao, F.; Kulla, H.; Kovalenko, S.; Brouwer, A. M.; Saalfrank, P.; Hecht, S.; Bléger, D. ortho-Fluoroazobenzenes: Visible Light Switches with Very Long-Lived Z Isomers. Chem. Eur. J. 2014, 20 (50), 16492– 16501, DOI: 10.1002/chem.201404649Google ScholarThere is no corresponding record for this reference.
- 444Liang, X.; Mochizuki, T.; Asanuma, H. A Supra-Photoswitch Involving Sandwiched DNA Base Pairs and Azobenzenes for Light-Driven Nanostructures and Nanodevices. Small 2009, 5 (15), 1761– 1768, DOI: 10.1002/smll.200900223Google Scholar444A Supra-photoswitch Involving Sandwiched DNA Base Pairs and Azobenzenes for Light-Driven Nanostructures and NanodevicesLiang, Xingguo; Mochizuki, Toshio; Asanuma, HiroyukiSmall (2009), 5 (15), 1761-1768CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)A supra-photoswitch is designed for complete ON/OFF switching of DNA hybridization by light irradn. for the purpose of using DNA as a material for building nanostructures. Azobenzenes, attached to D-threoninols that function as scaffolds, are introduced into each DNA strand after every two natural nucleotides (in the form (NNX)n where N and X represent the natural nucleotide and the azobenzene moiety, resp.). Hybridization of these two modified strands forms a supra-photoswitch consisting of alternating natural base pairs and azobenzene moieties. In this newly designed sequence, each base pair is sandwiched between two azobenzene moieties and all the azobenzene moieties are sepd. by base pairs. When the duplex is irradiated by visible light, the azobenzene moieties take the trans form and this duplex is surprisingly stable compared to the corresponding native duplex composed of only natural oligonucleotides. On the other hand, when the azobenzene moieties are isomerized to the cis form by UV light irradn., the duplex is completely dissocd. Based on this design, a DNA hairpin structure is synthesized that should be closed by visible light irradn. and opened by UV light irradn. at the level of a single mol. Indeed, perfect ON/OFF photoregulation is attained. This is a promising strategy for the design of supra-photoswitches such as photoresponsive sticky ends on DNA nanodevices and other nanostructures.
- 445Samanta, S.; Beharry, A. A.; Sadovski, O.; McCormick, T. M.; Babalhavaeji, A.; Tropepe, V.; Woolley, G. A. Photoswitching Azo Compounds in vivo with Red Light. J. Am. Chem. Soc. 2013, 135 (26), 9777– 9784, DOI: 10.1021/ja402220tGoogle Scholar445Photoswitching Azo Compounds in Vivo with Red LightSamanta, Subhas; Beharry, Andrew A.; Sadovski, Oleg; McCormick, Theresa M.; Babalhavaeji, Amirhossein; Tropepe, Vince; Woolley, G. AndrewJournal of the American Chemical Society (2013), 135 (26), 9777-9784CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The photoisomerization of azobenzenes provides a general means for the photocontrol of mol. structure and function. For applications in vivo, however, the wavelength of irradn. required for trans-to-cis isomerization of azobenzenes is crit. since UV and most visible wavelengths are strongly scattered by cells and tissues. We report here that azobenzene compds. in which all four positions ortho to the azo group are substituted with bulky electron-rich substituents can be effectively isomerized with red light (630-660 nm), a wavelength range that is orders of magnitude more penetrating through tissue than other parts of the visible spectrum. When the ortho substituent is chloro, the compds. also exhibit stability to redn. by glutathione, enabling their use in intracellular environments in vivo.
- 446Schierling, B.; Noel, A.-J.; Wende, W.; Hien, L. T.; Volkov, E.; Kubareva, E.; Oretskaya, T.; Kokkinidis, M.; Rompp, A.; Spengler, B.; Pingoud, A. Controlling the Enzymatic Activity of a Restriction Enzyme by Light. Proc. Natl. Acad. Sci. U.S.A. 2010, 107 (4), 1361– 1366, DOI: 10.1073/pnas.0909444107Google ScholarThere is no corresponding record for this reference.
- 447Zhang, F.; Zarrine-Afsar, A.; Al-Abdul-Wahid, M. S.; Prosser, R. S.; Davidson, A. R.; Woolley, G. A. Structure-Based Approach to the Photocontrol of Protein Folding. J. Am. Chem. Soc. 2009, 131 (6), 2283– 2289, DOI: 10.1021/ja807938vGoogle Scholar447Structure-Based Approach to the Photocontrol of Protein FoldingZhang, Fuzhong; Zarrine-Afsar, Arash; Al-Abdul-Wahid, M. Sameer; Prosser, R. Scott; Davidson, Alan R.; Woolley, G. AndrewJournal of the American Chemical Society (2009), 131 (6), 2283-2289CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Photoswitchable proteins offer exciting prospects for remote control of biochem. processes. We propose a general approach to the design of photoswitchable proteins based on the introduction of a photoswitchable intramol. cross-linker. We chose, as a model, a FynSH3 domain for which the free energy of folding is less than the energy available from photoisomerization of the cross-linker. Taking the exptl. detd. structure of the folded protein as a starting point, mutations were made to introduce pairs of Cys residues so that the distance between Cys sulfur atoms matches the ideal length of the cis form, but not the trans form, of the cross-linker. When the trans cross-linker was introduced into this L3C-L29C-T47AFynSH3 mutant, the protein was destabilized so that folded and unfolded forms coexisted. Irradn. of the cross-linker to produce the cis isomer recovered the folded, active state of the protein. This work shows that structure-based introduction of switchable cross-linkers is a feasible approach for photocontrol of folding/unfolding of globular proteins.
- 448Bose, M.; Groff, D.; Xie, J.; Brustad, E.; Schultz, P. G. The Incorporation of a Photoisomerizable Amino Acid into Proteins in E. c oli. J. Am. Chem. Soc. 2006, 128 (2), 388– 389, DOI: 10.1021/ja055467uGoogle Scholar448The incorporation of a photoisomerizable amino acid into proteins in E. coliBose, Mohua; Groff, Dan; Xie, Jianming; Brustad, Eric; Schultz, Peter G.Journal of the American Chemical Society (2006), 128 (2), 388-389CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)An orthogonal aminoacyl tRNA synthetase/tRNA pair has been evolved that allows the incorporation of the photoisomerizable amino acid phenylalanine-4'-azobenzene (AzoPhe) into proteins in Escherichia coli in response to the amber nonsense codon. Further, we show that AzoPhe can be used to photoregulate the binding affinity of catabolite activator protein to its promoter. The ability to selectively incorporate AzoPhe into proteins at defined sites should make it possible to regulate a variety of biol. processes with light, including enzyme, receptor, and ion channel activity.
- 449Hoppmann, C.; Lacey, V. K.; Louie, G. V.; Wei, J.; Noel, J. P.; Wang, L. Genetically Encoding Photoswitchable Click Amino Acids in Escherichia coli and Mammalian Cells. Angew. Chem. Int. Ed. 2014, 53 (15), 3932– 3936, DOI: 10.1002/anie.201400001Google Scholar449Genetically Encoding Photoswitchable Click Amino Acids in Escherichia coli and Mammalian CellsHoppmann, Christian; Lacey, Vanessa K.; Louie, Gordon V.; Wei, Jing; Noel, Joseph P.; Wang, LeiAngewandte Chemie, International Edition (2014), 53 (15), 3932-3936CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The ability to reversibly control protein structure and function with light would offer high spatiotemporal resoln. for investigating biol. processes. To confer photoresponsiveness on general proteins, we genetically incorporated a set of photoswitchable click amino acids (PSCaas), which contain both a reversible photoswitch and an addnl. click functional group for further modifications. Orthogonal tRNA-synthetases were evolved to genetically encode PSCaas bearing azobenzene with an alkene, keto, or benzyl chloride group in E. coli and in mammalian cells. After incorporation into calmodulin, the benzyl chloride PSCaa spontaneously generated a covalent protein bridge by reacting with a nearby cysteine residue through proximity-enabled bioreactivity. The resultant azobenzene bridge isomerized in response to light, thereby changing the conformation of calmodulin. These genetically encodable PSCaas will prove valuable for engineering photoswitchable bridges into proteins for reversible optogenetic regulation.
- 450Hoppmann, C.; Maslennikov, I.; Choe, S.; Wang, L. In situ Formation of an Azo Bridge on Proteins Controllable by Visible Light. J. Am. Chem. Soc. 2015, 137 (35), 11218– 11221, DOI: 10.1021/jacs.5b06234Google Scholar450In Situ Formation of an Azo Bridge on Proteins Controllable by Visible LightHoppmann, Christian; Maslennikov, Innokentiy; Choe, Senyon; Wang, LeiJournal of the American Chemical Society (2015), 137 (35), 11218-11221CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Optical modulation of proteins provides superior spatiotemporal resoln. for understanding biol. processes, and photoswitches built on light-sensitive proteins have been significantly advancing neuronal and cellular studies. Small mol. photoswitches could complement protein-based switches by mitigating potential interference and affording high specificity for modulation sites. However, genetic encodability and responsiveness to nonultraviolet light, two desired properties possessed by protein photoswitches, are challenging to be engineered into small mol. photoswitches. Here the authors developed a small mol. photoswitch that can be genetically installed onto proteins in situ and controlled by visible light. A pentafluoro azobenzene-based photoswitchable click amino acid (F-PSCaa) was designed to isomerize in response to visible light. After genetic incorporation into proteins via the expansion of the genetic code, F-PSCaa reacts with a nearby cysteine within the protein generating an azo bridge in situ. The resultant bridge is switchable by visible light and allows conformation and binding of CaM to be regulated by such light. This photoswitch should prove valuable in optobiol. for its minimal interference, site flexibility, genetic encodability, and response to the more biocompatible visible light.
- 451John, A. A.; Ramil, C. P.; Tian, Y.; Cheng, G.; Lin, Q. Synthesis and Site-Specific Incorporation of Red-Shifted Azobenzene Amino Acids into Proteins. Org. Lett. 2015, 17 (24), 6258– 6261, DOI: 10.1021/acs.orglett.5b03268Google Scholar451Synthesis and Site-Specific Incorporation of Red-Shifted Azobenzene Amino Acids into ProteinsJohn, Alford A.; Ramil, Carlo P.; Tian, Yulin; Cheng, Gang; Lin, QingOrganic Letters (2015), 17 (24), 6258-6261CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)A series of red-shifted azobenzene amino acids were synthesized in moderate-to-excellent yields via a two-step procedure in which tyrosine derivs. were first oxidized to the corresponding quinonoidal spirolactones followed by ceric ammonium nitrate-catalyzed azo formation with the substituted phenylhydrazines. The resulting azobenzene-alanine derivs. exhibited efficient trans/cis photoswitching upon irradn. with a blue (448 nm) or green (530 nm) LED light. Moreover, nine superfolder green fluorescent protein (sfGFP) mutants carrying the azobenzene-alanine analogs were expressed in E. coli in good yields via amber codon suppression with an orthogonal tRNA/PylRS pair, and one of the mutants showed durable photoswitching with the LED light.
- 452Klippenstein, V.; Hoppmann, C.; Ye, S.; Wang, L.; Paoletti, P. Optocontrol of Glutamate Receptor Activity by Single Side-Chain Photoisomerization. Elife 2017, 6, e25808 DOI: 10.7554/eLife.25808Google Scholar452Optocontrol of glutamate receptor activity by single side-chain photoisomerizationKlippenstein, Viktoria; Hoppmann, Christian; Ye, Shixin; Wang, Lei; Paoletti, PierreeLife (2017), 6 (), e25808/1-e25808/29CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)Engineering light-sensitivity into proteins has wide ranging applications in mol. studies and neuroscience. Commonly used tethered photoswitchable ligands, however, require solvent-accessible protein labeling, face structural constrains, and are bulky. Here, we designed a set of optocontrollable NMDA receptors by directly incorporating single photoswitchable amino acids (PSAAs) providing genetic encodability, reversibility, and site tolerance. We identified several positions within the multi-domain receptor endowing robust photomodulation. PSAA photoisomerization at the GluN1 clamshell hinge is sufficient to control glycine sensitivity and activation efficacy. Strikingly, in the pore domain, flipping of a M3 residue within a conserved transmembrane cavity impacts both gating and permeation properties. Our study demonstrates the first detection of mol. rearrangements in real-time due to the reversible light-switching of single amino acid side-chains, adding a dynamic dimension to protein site-directed mutagenesis. This novel approach to interrogate neuronal protein function has general applicability in the fast expanding field of optopharmacol.
- 453Kneuttinger, A. C.; Straub, K.; Bittner, P.; Simeth, N. A.; Bruckmann, A.; Busch, F.; Rajendran, C.; Hupfeld, E.; Wysocki, V. H.; Horinek, D. Light Regulation of Enzyme Allostery through Photo-responsive Unnatural Amino Acids. Cell Chem. Biol. 2019, 26 (11), 1501– 1514, DOI: 10.1016/j.chembiol.2019.08.006Google Scholar453Light Regulation of Enzyme Allostery through Photo-responsive Unnatural Amino AcidsKneuttinger, Andrea C.; Straub, Kristina; Bittner, Philipp; Simeth, Nadja A.; Bruckmann, Astrid; Busch, Florian; Rajendran, Chitra; Hupfeld, Enrico; Wysocki, Vicki H.; Horinek, Dominik; Koenig, Burkhard; Merkl, Rainer; Sterner, ReinhardCell Chemical Biology (2019), 26 (11), 1501-1514.e9CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)Imidazole glycerol phosphate synthase (ImGPS) is an allosteric bienzyme complex in which substrate binding to the synthase subunit HisF stimulates the glutaminase subunit HisH. To control this stimulation with light, we have incorporated the photo-responsive unnatural amino acids phenylalanine-4'-azobenzene (AzoF), o-nitropiperonyl-O-tyrosine (NPY), and methyl-o-nitropiperonyllysine (mNPK) at strategic positions of HisF. The light-mediated isomerization of AzoF at position 55 (fS55AzoFE ↔ fS55AzoFZ) resulted in a reversible 10-fold regulation of HisH activity. The light-mediated decaging of NPY at position 39 (fY39NPY → fY39) and of mNPK at position 99 (fK99mNPK → fK99) led to a 4- to 6-fold increase of HisH activity. Mol. dynamics simulations explained how the unnatural amino acids interfere with the allosteric machinery of ImGPS and revealed addnl. aspects of HisH stimulation in wild-type ImGPS. Our findings show that unnatural amino acids can be used as a powerful tool for the spatiotemporal control of a central metabolic enzyme complex by light.
- 454Luo, J.; Samanta, S.; Convertino, M.; Dokholyan, N. V.; Deiters, A. Reversible and Tunable Photoswitching of Protein Function through Genetic Encoding of Azobenzene Amino Acids in Mammalian Cells. ChemBioChem 2018, 19 (20), 2178– 2185, DOI: 10.1002/cbic.201800226Google Scholar454Reversible and Tunable Photoswitching of Protein Function through Genetic Encoding of Azobenzene Amino Acids in Mammalian CellsLuo, Ji; Samanta, Subhas; Convertino, Marino; Dokholyan, Nikolay V.; Deiters, AlexanderChemBioChem (2018), 19 (20), 2178-2185CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)The genetic encoding of three different azobenzene phenylalanines with different photochem. properties was achieved in human cells by using an engineered pyrrolysyl tRNA/tRNA synthetase pair. In order to demonstrate reversible light control of protein function, azobenzenes were site-specifically introduced into firefly luciferase. Computational strategies were applied to guide the selection of potential photoswitchable sites that lead to a reversibly controlled luciferase enzyme. In addn., the new azobenzene analogs provide enhanced thermal stability, high photoconversion, and responsiveness to visible light. These small-mol. photoswitches can reversibly photocontrol protein function with excellent spatiotemporal resoln., and preferred sites for incorporation can be computationally detd., thus providing a new tool for investigating biol. processes.
- 455Kneuttinger, A. C.; Winter, M.; Simeth, N. A.; Heyn, K.; Merkl, R.; König, B.; Sterner, R. Artificial Light Regulation of an Allosteric Bienzyme Complex by a Photosensitive Ligand. ChemBioChem 2018, 19 (16), 1750– 1757, DOI: 10.1002/cbic.201800219Google Scholar455Artificial Light Regulation of an Allosteric Bienzyme Complex by a Photosensitive LigandKneuttinger, Andrea C.; Winter, Martin; Simeth, Nadja A.; Heyn, Kristina; Merkl, Rainer; Koenig, Burkhard; Sterner, ReinhardChemBioChem (2018), 19 (16), 1750-1757CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)The artificial regulation of proteins by light is an emerging subdiscipline of synthetic biol. Here, we used this concept to photocontrol both catalysis and allostery within the heterodimeric enzyme complex imidazole glycerol phosphate synthase (ImGP-S). ImGP-S consists of the cyclase subunit HisF and the glutaminase subunit HisH, which is allosterically stimulated by substrate binding to HisF. We show that a light-sensitive diarylethene (1,2-dithienylethene, DTE)-based competitive inhibitor in its ring-open state binds with low micromolar affinity to the cyclase subunit and displaces its substrate from the active site. As a consequence, catalysis by HisF and allosteric stimulation of HisH are impaired. Following UV-light irradn., the DTE ligand adopts its ring-closed state and loses affinity for HisF, restoring activity and allostery. Our approach allows for the switching of ImGP-S activity and allostery during catalysis and appears to be generally applicable for the light regulation of other multienzyme complexes.
- 456Kneuttinger, A. C.; Rajendran, C.; Simeth, N. A.; Bruckmann, A.; König, B.; Sterner, R. Significance of the Protein Interface Configuration for Allostery in Imidazole Glycerol Phosphate Synthase. Biochemistry 2020, 59 (29), 2729– 2742, DOI: 10.1021/acs.biochem.0c00332Google Scholar456Significance of the Protein Interface Configuration for Allostery in Imidazole Glycerol Phosphate SynthaseKneuttinger, Andrea C.; Rajendran, Chitra; Simeth, Nadja A.; Bruckmann, Astrid; Koenig, Burkhard; Sterner, ReinhardBiochemistry (2020), 59 (29), 2729-2742CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Imidazole glycerol phosphate synthase (ImGPS) from Thermotoga maritima is a model enzyme for studying allostery. The ImGPS complex consists of the cyclase subunit HisF and the glutaminase subunit HisH whose activity is stimulated by substrate binding to HisF in a V-type manner. To investigate the significance of a putative closing hinge motion at the cyclase:glutaminase interface for HisH activity, we replaced residue W123 in HisH with the light-switchable unnatural amino acid phenylalanine-4'-azobenzene (AzoF). Crystal structure anal. employing angle, buried surface area, and distance measurements showed that incorporation of AzoF at this position causes a closing of the interface by ~ 18 ± 3%. This slightly different interface configuration results in a much higher catalytic efficiency in unstimulated HisH due to an elevated turnover no. Moreover, the catalytic efficiency of HisH when stimulated by binding of a substrate to HisF was also significantly increased by AzoF incorporation. This was caused by a K-type stimulation that led to a decrease in the apparent dissocn. const. for its substrate, glutamine. In addn., AzoF improved the apparent binding of a substrate analog at the HisF active site. Remarkably, light-induced isomerization of AzoF considerably enhanced these effects. In conclusion, our findings confirm that signal transduction from HisF to HisH in ImGPS involves the closing of the cyclase:glutaminase subunit interface and that incorporation of AzoF at a hinge position reinforces this catalytically relevant conformational change.
- 457Zubi, Y. S.; Seki, K.; Li, Y.; Hunt, A. C.; Liu, B.; Roux, B.; Jewett, M. C.; Lewis, J. C. Metal-Responsive Regulation of Enzyme Catalysis Using Genetically Encoded Chemical Switches. Nature Commun. 2022, 13 (1), 1864, DOI: 10.1038/s41467-022-29239-yGoogle ScholarThere is no corresponding record for this reference.
- 458Yang, H.; Swartz, A. M.; Park, H. J.; Srivastava, P.; Ellis-Guardiola, K.; Upp, D. M.; Lee, G.; Belsare, K.; Gu, Y.; Zhang, C. Evolving Artificial Metalloenzymes via Random Mutagenesis. Nat. Chem. 2018, 10 (3), 318– 324, DOI: 10.1038/nchem.2927Google Scholar458Evolving artificial metalloenzymes via random mutagenesisYang, Hao; Swartz, Alan M.; Park, Hyun June; Srivastava, Poonam; Ellis-Guardiola, Ken; Upp, David M.; Lee, Gihoon; Belsare, Ketaki; Gu, Yifan; Zhang, Chen; Moellering, Raymond E.; Lewis, Jared C.Nature Chemistry (2018), 10 (3), 318-324CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Random mutagenesis has the potential to optimize the efficiency and selectivity of protein catalysts without requiring detailed knowledge of protein structure; however, introducing synthetic metal cofactors complicates the expression and screening of enzyme libraries, and activity arising from free cofactor must be eliminated. Here we report an efficient platform to create and screen libraries of artificial metalloenzymes (ArMs) via random mutagenesis, which we use to evolve highly selective dirhodium cyclopropanases. Error-prone PCR and combinatorial codon mutagenesis enabled multiplexed anal. of random mutations, including at sites distal to the putative ArM active site that are difficult to identify using targeted mutagenesis approaches. Variants that exhibited significantly improved selectivity for each of the cyclopropane product enantiomers were identified, and higher activity than previously reported ArM cyclopropanases obtained via targeted mutagenesis was also obsd. This improved selectivity carried over to other dirhodium-catalyzed transformations, including N-H, S-H and Si-H insertion, demonstrating that ArMs evolved for one reaction can serve as starting points to evolve catalysts for others.
- 459Leveson-Gower, R. B.; Zhou, Z.; Drienovská, I.; Roelfes, G. Unlocking Iminium Catalysis in Artificial Enzymes to Create a Friedel-Crafts Alkylase. ACS Catal. 2021, 11 (12), 6763– 6770, DOI: 10.1021/acscatal.1c00996Google Scholar459Unlocking Iminium Catalysis in Artificial Enzymes to Create a Friedel-Crafts AlkylaseLeveson-Gower, Reuben B.; Zhou, Zhi; Drienovska, Ivana; Roelfes, GerardACS Catalysis (2021), 11 (12), 6763-6770CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)The construction and engineering of artificial enzymes consisting of abiol. catalytic moieties incorporated into protein scaffolds is a promising strategy to realize non-natural mechanisms in biocatalysis. Here, incorporation of the noncanonical amino acid para-aminophenylalanine (pAF) into the nonenzymic protein scaffold LmrR creates a proficient and stereoselective artificial enzyme (LmrR_pAF) for the vinylogous Friedel-Crafts alkylation between α,β-unsatd. aldehydes and indoles. PAF acts as a catalytic residue, activating enal substrates toward conjugate addn. via the formation of intermediate iminium ion species, while the protein scaffold provides rate acceleration and stereoinduction. Improved LmrR_pAF variants were identified by low-throughput directed evolution advised by alanine-scanning to obtain a triple mutant that provided higher yields and enantioselectivities for a range of aliph. enals and substituted indoles. Anal. of Michaelis-Menten kinetics of LmrR_pAF and evolved mutants reveals that different activities emerge via evolutionary pathways that diverge from one another and specialize catalytic reactivity. Translating this iminium-based catalytic mechanism into an enzymic context will enable many more biocatalytic transformations inspired by organocatalysis.
- 460Mayer, C.; Dulson, C.; Reddem, E.; Thunnissen, A.-M. W. H.; Roelfes, G. Directed Evolution of a Designer Enzyme Featuring an Unnatural Catalytic Amino Acid. Angew. Chem. Int. Ed. 2019, 58 (7), 2083– 2087, DOI: 10.1002/anie.201813499Google Scholar460Directed Evolution of a Designer Enzyme Featuring an Unnatural Catalytic Amino AcidMayer, Clemens; Dulson, Christopher; Reddem, Eswar; Thunnissen, Andy-Mark W. H.; Roelfes, GerardAngewandte Chemie, International Edition (2019), 58 (7), 2083-2087CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The impressive rate accelerations that enzymes display in nature often result from boosting the inherent catalytic activities of side chains by their precise positioning inside a protein binding pocket. Such fine-tuning is also possible for catalytic unnatural amino acids. Specifically, the directed evolution of a recently described designer enzyme, which utilizes an aniline side chain to promote a model hydrazone formation reaction, is reported. Consecutive rounds of directed evolution identified several mutations in the promiscuous binding pocket, in which the unnatural amino acid is embedded in the starting catalyst. When combined, these mutations boost the turnover frequency (kcat) of the designer enzyme by almost 100-fold. This results from strengthening the catalytic contribution of the unnatural amino acid, as the engineered designer enzymes outperform variants, in which the aniline side chain is replaced with a catalytically inactive tyrosine residue, by more than 200-fold.
- 461Kaes, C.; Katz, A.; Hosseini, M. W. Bipyridine: The Most Widely Used Ligand. A Review of Molecules Comprising at Least Two 2,2‘-Bipyridine Units. Chem. Rev. 2000, 100 (10), 3553– 3590, DOI: 10.1021/cr990376zGoogle Scholar461Bipyridine: The Most Widely Used Ligand. A Review of Molecules Comprising at Least Two 2,2'-Bipyridine UnitsKaes, Christian; Katz, Alexander; Hosseini, Mir WaisChemical Reviews (Washington, D. C.) (2000), 100 (10), 3553-3590CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review with 256 refs. which presents an overview of the most explored chelate system in coordination chem.
- 462Xie, J.; Liu, W.; Schultz, P. G. A Genetically Encoded Bidentate, Metal-Binding Amino Acid. Angew. Chem. Int. Ed. 2007, 46 (48), 9239– 9242, DOI: 10.1002/anie.200703397Google Scholar462A genetically encoded bidentate, metal-binding amino acidXie, Jianming; Liu, Wenshen; Schultz, Peter G.Angewandte Chemie, International Edition (2007), 46 (48), 9239-9242CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)To facilitate the design of metalloproteins, the bidentate, metal-binding amino acid bipyridylalanine (BpyAla) was genetically encoded in E. coli in response to the amber nonsense codon with high fidelity and yield. The incorporation of BpyAla requires a BpyAla-specific aminoacyl-tRNA synthetase, which was evolved in a stepwise fashion. The structural basis of selective recognition of BpyAla by this synthetase was also detd.
- 463Lee, H. S.; Schultz, P. G. Biosynthesis of a Site-Specific DNA Cleaving Protein. J. Am. Chem. Soc. 2008, 130 (40), 13194– 13195, DOI: 10.1021/ja804653fGoogle Scholar463Biosynthesis of a Site-Specific DNA Cleaving ProteinLee, Hyun Soo; Schultz, Peter G.Journal of the American Chemical Society (2008), 130 (40), 13194-13195CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)An E. coli catabolite activator protein (CAP) has been converted into a sequence-specific DNA cleaving protein by genetically introducing (2,2'-bipyridin-5-yl)alanine (Bpy-Ala) into the protein. The mutant CAP (CAP-K26Bpy-Ala) showed comparable binding affinity to CAP-WT for the consensus operator sequence. In the presence of Cu(II) and 3-mercaptopropionic acid, CAP-K26Bpy-Ala cleaves double-stranded DNA with high sequence specificity. This method should provide a useful tool for mapping the mol. details of protein-nucleic acid interactions.
- 464Roelfes, G. LmrR: A Privileged Scaffold for Artificial Metalloenzymes. Acc. Chem. Res. 2019, 52 (3), 545– 556, DOI: 10.1021/acs.accounts.9b00004Google Scholar464LmrR: A Privileged Scaffold for Artificial MetalloenzymesRoelfes, GerardAccounts of Chemical Research (2019), 52 (3), 545-556CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. The biotechnol. revolution has made it possible to create enzymes for many reactions by directed evolution. However, because of the immense no. of possibilities, the availability of enzymes that possess a basal level of the desired catalytic activity is a prerequisite for success. For new-to-nature reactions, artificial metalloenzymes (ARMs), which are rationally designed hybrids of proteins and catalytically active transition-metal complexes, can be such a starting point. This Account details our efforts toward the creation of ARMs for the catalysis of new-to-nature reactions. Key to our approach is the notion that the binding of substrates, i.e., effective molarity, is a key component to achieving large accelerations in catalysis. For this reason, our designs are based on the multidrug resistance regulator LmrR, a dimeric transcription factor with a large, hydrophobic binding pocket at its dimer interface. In this pocket, there are two tryptophan moieties, which are important for promiscuous binding of planar hydrophobic conjugated compds. by π-stacking. The catalytic machinery is introduced either by the covalent linkage of a catalytically active metal complex or via the ligand or supramol. assembly, taking advantage of the two central tryptophan moieties for noncovalent binding of transition-metal complexes. Designs based on the chem. modification of LmrR were successful in catalysis, but this approach proved too laborious to be practical. Therefore, expanded genetic code methodologies were used to introduce metal binding unnatural amino acids during LmrR biosynthesis in vivo. These ARMs have been successfully applied in Cu(II) catalyzed Friedel-Crafts alkylation of indoles. The extension to MDRs from the TetR family resulted in ARMs capable of providing the opposite enantiomer of the Friedel-Crafts product. We have employed a computationally assisted redesign of these ARMs to create a more active and selective artificial hydratase, introducing a glutamate as a general base at a judicious position so it can activate and direct the incoming water nucleophile. A supramolecularly assembled ARM from LmrR and copper(II)-phenanthroline was successful in Friedel-Crafts alkylation reactions, giving rise to up to 94% ee. Also, hemin was bound, resulting in an artificial heme enzyme for enantioselective cyclopropanation reactions. The importance of structural dynamics of LmrR was suggested by computational studies, which showed that the pore can open up to allow access of substrates to the catalytic iron center, which, according to the crystal structure, is deeply buried inside the protein. Finally, the assembly approaches were combined to introduce both a catalytic and a regulatory domain, resulting in an ARM that was specifically activated in the presence of Fe(II) salts but not Zn(II) salts. Our work demonstrates that LmrR is a privileged scaffold for ARM design: It allows for multiple assembly methods and even combinations of these, it can be applied in a variety of different catalytic reactions, and it shows significant structural dynamics that contribute to achieving the desired catalytic activity. Moreover, both the creation via expanded genetic code methods as well as the supramol. assembly make LmrR-based ARMs highly suitable for achieving the ultimate goal of the integration of ARMs in biosynthetic pathways in vivo to create a hybrid metab.
- 465Drienovská, I.; Rioz-Martínez, A.; Draksharapu, A.; Roelfes, G. Novel Artificial Metalloenzymes by in vivo Incorporation of Metal-Binding Unnatural Amino Acids. Chem. Sci. 2015, 6 (1), 770– 776, DOI: 10.1039/C4SC01525HGoogle Scholar465Novel artificial metalloenzymes by in vivo incorporation of metal-binding unnatural amino acidsDrienovska, Ivana; Rioz-Martinez, Ana; Draksharapu, Apparao; Roelfes, GerardChemical Science (2015), 6 (1), 770-776CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Artificial metalloenzymes have emerged as an attractive new approach to enantioselective catalysis. Herein, we introduce a novel strategy for prepn. of artificial metalloenzymes utilizing amber stop codon suppression methodol. for the in vivo incorporation of metal-binding unnatural amino acids. The resulting artificial metalloenzymes were applied in catalytic asym. Friedel-Crafts alkylation reactions and up to 83% ee for the product was achieved.
- 466Drienovská, I.; Alonso-Cotchico, L.; Vidossich, P.; Lledós, A.; Maréchal, J.-D.; Roelfes, G. Design of an Enantioselective Artificial Metallo-Hydratase Enzyme Containing an Unnatural Metal-Binding Amino Acid. Chem. Sci. 2017, 8 (10), 7228– 7235, DOI: 10.1039/C7SC03477FGoogle ScholarThere is no corresponding record for this reference.
- 467Ségaud, N.; Drienovská, I.; Chen, J.; Browne, W. R.; Roelfes, G. Artificial Metalloproteins for Binding and Stabilization of a Semiquinone Radical. Inorg. Chem. 2017, 56 (21), 13293– 13299, DOI: 10.1021/acs.inorgchem.7b02073Google ScholarThere is no corresponding record for this reference.
- 468Bersellini, M.; Roelfes, G. Multidrug Resistance Regulators (MDRs) as Scaffolds for the Design of Artificial Metalloenzymes. Org. Biomol. Chem. 2017, 15 (14), 3069– 3073, DOI: 10.1039/C7OB00390KGoogle Scholar468Multidrug resistance regulators (MDRs) as scaffolds for the design of artificial metalloenzymesBersellini, Manuela; Roelfes, GerardOrganic & Biomolecular Chemistry (2017), 15 (14), 3069-3073CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)The choice of protein scaffolds is an important element in the design of artificial metalloenzymes. Here, we introduced multidrug resistance regulators (MDRs) from the TetR family as a viable class of protein scaffolds for artificial metalloenzyme design. The in vivo incorporation of the metal-binding amino acid, (2,2-bipyridin-5yl)alanine (BpyA), by stop codon suppression methods was used to create artificial metalloenzymes from 3 members of the TetR family of MDRs: QacR, CgmR, and RamR. Excellent results were achieved with QacR Y123BpyA in the Cu2+-catalyzed enantioselective vinylogous Friedel-Crafts alkylation reaction with ee's up to 94% of the opposite enantiomer that was achieved with other mutants and the previously reported LmrR-based artificial metalloenzymes.
- 469Jung, S.-M.; Yang, M.; Song, W. J. Symmetry-Adapted Synthesis of Dicopper Oxidases with Divergent Dioxygen Reactivity. Inorg. Chem. 2022, 61 (31), 12433– 12441, DOI: 10.1021/acs.inorgchem.2c01898Google ScholarThere is no corresponding record for this reference.
- 470Drienovská, I.; Scheele, R. A.; Gutiérrez de Souza, C.; Roelfes, G. A Hydroxyquinoline-Based Unnatural Amino Acid for the Design of Novel Artificial Metalloenzymes. ChemBioChem 2020, 21 (21), 3077– 3081, DOI: 10.1002/cbic.202000306Google Scholar470A Hydroxyquinoline-Based Unnatural Amino Acid for the Design of Novel Artificial MetalloenzymesDrienovska, Ivana; Scheele, Remkes A.; Gutierrez de Souza, Cora; Roelfes, GerardChemBioChem (2020), 21 (21), 3077-3081CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)We have examd. the potential of the noncanonical amino acid (8-hydroxyquinolin-3-yl)alanine (HQAla) for the design of artificial metalloenzymes. HQAla, a versatile chelator of late transition metals, was introduced into the lactococcal multidrug-resistance regulator (LmrR) by stop codon suppression methodol. LmrR_HQAla was shown to complex efficiently with three different metal ions, CuII, ZnII and RhIII to form unique artificial metalloenzymes. The catalytic potential of the CuII-bound LmrR_HQAla enzyme was shown through its ability to catalyze asym. Friedel-Craft alkylation and water addn., whereas the ZnII-coupled enzyme was shown to mimic natural Zn hydrolase activity.
- 471Stein, A.; Liang, A. D.; Sahin, R.; Ward, T. R. Incorporation of Metal-Chelating Unnatural Amino Acids into Halotag for Allylic Deamination. J. Organomet. Chem. 2022, 962, 122272, DOI: 10.1016/j.jorganchem.2022.122272Google ScholarThere is no corresponding record for this reference.
- 472Los, G. V.; Encell, L. P.; McDougall, M. G.; Hartzell, D. D.; Karassina, N.; Zimprich, C.; Wood, M. G.; Learish, R.; Ohana, R. F.; Urh, M. HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis. ACS Chem. Biol. 2008, 3 (6), 373– 382, DOI: 10.1021/cb800025kGoogle Scholar472HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein AnalysisLos, Georgyi V.; Encell, Lance P.; McDougall, Mark G.; Hartzell, Danette D.; Karassina, Natasha; Zimprich, Chad; Wood, Monika G.; Learish, Randy; Ohana, Rachel Friedman; Urh, Marjeta; Simpson, Dan; Mendez, Jacqui; Zimmerman, Kris; Otto, Paul; Vidugiris, Gediminas; Zhu, Ji; Darzins, Aldis; Klaubert, Dieter H.; Bulleit, Robert F.; Wood, Keith V.ACS Chemical Biology (2008), 3 (6), 373-382CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)We have designed a modular protein tagging system that allows different functionalities to be linked onto a single genetic fusion, either in soln., in living cells, or in chem. fixed cells. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful mols., such as fluorescent dyes, affinity handles, or solid surfaces. Covalent bond formation between the protein tag and the chloroalkane linker is highly specific, occurs rapidly under physiol. conditions, and is essentially irreversible. We demonstrate the utility of this system for cellular imaging and protein immobilization by analyzing multiple mol. processes assocd. with NF-κB-mediated cellular physiol., including imaging of subcellular protein translocation and capture of protein-protein and protein-DNA complexes.
- 473Coquière, D.; Bos, J.; Beld, J.; Roelfes, G. Enantioselective Artificial Metalloenzymes Based on a Bovine Pancreatic Polypeptide Scaffold. Angew. Chem. Int. Ed. 2009, 48 (28), 5159– 5162, DOI: 10.1002/anie.200901134Google ScholarThere is no corresponding record for this reference.
- 474Madoori, P. K.; Agustiandari, H.; Driessen, A. J. M.; Thunnissen, A. M. W. H. Structure of the Transcriptional Regulator LmrR and Its Mechanism of Multidrug Recognition. EMBO J. 2009, 28 (2), 156– 166, DOI: 10.1038/emboj.2008.263Google Scholar474Structure of the transcriptional regulator LmrR and its mechanism of multidrug recognitionMadoori, Pramod Kumar; Agustiandari, Herfita; Driessen, Arnold J. M.; Thunnissen, Andy-Mark W. H.EMBO Journal (2009), 28 (2), 156-166CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)LmrR is a PadR-related transcriptional repressor that regulates the prodn. of LmrCD, a major multidrug ABC transporter in Lactococcus lactis. Transcriptional regulation is presumed to follow a drug-sensitive induction mechanism involving the direct binding of transporter ligands to LmrR. Here, we present crystal structures of LmrR in an apo state and in two drug-bound states complexed with Hoechst 33342 and daunomycin. LmrR shows a common topol. contg. a typical β-winged helix-turn-helix domain with an addnl. C-terminal helix involved in dimerization. Its dimeric organization is highly unusual with a flat-shaped hydrophobic pore at the dimer center serving as a multidrug-binding site. The drugs bind in a similar manner with their arom. rings sandwiched in between the indole groups of two dimer-related tryptophan residues. Multidrug recognition is facilitated by conformational plasticity and the absence of drug-specific hydrogen bonds. Combined analyses using site-directed mutagenesis, fluorescence-based drug binding and protein-DNA gel shift assays reveal an allosteric coupling between the multidrug- and DNA-binding sites of LmrR that most likely has a function in the induction mechanism.
- 475Yu, Y.; Hu, C.; Xia, L.; Wang, J. Artificial Metalloenzyme Design with Unnatural Amino Acids and Non-Native Cofactors. ACS Catal. 2018, 8 (3), 1851– 1863, DOI: 10.1021/acscatal.7b03754Google Scholar475Artificial Metalloenzyme Design with Unnatural Amino Acids and Non-Native CofactorsYu, Yang; Hu, Cheng; Xia, Lin; Wang, JiangyunACS Catalysis (2018), 8 (3), 1851-1863CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)A review. There are 20 proteinogenic amino acids and a limited no. of cofactors naturally available to build enzymes. Genetic codon expansion enables us to incorporate more than 200 unnatural amino acids into proteins using cell translation machinery, greatly expanding structures available to protein chemists. Such tools enable scientists to mimic the active site of an enzyme to tune enzymic activity, anchor cofactors, and immobilize enzymes on electrode surfaces. Non-native cofactors can be incorporated into the protein through covalent or noncovalent interactions, expanding the reaction scope of existing enzymes. The review discusses strategies to incorporate unnatural amino acids and non-native cofactors and their applications in tuning and expanding enzymic activities of artificial metalloenzymes.
- 476Yang, H.; Srivastava, P.; Zhang, C.; Lewis, J. C. A General Method for Artificial Metalloenzyme Formation through Strain-Promoted Azide-Alkyne Cycloaddition. ChemBioChem 2014, 15 (2), 223– 227, DOI: 10.1002/cbic.201300661Google ScholarThere is no corresponding record for this reference.
- 477Srivastava, P.; Yang, H.; Ellis-Guardiola, K.; Lewis, J. C. Engineering a Dirhodium Artificial Metalloenzyme for Selective Olefin Cyclopropanation. Nature Commun. 2015, 6 (1), 7789, DOI: 10.1038/ncomms8789Google ScholarThere is no corresponding record for this reference.
- 478Upp, D. M.; Huang, R.; Li, Y.; Bultman, M. J.; Roux, B.; Lewis, J. C. Engineering Dirhodium Artificial Metalloenzymes for Diazo Coupling Cascade Reactions. Angew. Chem. Int. Ed. 2021, 60 (44), 23672– 23677, DOI: 10.1002/anie.202107982Google Scholar478Engineering Dirhodium Artificial Metalloenzymes for Diazo Coupling Cascade ReactionsUpp, David M.; Huang, Rui; Li, Ying; Bultman, Max J.; Roux, Benoit; Lewis, Jared C.Angewandte Chemie, International Edition (2021), 60 (44), 23672-23677CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Artificial metalloenzymes (ArMs) are commonly used to control the stereoselectivity of catalytic reactions, but controlling chemoselectivity remains challenging. In this study, we engineer a dirhodium ArM to catalyze diazo cross-coupling to form an alkene that, in a one-pot cascade reaction, is reduced to an alkane with high enantioselectivity (typically >99% ee) by an alkene reductase. The numerous protein and small mol. components required for the cascade reaction had minimal effect on ArM catalysis. Directed evolution of the ArM led to improved yields and E/Z selectivities for a variety of substrates, which translated to cascade reaction yields. MD simulations of ArM variants were used to understand the structural role of the cofactor on ArM conformational dynamics. These results highlight the ability of ArMs to control both catalyst stereoselectivity and chemoselectivity to enable reactions in complex media that would otherwise lead to undesired side reactions.
- 479Ellis-Guardiola, K.; Rui, H.; Beckner, R. L.; Srivastava, P.; Sukumar, N.; Roux, B.; Lewis, J. C. Crystal Structure and Conformational Dynamics of Pyrococcus furiosus Prolyl Oligopeptidase. Biochemistry 2019, 58 (12), 1616– 1626, DOI: 10.1021/acs.biochem.9b00031Google ScholarThere is no corresponding record for this reference.
- 480Brady, L.; Brzozowski, A. M.; Derewenda, Z. S.; Dodson, E.; Dodson, G.; Tolley, S.; Turkenburg, J. P.; Christiansen, L.; Huge-Jensen, B.; Norskov, L. A Serine Protease Triad Forms the Catalytic Centre of a Triacylglycerol Lipase. Nature 1990, 343 (6260), 767– 770, DOI: 10.1038/343767a0Google Scholar480A serine protease triad forms the catalytic center of a triacylglycerol lipaseBrady, Leo; Brzozowski, Andrzej M.; Derewenda, Zygmunt S.; Dodson, Eleanor; Dodson, Guy; Tolley, Shirley; Turkenburg, Johan P.; Christiansen, Lars; Huge-Jensen, Birgitte; et al.Nature (London, United Kingdom) (1990), 343 (6260), 767-70CODEN: NATUAS; ISSN:0028-0836.The x-ray structure of the Mucor miehei triglyceride lipase, is reported and the at. model obtained at 3.1 Å resoln. and refined to 1.9 Å resoln. is described. It reveals a serine...histidine...aspartate trypsin-like catalytic triad with an active serine buried under a short helical fragment of a long surface loop.
- 481Buller, A. R.; Townsend, C. A. Intrinsic Evolutionary Constraints on Protease Structure, Enzyme Acylation, and the Identity of the Catalytic Triad. Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (8), E653-E661 DOI: 10.1073/pnas.1221050110Google ScholarThere is no corresponding record for this reference.
- 482Smith, A. J. T.; Müller, R.; Toscano, M. D.; Kast, P.; Hellinga, H. W.; Hilvert, D.; Houk, K. N. Structural Reorganization and Preorganization in Enzyme Active Sites: Comparisons of Experimental and Theoretically Ideal Active Site Geometries in the Multistep Serine Esterase Reaction Cycle. J. Am. Chem. Soc. 2008, 130 (46), 15361– 15373, DOI: 10.1021/ja803213pGoogle Scholar482Structural Reorganization and Preorganization in Enzyme Active Sites: Comparisons of Experimental and Theoretically Ideal Active Site Geometries in the Multistep Serine Esterase Reaction CycleSmith, Adam J. T.; Muller, Roger; Toscano, Miguel D.; Kast, Peter; Hellinga, Homme W.; Hilvert, Donald; Houk, K. N.Journal of the American Chemical Society (2008), 130 (46), 15361-15373CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Many enzymes catalyze reactions with multiple chem. steps, requiring the stabilization of multiple transition states during catalysis. Such enzymes must strike a balance between the conformational reorganization required to stabilize multiple transition states of a reaction and the confines of a preorganized active site in the polypeptide tertiary structure. Here we investigate the compromise between structural reorganization during the catalytic process and preorganization of the active site for a multistep enzyme-catalyzed reaction, the hydrolysis of esters by the Ser-His-Asp/Glu catalytic triad. Quantum mech. transition states were used to generate ensembles of geometries that can catalyze each individual step in the mechanism. These geometries are compared to each other by superpositions of catalytic atoms to find "consensus" geometries that can catalyze all steps with minimal rearrangement. These consensus geometries are found to be excellent matches for the natural active site. Preorganization is therefore found to be the major defining characteristic of the active site, and reorganizational motions often proposed to promote catalysis have been minimized. The variability of enzyme active sites obsd. by x-ray crystallog. was also investigated empirically. A catalog of geometrical parameters relating active site residues to each other and to bound inhibitors was collected from a set of crystal structures. The crystal-structure-derived values were then compared to the ranges found in quantum mech. optimized structures along the entire reaction coordinate. The empirical ranges are found to encompass the theor. ranges when thermal fluctuations are taken into account. Therefore, the active sites are preorganized to a geometry that can be objectively and quant. defined as minimizing conformational reorganization while maintaining optimal transition state stabilization for every step during catalysis. The results provide a useful guiding principle for de novo design of enzymes with multistep mechanisms.
- 483Burton, A. J.; Thomson, A. R.; Dawson, W. M.; Brady, R. L.; Woolfson, D. N. Installing Hydrolytic Activity into a Completely De Novo Protein Framework. Nat. Chem. 2016, 8 (9), 837– 844, DOI: 10.1038/nchem.2555Google Scholar483Installing hydrolytic activity into a completely de novo protein frameworkBurton, Antony J.; Thomson, Andrew R.; Dawson, William M.; Brady, R. Leo; Woolfson, Derek N.Nature Chemistry (2016), 8 (9), 837-844CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)The design of enzyme-like catalysts tests the understanding of sequence-to-structure/function relations in proteins. Here, the authors installed hydrolytic activity predictably into a completely de novo and thermostable α-helical barrel, which comprised 7 helixes arranged around an accessible channel. The authors showed that the lumen of the barrel accepted 21 mutations to functional polar residues. The resulting variant, which had Cys-His-Glu triads on each helix, hydrolyzed p-nitrophenyl acetate with catalytic efficiencies that matched the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the 1st report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of this system also allowed the facile incorporation of unnatural side-chains to improve activity and probe the catalytic mechanism. Such a predictable and robust construction of truly de novo biocatalysts holds promise for applications in chem. and biochem. synthesis.
- 484Rajagopalan, S.; Wang, C.; Yu, K.; Kuzin, A. P.; Richter, F.; Lew, S.; Miklos, A. E.; Matthews, M. L.; Seetharaman, J.; Su, M. Design of Activated Serine-Containing Catalytic Triads with Atomic-Level Accuracy. Nat. Chem. Biol. 2014, 10 (5), 386– 391, DOI: 10.1038/nchembio.1498Google ScholarThere is no corresponding record for this reference.
- 485Richter, F.; Blomberg, R.; Khare, S. D.; Kiss, G.; Kuzin, A. P.; Smith, A. J. T.; Gallaher, J.; Pianowski, Z.; Helgeson, R. C.; Grjasnow, A. Computational Design of Catalytic Dyads and Oxyanion Holes for Ester Hydrolysis. J. Am. Chem. Soc. 2012, 134 (39), 16197– 16206, DOI: 10.1021/ja3037367Google Scholar485Computational Design of Catalytic Dyads and Oxyanion Holes for Ester HydrolysisRichter, Florian; Blomberg, Rebecca; Khare, Sagar D.; Kiss, Gert; Kuzin, Alexandre P.; Smith, Adam J. T.; Gallaher, Jasmine; Pianowski, Zbigniew; Helgeson, Roger C.; Grjasnow, Alexej; Xiao, Rong; Seetharaman, Jayaraman; Su, Min; Vorobiev, Sergey; Lew, Scott; Forouhar, Farhad; Kornhaber, Gregory J.; Hunt, John F.; Montelione, Gaetano T.; Tong, Liang; Houk, K. N.; Hilvert, Donald; Baker, DavidJournal of the American Chemical Society (2012), 134 (39), 16197-16206CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Nucleophilic catalysis is a general strategy for accelerating ester and amide hydrolysis. In natural active sites, nucleophilic elements such as catalytic dyads and triads are usually paired with oxyanion holes for substrate activation, but it is difficult to parse out the independent contributions of these elements or to understand how they emerged in the course of evolution. Here we explore the minimal requirements for esterase activity by computationally designing artificial catalysts using catalytic dyads and oxyanion holes. We found much higher success rates using designed oxyanion holes formed by backbone NH groups rather than by side chains or bridging water mols. and obtained four active designs in different scaffolds by combining this motif with a Cys-His dyad. Following active site optimization, the most active of the variants exhibited a catalytic efficiency (kcat/KM) of 400 M-1 s-1 for the cleavage of a p-nitrophenyl ester. Kinetic expts. indicate that the active site cysteines are rapidly acylated as programmed by design, but the subsequent slow hydrolysis of the acyl-enzyme intermediate limits overall catalytic efficiency. Moreover, the Cys-His dyads are not properly formed in crystal structures of the designed enzymes. These results highlight the challenges that computational design must overcome to achieve high levels of activity.
- 486Burke, A. J.; Lovelock, S. L.; Frese, A.; Crawshaw, R.; Ortmayer, M.; Dunstan, M.; Levy, C.; Green, A. P. Design and Evolution of an Enzyme with a Non-Canonical Organocatalytic Mechanism. Nature 2019, 570 (7760), 219– 223, DOI: 10.1038/s41586-019-1262-8Google Scholar486Design and evolution of an enzyme with a non-canonical organocatalytic mechanismBurke, Ashleigh J.; Lovelock, Sarah L.; Frese, Amina; Crawshaw, Rebecca; Ortmayer, Mary; Dunstan, Mark; Levy, Colin; Green, Anthony P.Nature (London, United Kingdom) (2019), 570 (7760), 219-223CODEN: NATUAS; ISSN:0028-0836. (Nature Research)The combination of computational design and lab. evolution is a powerful and potentially versatile strategy for the development of enzymes with new functions1-4. However, the limited functionality presented by the genetic code restricts the range of catalytic mechanisms that are accessible in designed active sites. Inspired by mechanistic strategies from small-mol. organocatalysis5, here we report the generation of a hydrolytic enzyme that uses Nδ-methylhistidine as a non-canonical catalytic nucleophile. Histidine methylation is essential for catalytic function because it prevents the formation of unreactive acyl-enzyme intermediates, which has been a long-standing challenge when using canonical nucleophiles in enzyme design6-10. Enzyme performance was optimized using directed evolution protocols adapted to an expanded genetic code, affording a biocatalyst capable of accelerating ester hydrolysis with greater than 9,000-fold increased efficiency over free Nδ-methylhistidine in soln. Crystallog. snapshots along the evolutionary trajectory highlight the catalytic devices that are responsible for this increase in efficiency. Nδ-methylhistidine can be considered to be a genetically encodable surrogate of the widely employed nucleophilic catalyst dimethylaminopyridine11, and its use will create opportunities to design and engineer enzymes for a wealth of valuable chem. transformations.
- 487Bjelic, S.; Nivón, L. G.; Çelebi-Ölçüm, N.; Kiss, G.; Rosewall, C. F.; Lovick, H. M.; Ingalls, E. L.; Gallaher, J. L.; Seetharaman, J.; Lew, S. Computational Design of Enone-Binding Proteins with Catalytic Activity for the Morita-Baylis-Hillman Reaction. ACS Chem. Biol. 2013, 8 (4), 749– 757, DOI: 10.1021/cb3006227Google Scholar487Computational Design of Enone-Binding Proteins with Catalytic Activity for the Morita-Baylis-Hillman ReactionBjelic, Sinisa; Nivon, Lucas G.; Celebi-Olcum, Nihan; Kiss, Gert; Rosewall, Carolyn F.; Lovick, Helena M.; Ingalls, Erica L.; Gallaher, Jasmine Lynn; Seetharaman, Jayaraman; Lew, Scott; Montelione, Gaetano Thomas; Hunt, John Francis; Michael, Forrest Edwin; Houk, K. N.; Baker, DavidACS Chemical Biology (2013), 8 (4), 749-757CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)The Morita-Baylis-Hillman reaction forms a carbon-carbon bond between the α-carbon of a conjugated carbonyl compd. and a carbon electrophile. The reaction mechanism involves Michael addn. of a nucleophile catalyst at the carbonyl β-carbon, followed by bond formation with the electrophile and catalyst disassocn. to release the product. We used Rosetta to design 48 proteins contg. active sites predicted to carry out this mechanism, of which two show catalytic activity by mass spectrometry (MS). Substrate labeling measured by MS and site-directed mutagenesis expts. show that the designed active-site residues are responsible for activity, although rate acceleration over background is modest. To characterize the designed proteins, we developed a fluorescence-based screen for intermediate formation in cell lysates, carried out microsecond mol. dynamics simulations, and solved X-ray crystal structures. These data indicate a partially formed active site and suggest several clear avenues for designing more active catalysts.
- 488Hutton, A. E.; Foster, J.; Crawshaw, R.; Hardy, F. J.; Johannissen, L. O.; Lister, T. M.; Gerard, E. F.; Birch-Price, Z.; Obexer, R.; Hay, S.; Green, A. P. A Non-Canonical Nucleophile Unlocks a New Mechanistic Pathway in a Designed Enzyme. Nat Commun 2024, DOI: 10.1038/s41467-024-46123-zGoogle ScholarThere is no corresponding record for this reference.
- 489Crawshaw, R.; Crossley, A.; Johannissen, L.; Burke, A.; Hay, S.; Levy, C.; Baker, D.; Lovelock, S.; Green, A. Engineering an Efficient and Enantioselective Enzyme for the Morita-Baylis-Hillman Reaction. Nat. Chem. 2022, 14, 313, DOI: 10.1038/s41557-021-00833-9Google Scholar489Engineering an efficient and enantioselective enzyme for the Morita-Baylis-Hillman reactionCrawshaw, Rebecca; Crossley, Amy E.; Johannissen, Linus; Burke, Ashleigh J.; Hay, Sam; Levy, Colin; Baker, David; Lovelock, Sarah L.; Green, Anthony P.Nature Chemistry (2022), 14 (3), 313-320CODEN: NCAHBB; ISSN:1755-4330. (Nature Portfolio)Abstr.: The combination of computational design and directed evolution could offer a general strategy to create enzymes with new functions. So far, this approach has delivered enzymes for a handful of model reactions. Here we show that new catalytic mechanisms can be engineered into proteins to accelerate more challenging chem. transformations. Evolutionary optimization of a primitive design afforded an efficient and enantioselective enzyme (BH32.14) for the Morita-Baylis-Hillman (MBH) reaction. BH32.14 is suitable for preparative-scale transformations, accepts a broad range of aldehyde and enone coupling partners and is able to promote selective monofunctionalizations of dialdehydes. Crystallog., biochem. and computational studies reveal that BH32.14 operates via a sophisticated catalytic mechanism comprising a His23 nucleophile paired with a judiciously positioned Arg124. This catalytic arginine shuttles between conformational states to stabilize multiple oxyanion intermediates and serves as a genetically encoded surrogate of privileged bidentate hydrogen-bonding catalysts (for example, thioureas). This study demonstrates that elaborate catalytic devices can be built from scratch to promote demanding multi-step processes not obsd. in nature. [graphic not available: see fulltext].
- 490Agten, S. M.; Dawson, P. E.; Hackeng, T. M. Oxime Conjugation in Protein Chemistry: From Carbonyl Incorporation to Nucleophilic Catalysis. J. Pept. Sci. 2016, 22 (5), 271– 279, DOI: 10.1002/psc.2874Google Scholar490Oxime conjugation in protein chemistry: from carbonyl incorporation to nucleophilic catalysisAgten, Stijn M.; Dawson, Philip E.; Hackeng, Tilman M.Journal of Peptide Science (2016), 22 (5), 271-279CODEN: JPSIEI; ISSN:1075-2617. (John Wiley & Sons Ltd.)A review. Use of oxime forming reactions has become a widely applied strategy for peptide and protein bioconjugation. The efficiency of the reaction and robust stability of the oxime product led to the development of a growing list of methods to introduce the required ketone or aldehyde functionality site specifically into proteins. Early methods focused on site-specific oxidn. of an N-terminal serine or threonine and more recently transamination methods have been developed to convert a broader set of N-terminal amino acids into a ketone or aldehyde. More recently, site-specific modification of protein has been attained through engineering enzymes involved in posttranslational modifications to accommodate aldehyde-contg. substrates. Similarly, a growing list of unnatural amino acids can be introduced through development of selective amino-acyl tRNA synthetase/tRNA pairs combined with codon reassignment. In the case of glycoproteins, glycans can be selectively modified chem. or enzymically to introduce aldehyde functional groups. Finally, the total chem. synthesis of proteins complements these biol. and chemoenzymic approaches. Once introduced, the oxime ligation of these aldehyde and ketone groups can be catalyzed by aniline or a variety of aniline derivs. to tune the activity, pH preference, stability and soly. of the catalyst.
- 491Kölmel, D. K.; Kool, E. T. Oximes and Hydrazones in Bioconjugation: Mechanism and Catalysis. Chem. Rev. 2017, 117 (15), 10358– 10376, DOI: 10.1021/acs.chemrev.7b00090Google Scholar491Oximes and Hydrazones in Bioconjugation: Mechanism and CatalysisKolmel, Dominik K.; Kool, Eric T.Chemical Reviews (Washington, DC, United States) (2017), 117 (15), 10358-10376CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. The formation of oximes and hydrazones is employed in numerous scientific fields as a simple and versatile conjugation strategy. This imine-forming reaction is applied in fields as diverse as polymer chem., biomaterials and hydrogels, dynamic combinatorial chem., org. synthesis, and chem. biol. Here we outline chem. developments in this field, with special focus on the past ∼10 years of developments. Recent strategies for installing reactive carbonyl groups and α-nucleophiles into biomols. are described. The basic chem. properties of reactants and products in this reaction are then reviewed, with an eye to understanding the reaction's mechanism and how reactant structure controls rates and equil. in the process. Recent work that has uncovered structural features and new mechanisms for speeding the reaction, sometimes by orders of magnitude, is discussed. We describe recent studies that have identified esp. fast reacting aldehyde/ketone substrates and structural effects that lead to rapid-reacting α-nucleophiles as well. Among the most effective new strategies has been the development of substituents near the reactive aldehyde group that either transfer protons at the transition state or trap the initially formed tetrahedral intermediates. In addn., the recent development of efficient nucleophilic catalysts for the reaction is outlined, improving greatly upon aniline, the classical catalyst for imine formation. A no. of uses of such second- and third-generation catalysts in bioconjugation and in cellular applications are highlighted. While formation of hydrazone and oxime has been traditionally regarded as being limited by slow rates, developments in the past 5 years have resulted in completely overturning this limitation; indeed, the reaction is now one of the fastest and most versatile reactions available for conjugations of biomols. and biomaterials.
- 492Drienovská, I.; Mayer, C.; Dulson, C.; Roelfes, G. A Designer Enzyme for Hydrazone and Oxime Formation Featuring an Unnatural Catalytic Aniline Residue. Nat. Chem. 2018, 10 (9), 946– 952, DOI: 10.1038/s41557-018-0082-zGoogle Scholar492A designer enzyme for hydrazone and oxime formation featuring an unnatural catalytic aniline residueDrienovska, Ivana; Mayer, Clemens; Dulson, Christopher; Roelfes, GerardNature Chemistry (2018), 10 (9), 946-952CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Creating designer enzymes with the ability to catalyze abiol. transformations is a formidable challenge. Efforts toward this goal typically consider only canonical amino acids in the initial design process. However, incorporating unnatural amino acids that feature uniquely reactive side chains could significantly expand the catalytic repertoire of designer enzymes. To explore the potential of such artificial building blocks for enzyme design, here we selected p-aminophenylalanine (pAF) as a potentially novel catalytic residue. We demonstrate that the catalytic activity of the aniline side chain for hydrazone and oxime formation reactions is increased by embedding p-aminophenylalanine into the hydrophobic pore of the multidrug transcriptional regulator LmrR from Lactococcus lactis. Both the recruitment of reactants by the promiscuous binding pocket and a judiciously placed aniline that functions as a catalytic residue contribute to the success of the identified artificial enzyme. We anticipate that our design strategy will prove rewarding to significantly expand the catalytic repertoire of designer enzymes in the future.
- 493Ofori Atta, L.; Zhou, Z.; Roelfes, G. In vivo Biocatalytic Cascades Featuring an Artificial-Enzyme-Catalysed New-to-Nature Reaction. Angew. Chem. Int. Ed. 2023, 62 (1), e202214191 DOI: 10.1002/anie.202214191Google ScholarThere is no corresponding record for this reference.
- 494Leveson-Gower, R. B.; de Boer, R. M.; Roelfes, G. Tandem Friedel-Crafts-Alkylation-Enantioselective-Protonation by Artificial Enzyme Iminium Catalysis. ChemCatChem 2022, 14 (8), e202101875 DOI: 10.1002/cctc.202101875Google Scholar494Tandem Friedel-Crafts-Alkylation-Enantioselective-Protonation by Artificial Enzyme Iminium CatalysisLeveson-Gower, Reuben B.; de Boer, Ruben M.; Roelfes, GerardChemCatChem (2022), 14 (8), e202101875CODEN: CHEMK3; ISSN:1867-3880. (Wiley-VCH Verlag GmbH & Co. KGaA)The incorporation of organocatalysts into protein scaffolds holds the promise of overcoming some of the limitations of this powerful catalytic approach. Previously, we showed that incorporation of the non-canonical amino acid para-aminophenylalanine into the non-enzymic protein scaffold LmrR forms a proficient and enantioselective artificial enzyme (LmrR_pAF) for the Friedel-Crafts alkylation of indoles with enals. The unnatural aniline side-chain is directly involved in catalysis, operating via a well-known organocatalytic iminium-based mechanism. In this study, we show that LmrR_pAF can enantioselectively form tertiary carbon centers not only during C-C bond formation, but also by enantioselective protonation, delivering a proton to one face of a prochiral enamine intermediate. The importance of various side-chains in the pocket of LmrR is distinct from the Friedel-Crafts reaction without enantioselective protonation, and two particularly important residues were probed by exhaustive mutagenesis.
- 495Zhou, Z.; Roelfes, G. Synergistic Catalysis in an Artificial Enzyme by Simultaneous Action of Two Abiological Catalytic Sites. Nat. Catal. 2020, 3 (3), 289– 294, DOI: 10.1038/s41929-019-0420-6Google Scholar495Synergistic catalysis in an artificial enzyme by simultaneous action of two abiological catalytic sitesZhou, Zhi; Roelfes, GerardNature Catalysis (2020), 3 (3), 289-294CODEN: NCAACP; ISSN:2520-1158. (Nature Research)Abstr.: Artificial enzymes, which are hybrids of proteins with abiol. catalytic groups, have emerged as a powerful approach towards the creation of enzymes for new-to-nature reactions. Typically, only a single abiol. catalytic moiety is incorporated. Here we introduce a design of an artificial enzyme that comprises two different abiol. catalytic moieties and show that these can act synergistically to achieve high activity and enantioselectivity (up to >99% e.e.) in the catalyzed Michael addn. reaction. The design is based on the lactococcal multidrug resistance regulator as the protein scaffold and combines a genetically encoded unnatural p-aminophenylalanine residue (which activates an enal through iminium ion formation) and a supramolecularly bound Lewis acidic Cu(II) complex (which activates the Michael donor by enolization and delivers it to one preferred prochiral face of the activated enal). This study demonstrates that synergistic combination of abiol. catalytic groups is a robust way to achieve catalysis that is normally outside of the realm of artificial enzymes.
- 496Zhou, Z.; Roelfes, G. Synergistic Catalysis of Tandem Michael Addition/Enantioselective Protonation Reactions by an Artificial Enzyme. ACS Catal. 2021, 11 (15), 9366– 9369, DOI: 10.1021/acscatal.1c02298Google ScholarThere is no corresponding record for this reference.
- 497Gran-Scheuch, A.; Bonandi, E.; Drienovská, I. Expanding the Genetic Code: Incorporation of Functional Secondary Amines via Stop Codon Suppression. ChemCatChem 2024, 16, e202301004 DOI: 10.1002/cctc.202301004Google ScholarThere is no corresponding record for this reference.
- 498Longwitz, L.; Leveson-Gower, R. B.; Rozeboom, H. J.; Thunnissen, A.-M. W. H.; Roelfes, G. Boron Catalysis in a Designer Enzyme. Nature 2024, 629, 824, DOI: 10.1038/s41586-024-07391-3Google ScholarThere is no corresponding record for this reference.
- 499Garrido-Castro, A. F.; Maestro, M. C.; Alemán, J. Asymmetric Induction in Photocatalysis - Discovering a New Side to Light-Driven Chemistry. Tetrahedron Lett. 2018, 59 (14), 1286– 1294, DOI: 10.1016/j.tetlet.2018.02.040Google ScholarThere is no corresponding record for this reference.
- 500Yao, W.; Bazan-Bergamino, E. A.; Ngai, M.-Y. Asymmetric Photocatalysis Enabled by Chiral Organocatalysts. ChemCatChem 2022, 14 (1), e202101292 DOI: 10.1002/cctc.202101292Google Scholar500Asymmetric Photocatalysis Enabled by Chiral OrganocatalystsYao, Wang; Bazan-Bergamin, Emmanuel A.; Ngai, Ming-YuChemCatChem (2022), 14 (1), e202101292CODEN: CHEMK3; ISSN:1867-3880. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Visible-light photocatalysis has advanced as a versatile tool in org. synthesis. However, attaining precise stereocontrol in photocatalytic reactions has been a longstanding challenge due to undesired photochem. background reactions and the involvement of highly reactive radicals or radical ion intermediates generated under photocatalytic conditions. To address this problem and expand the synthetic utility of photocatalytic reactions, a no. of innovative strategies, including mono- and dual-catalytic approaches, have recently emerged. Of these, exploiting chiral organocatalysis, such as enamine catalysis, iminium-ion catalysis, Broensted acid/base catalysis, and N-heterocyclic carbene catalysis, to induce chirality transfer of photocatalytic reactions has been widely explored. This Review aims to provide a current, comprehensive overview of asym. photocatalytic reactions enabled by chiral organocatalysts published through June 2021. The substrate scope, advantages, limitations, and proposed reaction mechanisms of each reaction are discussed. This review should serve as a ref. for the development of visible-light-induced asym. photocatalysis and promote the improvement of the chem. reactivity and stereoselectivity of these reactions.
- 501Heyes, D. J.; Lakavath, B.; Hardman, S. J. O.; Sakuma, M.; Hedison, T. M.; Scrutton, N. S. Photochemical Mechanism of Light-Driven Fatty Acid Photodecarboxylase. ACS Catal. 2020, 10 (12), 6691– 6696, DOI: 10.1021/acscatal.0c01684Google Scholar501Photochemical Mechanism of Light-Driven Fatty Acid PhotodecarboxylaseHeyes, Derren J.; Lakavath, Balaji; Hardman, Samantha J. O.; Sakuma, Michiyo; Hedison, Tobias M.; Scrutton, Nigel S.ACS Catalysis (2020), 10 (12), 6691-6696CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Fatty acid photodecarboxylase (FAP) is a promising target for the prodn. of biofuels and fine chems. It contains a FAD cofactor and catalyzes the blue-light-dependent decarboxylation of fatty acids to generate the corresponding alkane. However, little is known about the catalytic mechanism of FAP, or how light is used to drive enzymic decarboxylation. Here, we have used a combination of time-resolved and cryogenic trapping UV-visible absorption spectroscopy to characterize a red-shifted flavin intermediate obsd. in the catalytic cycle of FAP. We show that this intermediate can form below the "glass transition" temp. of proteins, whereas the subsequent decay of the species proceeds only at higher temps., implying a role for protein motions in the decay of the intermediate. Solvent isotope effect measurements, combined with analyses of selected site-directed variants of FAP, suggest that the formation of the red-shifted flavin species is directly coupled with hydrogen atom transfer from a nearby active site cysteine residue, yielding the final alkane product. Our study suggests that this cysteine residue forms a thiolate-flavin charge-transfer species, which is assigned as the red-shifted flavin intermediate. Taken together, our data provide insights into light-dependent decarboxylase mechanisms catalyzed by FAP and highlight important considerations in the (re)design of flavin-based photoenzymes.
- 502Heyes, D. J.; Zhang, S.; Taylor, A.; Johannissen, L. O.; Hardman, S. J. O.; Hay, S.; Scrutton, N. S. Photocatalysis as the ‘Master Switch’ of Photomorphogenesis in Early Plant Development. Nature Plants 2021, 7 (3), 268– 276, DOI: 10.1038/s41477-021-00866-5Google ScholarThere is no corresponding record for this reference.
- 503Tan, C.; Liu, Z.; Li, J.; Guo, X.; Wang, L.; Sancar, A.; Zhong, D. The Molecular Origin of High DNA-Repair Efficiency by Photolyase. Nature Commun. 2015, 6 (1), 7302, DOI: 10.1038/ncomms8302Google ScholarThere is no corresponding record for this reference.
- 504Black, M. J.; Biegasiewicz, K. F.; Meichan, A. J.; Oblinsky, D. G.; Kudisch, B.; Scholes, G. D.; Hyster, T. K. Asymmetric Redox-Neutral Radical Cyclization Catalysed by Flavin-Dependent ‘Ene’-Reductases. Nat. Chem. 2020, 12 (1), 71– 75, DOI: 10.1038/s41557-019-0370-2Google Scholar504Asymmetric redox-neutral radical cyclization catalysed by flavin-dependent 'ene'-reductasesBlack Michael J; Biegasiewicz Kyle F; Meichan Andrew J; Oblinsky Daniel G; Kudisch Bryan; Scholes Gregory D; Hyster Todd KNature chemistry (2020), 12 (1), 71-75 ISSN:.Flavin-dependent 'ene'-reductases (EREDs) are exquisite catalysts for effecting stereoselective reductions. Although these reactions typically proceed through a hydride transfer mechanism, we recently found that EREDs can also catalyse reductive dehalogenations and cyclizations via single electron transfer mechanisms. Here, we demonstrate that these enzymes can catalyse redox-neutral radical cyclizations to produce enantioenriched oxindoles from α-haloamides. This transformation is a C-C bond-forming reaction currently unknown in nature and one for which there are no catalytic asymmetric examples. Mechanistic studies indicate the reaction proceeds via the flavin semiquinone/quinone redox couple, where ground-state flavin semiquinone provides the electron for substrate reduction and flavin quinone oxidizes the vinylogous α-amido radical formed after cyclization. This mechanistic manifold was previously unknown for this enzyme family, highlighting the versatility of EREDs in asymmetric synthesis.
- 505Emmanuel, M. A.; Greenberg, N. R.; Oblinsky, D. G.; Hyster, T. K. Accessing Non-Natural Reactivity by Irradiating Nicotinamide-Dependent Enzymes with Light. Nature 2016, 540 (7633), 414– 417, DOI: 10.1038/nature20569Google Scholar505Accessing non-natural reactivity by irradiating nicotinamide-dependent enzymes with lightEmmanuel, Megan A.; Greenberg, Norman R.; Oblinsky, Daniel G.; Hyster, Todd K.Nature (London, United Kingdom) (2016), 540 (7633), 414-417CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Enzymes are ideal for use in asym. catalysis by the chem. industry, because their chem. compns. can be tailored to a specific substrate and selectivity pattern while providing efficiencies and selectivities that surpass those of classical synthetic methods. However, enzymes are limited to reactions that are found in nature and, as such, facilitate fewer types of transformation than do other forms of catalysis. Thus, a longstanding challenge in the field of biol. mediated catalysis has been to develop enzymes with new catalytic functions. Here we describe a method for achieving catalytic promiscuity that uses the photoexcited state of nicotinamide cofactors (mols. that assist enzyme-mediated catalysis). Under irradn. with visible light, the nicotinamide-dependent enzyme known as ketoreductase (KRED) can be transformed from a carbonyl reductase into an initiator of radical species and a chiral source of hydrogen atoms. We demonstrate this new reactivity through a highly enantioselective radical dehalogenation of lactones - a challenging transformation for small-mol. catalysts. Mechanistic expts. support the theory that a radical species acts as an intermediate in this reaction, with NADH and NADPH (the reduced forms of nicotinamide adenine nucleotide and NADP, resp.) serving as both a photoreductant and the source of hydrogen atoms. To our knowledge, this method represents the first example of photoinduced enzyme promiscuity, and highlights the potential for accessing new reactivity from existing enzymes simply by using the excited states of common biol. cofactors. This represents a departure from existing light-driven biocatalytic techniques, which are typically explored in the context of cofactor regeneration.
- 506Liu, X.; Kang, F.; Hu, C.; Wang, L.; Xu, Z.; Zheng, D.; Gong, W.; Lu, Y.; Ma, Y.; Wang, J. A Genetically Encoded Photosensitizer Protein Facilitates the Rational Design of a Miniature Photocatalytic CO2-Reducing Enzyme. Nat. Chem. 2018, 10 (12), 1201– 1206, DOI: 10.1038/s41557-018-0150-4Google Scholar506A genetically encoded photosensitizer protein facilitates the rational design of a miniature photocatalytic CO2-reducing enzymeLiu, Xiaohong; Kang, Fuying; Hu, Cheng; Wang, Li; Xu, Zhen; Zheng, Dandan; Gong, Weimin; Lu, Yi; Ma, Yanhe; Wang, JiangyunNature Chemistry (2018), 10 (12), 1201-1206CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Photosensitizers, which harness light energy to upgrade weak reductants to strong reductants, are pivotal components of the natural and artificial photosynthesis machineries. However, it has proved difficult to enhance and expand their functions through genetic engineering. Here the authors report a genetically encoded, 27 kDa photosensitizer protein (PSP), which facilitates the rational design of miniature photocatalytic CO2-reducing enzymes. Visible light drives PSP efficiently into a long-lived triplet excited state (PSP*), which reacts rapidly with reduced NAD to generate a super-reducing radical (PSP•), which is strong enough to reduce many CO2-reducing catalysts. The authors detd. the three-dimensional structure of PSP• at 1.8 Å resoln. by x-ray crystallog. Genetic engineering enabled the site-specific attachment of a nickel-terpyridine complex and the modular optimization of the photochem. properties of PSP, the chromophore/catalytic center distance and the catalytic center microenvironment, which culminated in a miniature photocatalytic CO2-reducing enzyme that has a CO2/CO conversion quantum efficiency of 2.6%.
- 507Siegel, J. B.; Zanghellini, A.; Lovick, H. M.; Kiss, G.; Lambert, A. R.; St. Clair, J. L.; Gallaher, J. L.; Hilvert, D.; Gelb, M. H.; Stoddard, B. L.; Houk, K. N.; Michael, F. E.; Baker, D. Computational Design of an Enzyme Catalyst for a Stereoselective Bimolecular Diels-Alder Reaction. Science 2010, 329, 309– 313, DOI: 10.1126/science.1190239Google Scholar507Computational Design of an Enzyme Catalyst for a Stereoselective Bimolecular Diels-Alder ReactionSiegel, Justin B.; Zanghellini, Alexandre; Lovick, Helena M.; Kiss, Gert; Lambert, Abigail R.; St. Clair, Jennifer L.; Gallaher, Jasmine L.; Hilvert, Donald; Gelb, Michael H.; Stoddard, Barry L.; Houk, Kendall N.; Michael, Forrest E.; Baker, DavidScience (Washington, DC, United States) (2010), 329 (5989), 309-313CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The Diels-Alder reaction is a cornerstone in org. synthesis, forming two carbon-carbon bonds and up to four new stereogenic centers in one step. No naturally occurring enzymes have been shown to catalyze bimol. Diels-Alder reactions. We describe the de novo computational design and exptl. characterization of enzymes catalyzing a bimol. Diels-Alder reaction with high stereoselectivity and substrate specificity. X-ray crystallog. confirms that the structure matches the design for the most active of the enzymes, and binding site substitutions reprogram the substrate specificity. Designed stereoselective catalysts for carbon-carbon bond-forming reactions should be broadly useful in synthetic chem.
- 508Sun, N.; Huang, J.; Qian, J.; Zhou, T.-P.; Guo, J.; Tang, L.; Zhang, W.; Deng, Y.; Zhao, W.; Wu, G. Enantioselective [2 + 2]-Cycloadditions with Triplet Photoenzymes. Nature 2022, 611 (7937), 715– 720, DOI: 10.1038/s41586-022-05342-4Google Scholar508Enantioselective [2+2]-cycloadditions with triplet photoenzymesSun, Ningning; Huang, Jianjian; Qian, Junyi; Zhou, Tai-Ping; Guo, Juan; Tang, Langyu; Zhang, Wentao; Deng, Yaming; Zhao, Weining; Wu, Guojiao; Liao, Rong-Zhen; Chen, Xi; Zhong, Fangrui; Wu, YuzhouNature (London, United Kingdom) (2022), 611 (7937), 715-720CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)Naturally evolved enzymes, despite their astonishingly large variety and functional diversity, operate predominantly through thermochem. activation. Integrating prominent photocatalysis modes into proteins, such as triplet energy transfer, could create artificial photoenzymes that expand the scope of natural biocatalysis1-3. Here, we exploit genetically reprogrammed, chem. evolved photoenzymes embedded with a synthetic triplet photosensitizer that are capable of excited-state enantio-induction4-6. Structural optimization through four rounds of directed evolution afforded proficient variants for the enantioselective intramol. [2+2]-photocycloaddn. of indole derivs. with good substrate generality and excellent enantioselectivities (up to 99% enantiomeric excess). A crystal structure of the photoenzyme-substrate complex elucidated the non-covalent interactions that mediate the reaction stereochem. This study expands the energy transfer reactivity7-10 of artificial triplet photoenzymes in a supramol. protein cavity and unlocks an integrated approach to valuable enantioselective photochem. synthesis that is not accessible with either the synthetic or the biol. world alone.
- 509Allen, A. R.; Noten, E. A.; Stephenson, C. R. J. Aryl Transfer Strategies Mediated by Photoinduced Electron Transfer. Chem. Rev. 2022, 122 (2), 2695– 2751, DOI: 10.1021/acs.chemrev.1c00388Google Scholar509Aryl Transfer Strategies Mediated by Photoinduced Electron TransferAllen, Anthony R.; Noten, Efrey A.; Stephenson, Corey R. J.Chemical Reviews (Washington, DC, United States) (2022), 122 (2), 2695-2751CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. This review encapsulates progress in radical aryl migration enabled by photochem. methods-particularly photoredox catalysis-since 2015. Special attention is paid to descriptions of scope, mechanism, and synthetic applications of each method.
- 510Dadashi-Silab, S.; Doran, S.; Yagci, Y. Photoinduced Electron Transfer Reactions for Macromolecular Syntheses. Chem. Rev. 2016, 116 (17), 10212– 10275, DOI: 10.1021/acs.chemrev.5b00586Google Scholar510Photoinduced Electron Transfer Reactions for Macromolecular SynthesesDadashi-Silab, Sajjad; Doran, Sean; Yagci, YusufChemical Reviews (Washington, DC, United States) (2016), 116 (17), 10212-10275CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)Photochem. reactions, particularly those involving photoinduced electron transfer processes, establish a substantial contribution to the modern synthetic chem., and the polymer community has been increasingly interested in exploiting and developing novel photochem. strategies. These reactions are efficiently utilized in almost every aspect of macromol. architecture synthesis, involving initiation, control of the reaction kinetics and mol. structures, functionalization, and decoration, etc. Merging with polymn. techniques, photochem. has opened up new intriguing and powerful avenues for macromol. synthesis. Construction of various polymers with incredibly complex structures and specific control over the chain topol., as well as providing the opportunity to manipulate the reaction course through spatiotemporal control, are one of the unique abilities of such photochem. reactions. This review paper provides a comprehensive account of the fundamentals and applications of photoinduced electron transfer reactions in polymer synthesis. Besides traditional photopolymn. methods, namely free radical and cationic polymns., step-growth polymns. involving electron transfer processes are included. In addn., controlled radical polymn. and "Click Chem." methods have significantly evolved over the last few decades allowing access to narrow mol. wt. distributions, efficient regulation of the mol. wt. and the monomer sequence and incredibly complex architectures, and polymer modifications and surface patterning are covered. Potential applications including synthesis of block and graft copolymers, polymer-metal nanocomposites, various hybrid materials and bioconjugates, and sequence defined polymers through photoinduced electron transfer reactions are also investigated in detail.
- 511Reid, B. G.; Flynn, G. C. Chromophore Formation in Green Fluorescent Protein. Biochemistry 1997, 36 (22), 6786– 6791, DOI: 10.1021/bi970281wGoogle Scholar511Chromophore formation in green fluorescent proteinReid, Brian G.; Flynn, Gregory C.Biochemistry (1997), 36 (22), 6786-6791CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The green fluorescent protein (GFP) from the jellyfish Aequorea victoria forms an intrinsic chromophore through cyclization and oxidn. of an internal tripeptide motif. Here, the authors monitored the formation of the chromophore in vitro using the S65T-GFP chromophore mutant. S65T-GFP recovered from inclusion bodies in Escherichia coli lacked the mature chromophore, suggesting that protein destined for inclusion bodies aggregated prior to productive folding. This material was used to follow the steps leading to chromophore formation. The process of chromophore formation in S65T-GFP was detd. to be an ordered reaction consisting of 3 distinct kinetic steps. Protein folding occurred fairly slowly (kf = 2.44 × 10-3 s-1) and prior to any chromophore modification. Next, an intermediate step occurred that included, but was not necessarily limited to, cyclization of the tripeptide chromophore motif (kc = 3.8 × 10-3 s-1). The final and slow step (kox = 1.51 × 10-4 s-1) in chromophore formation involved oxidn. of the cyclized chromophore. Since the chromophore formed de novo from purified denatured protein which was a 1st-order process, it was conclude that GFP chromophore formation is an autocatalytic process.
- 512Fu, Y.; Huang, J.; Wu, Y.; Liu, X.; Zhong, F.; Wang, J. Biocatalytic Cross-Coupling of Aryl Halides with a Genetically Engineered Photosensitizer Artificial Dehalogenase. J. Am. Chem. Soc. 2021, 143 (2), 617– 622, DOI: 10.1021/jacs.0c10882Google ScholarThere is no corresponding record for this reference.
- 513Gu, Y.; Ellis-Guardiola, K.; Srivastava, P.; Lewis, J. C. Preparation, Characterization, and Oxygenase Activity of a Photocatalytic Artificial Enzyme. ChemBioChem 2015, 16 (13), 1880– 1883, DOI: 10.1002/cbic.201500165Google ScholarThere is no corresponding record for this reference.
- 514Zubi, Y. S.; Liu, B.; Gu, Y.; Sahoo, D.; Lewis, J. C. Controlling the Optical and Catalytic Properties of Artificial Metalloenzyme Photocatalysts Using Chemogenetic Engineering. Chem. Sci. 2022, 13 (5), 1459– 1468, DOI: 10.1039/D1SC05792HGoogle ScholarThere is no corresponding record for this reference.
- 515Liu, B.; Zubi, Y. S.; Lewis, J. C. Iridium(iii) Polypyridine Artificial Metalloenzymes with Tunable Photophysical Properties: A New Platform for Visible Light Photocatalysis in Aqueous Solution. Dalton Trans. 2023, 52 (16), 5034– 5038, DOI: 10.1039/D3DT00932GGoogle ScholarThere is no corresponding record for this reference.
- 516Lee, J.; Song, W. J. Photocatalytic C-O Coupling Enzymes That Operate via Intramolecular Electron Transfer. J. Am. Chem. Soc. 2023, 145 (9), 5211– 5221, DOI: 10.1021/jacs.2c12226Google ScholarThere is no corresponding record for this reference.
- 517Mills, J. H.; Sheffler, W.; Ener, M. E.; Almhjell, P. J.; Oberdorfer, G.; Pereira, J. H.; Parmeggiani, F.; Sankaran, B.; Zwart, P. H.; Baker, D. Computational Design of a Homotrimeric Metalloprotein with a Trisbipyridyl Core. Proc. Natl. Acad. Sci. U.S.A. 2016, 113 (52), 15012– 15017, DOI: 10.1073/pnas.1600188113Google Scholar517Computational design of a homotrimeric metalloprotein with a trisbipyridyl coreMills, Jeremy H.; Sheffler, William; Ener, Maraia E.; Almhjell, Patrick J.; Oberdorfer, Gustav; Pereira, Jose Henrique; Parmeggiani, Fabio; Sankaran, Banumathi; Zwart, Peter H.; Baker, DavidProceedings of the National Academy of Sciences of the United States of America (2016), 113 (52), 15012-15017CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Metal-chelating heteroaryl small mols. have found widespread use as building blocks for coordination-driven, self-assembling nanostructures. The metal-chelating noncanonical amino acid (2,2'-bipyridin-5yl)alanine (Bpy-ala) could, in principle, be used to nucleate specific metalloprotein assemblies if introduced into proteins such that one assembly had much lower free energy than all alternatives. Here we describe the use of the Rosetta computational methodol. to design a self-assembling homotrimeric protein with [Fe(Bpy-ala)3]2+ complexes at the interface between monomers. X-ray crystallog. anal. of the homotrimer showed that the design process had near-at.-level accuracy: The all-atom rmsd between the design model and crystal structure for the residues at the protein interface is ∼1.4 Å. These results demonstrate that computational protein design together with genetically encoded noncanonical amino acids can be used to drive formation of precisely specified metal-mediated protein assemblies that could find use in a wide range of photophys. applications.
- 518Duan, H.-Z.; Hu, C.; Li, Y.-L.; Wang, S.-H.; Xia, Y.; Liu, X.; Wang, J.; Chen, Y.-X. Genetically Encoded Phosphine Ligand for Metalloprotein Design. J. Am. Chem. Soc. 2022, 144 (50), 22831– 22837, DOI: 10.1021/jacs.2c09683Google ScholarThere is no corresponding record for this reference.
- 519Beattie, A. T.; Dunkelmann, D. L.; Chin, J. W. Quintuply Orthogonal Pyrrolysyl-tRNA Synthetase/tRNAPyl Pairs. Nat. Chem. 2023, 15 (7), 948– 959, DOI: 10.1038/s41557-023-01232-yGoogle ScholarThere is no corresponding record for this reference.
- 520Dunkelmann, D. L.; Willis, J. C. W.; Beattie, A. T.; Chin, J. W. Engineered Triply Orthogonal Pyrrolysyl-tRNA Synthetase/tRNA Pairs Enable the Genetic Encoding of Three Distinct Non-Canonical Amino Acids. Nat. Chem. 2020, 12 (6), 535– 544, DOI: 10.1038/s41557-020-0472-xGoogle Scholar520Engineered triply orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs enable the genetic encoding of three distinct non-canonical amino acidsDunkelmann, Daniel L.; Willis, Julian C. W.; Beattie, Adam T.; Chin, Jason W.Nature Chemistry (2020), 12 (6), 535-544CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Expanding and reprogramming the genetic code of cells for the incorporation of multiple distinct non-canonical amino acids (ncAAs), and the encoded biosynthesis of non-canonical biopolymers, requires the discovery of multiple orthogonal aminoacyl-tRNA synthetase/tRNA pairs. These pairs must be orthogonal to both the host synthetases and tRNAs and to each other. Pyrrolysyl-tRNA synthetase (PylRS)/PyltRNA pairs are the most widely used system for genetic code expansion. Here, we reveal that the sequences of ΔNPylRS/ΔNPyltRNA pairs (which lack N-terminal domains) form two distinct classes. We show that the measured specificities of the ΔNPylRSs and ΔNPyltRNAs correlate with sequence-based clustering, and most ΔNPylRSs preferentially function with ΔNPyltRNAs from their class. We then identify 18 mutually orthogonal pairs from the 88 ΔNPylRS/ΔNPyltRNA combinations tested. Moreover, we generate a set of 12 triply orthogonal pairs, each composed of three new PylRS/PyltRNA pairs. Finally, we diverge the ncAA specificity and decoding properties of each pair, within a triply orthogonal set, and direct the incorporation of three distinct non-canonical amino acids into a single polypeptide.
- 521Italia, J. S.; Addy, P. S.; Erickson, S. B.; Peeler, J. C.; Weerapana, E.; Chatterjee, A. Mutually Orthogonal Nonsense-Suppression Systems and Conjugation Chemistries for Precise Protein Labeling at up to Three Distinct Sites. J. Am. Chem. Soc. 2019, 141 (15), 6204– 6212, DOI: 10.1021/jacs.8b12954Google Scholar521Mutually Orthogonal Nonsense-Suppression Systems and Conjugation Chemistries for Precise Protein Labeling at up to Three Distinct SitesItalia, James S.; Addy, Partha Sarathi; Erickson, Sarah B.; Peeler, Jennifer C.; Weerapana, Eranthie; Chatterjee, AbhishekJournal of the American Chemical Society (2019), 141 (15), 6204-6212CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Site-specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into a protein is an emerging technol. with tremendous potential. It relies on mutually orthogonal engineered aminoacyl-tRNA synthetase/tRNA pairs that suppress different nonsense/frameshift codons. So far, up to two distinct ncAAs have been incorporated into proteins expressed in E. coli, using archaea-derived tyrosyl and pyrrolysyl pairs. Here we report that the E. coli derived tryptophanyl pair can be combined with the archaeal tyrosyl or the pyrrolysyl pair in ATMW1 E. coli to incorporate two different ncAAs into one protein with high fidelity and efficiency. By combining all three orthogonal pairs, we further demonstrate simultaneous site-specific incorporation of three different ncAAs into one protein. To use this technol. for chemoselectively labeling proteins with multiple distinct entities at predefined sites, we also sought to identify different bioconjugation handles that can be coincorporated into proteins as ncAA-side chains and subsequently functionalized through mutually compatible labeling chemistries. To this end, we show that the recently developed chemoselective rapid azo-coupling reaction (CRACR) directed to 5-hydroxytryptophan (5HTP) is compatible with strain-promoted azide-alkyne cycloaddn. (SPAAC) targeted to p-azidophenylalanine (pAzF) and strain-promoted inverse electron-demand Diels-Alder cycloaddn. (SPIEDAC) targeted to cyclopropene-lysine (CpK) for rapid, catalyst-free protein labeling at multiple sites. Combining these mutually orthogonal nonsense suppression systems and the mutually compatible bioconjugation handles they incorporate, we demonstrate site-specific labeling of recombinantly expressed proteins at up to three distinct sites.
- 522Hashimoto, K.; Fischer, E. C.; Romesberg, F. E. Efforts toward Further Integration of an Unnatural Base Pair into the Biology of a Semisynthetic Organism. J. Am. Chem. Soc. 2021, 143 (23), 8603– 8607, DOI: 10.1021/jacs.1c03860Google ScholarThere is no corresponding record for this reference.
- 523Ledbetter, M. P.; Karadeema, R. J.; Romesberg, F. E. Reprograming the Replisome of a Semisynthetic Organism for the Expansion of the Genetic Alphabet. J. Am. Chem. Soc. 2018, 140 (2), 758– 765, DOI: 10.1021/jacs.7b11488Google Scholar523Reprograming the Replisome of a Semisynthetic Organism for the Expansion of the Genetic AlphabetLedbetter, Michael P.; Karadeema, Rebekah J.; Romesberg, Floyd E.Journal of the American Chemical Society (2018), 140 (2), 758-765CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Semi-synthetic organisms (SSOs) created from Escherichia coli can replicate a plasmid contg. an unnatural base pair (UBP) formed between the synthetic nucleosides dNaM and dTPT3 (dNaM-dTPT3) when the corresponding unnatural triphosphates are imported via expression of a nucleoside triphosphate transporter. The UBP can also be transcribed and used to translate proteins contg. unnatural amino acids. However, UBPs are not well retained in all sequences, limiting the information that can be encoded, and are invariably lost upon extended growth. Here we explore the contributions of the E. coli DNA replication and repair machinery to the propagation of DNA contg. dNaM-dTPT3 and show that replication by DNA polymerase III, supplemented with the activity of polymerase II and methyl-directed mismatch repair contribute to retention of the UBP and that recombinational repair of stalled forks is responsible for the majority of its loss. This work elucidates fundamental aspects of how bacteria replicate DNA and we use this information to reprogram the replisome of the SSO for increased UBP retention, which then allowed for the first time the construction of SSOs harboring a UBP in their chromosome.
- 524Stucki, A.; Vallapurackal, J.; Ward, T. R.; Dittrich, P. S. Droplet Microfluidics and Directed Evolution of Enzymes: An Intertwined Journey. Angew. Chem. Int. Ed. 2021, 60 (46), 24368– 24387, DOI: 10.1002/anie.202016154Google Scholar524Droplet Microfluidics and Directed Evolution of Enzymes: An Intertwined JourneyStucki, Ariane; Vallapurackal, Jaicy; Ward, Thomas R.; Dittrich, Petra S.Angewandte Chemie, International Edition (2021), 60 (46), 24368-24387CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Evolution is essential to the generation of complexity and ultimately life. It relies on the propagation of the properties, traits, and characteristics that allow an organism to survive in a challenging environment. It is evolution that shaped our world over about four billion years by slow and iterative adaptation. While natural evolution based on selection is slow and gradual, directed evolution allows the fast and streamlined optimization of a phenotype under selective conditions. The potential of directed evolution for the discovery and optimization of enzymes is mostly limited by the throughput of the tools and methods available for screening. Over the past twenty years, versatile tools based on droplet microfluidics have been developed to address the need for higher throughput. In this Review, we provide a chronol. overview of the intertwined development of microfluidics droplet-based compartmentalization methods and in vivo directed evolution of enzymes.
- 525Wicky, B. I. M.; Milles, L. F.; Courbet, A.; Ragotte, R. J.; Dauparas, J.; Kinfu, E.; Tipps, S.; Kibler, R. D.; Baek, M.; DiMaio, F. Hallucinating Symmetric Protein Assemblies. Science 2022, 378 (6615), 56– 61, DOI: 10.1126/science.add1964Google Scholar525Hallucinating symmetric protein assembliesWicky, B. I. M.; Milles, L. F.; Courbet, A.; Ragotte, R. J.; Dauparas, J.; Kinfu, E.; Tipps, S.; Kibler, R. D.; Baek, M.; DiMaio, F.; Li, X.; Carter, L.; Kang, A.; Nguyen, H.; Bera, A. K.; Baker, D.Science (Washington, DC, United States) (2022), 378 (6615), 56-61CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Deep learning generative approaches provide an opportunity to broadly explore protein structure space beyond the sequences and structures of natural proteins. Here, we use deep network hallucination to generate a wide range of sym. protein homo-oligomers given only a specification of the no. of protomers and the protomer length. Crystal structures of seven designs are very similar to the computational models (median root mean square deviation: 0.6 angstroms), as are three cryo-electron microscopy structures of giant 10-nm rings with up to 1550 residues and C33 symmetry; all differ considerably from previously solved structures. Our results highlight the rich diversity of new protein structures that can be generated using deep learning and pave the way for the design of increasingly complex components for nanomachines and biomaterials.
- 526Wang, J.; Lisanza, S.; Juergens, D.; Tischer, D.; Watson, J. L.; Castro, K. M.; Ragotte, R.; Saragovi, A.; Milles, L. F.; Baek, M. Scaffolding Protein Functional Sites Using Deep Learning. Science 2022, 377 (6604), 387– 394, DOI: 10.1126/science.abn2100Google Scholar526Scaffolding protein functional sites using deep learningWang, Jue; Lisanza, Sidney; Juergens, David; Tischer, Doug; Watson, Joseph L.; Castro, Karla M.; Ragotte, Robert; Saragovi, Amijai; Milles, Lukas F.; Baek, Minkyung; Anishchenko, Ivan; Yang, Wei; Hicks, Derrick R.; Exposit, Marc; Schlichthaerle, Thomas; Chun, Jung-Ho; Dauparas, Justas; Bennett, Nathaniel; Wicky, Basile I. M.; Muenks, Andrew; DiMaio, Frank; Correia, Bruno; Ovchinnikov, Sergey; Baker, DavidScience (Washington, DC, United States) (2022), 377 (6604), 387-394CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)A review. The binding and catalytic functions of proteins are generally mediated by a small no. of functional residues held in place by the overall protein structure. Here, we describe deep learning approaches for scaffolding such functional sites without needing to prespecify the fold or secondary structure of the scaffold. The first approach, "constrained hallucination," optimizes sequences such that their predicted structures contain the desired functional site. The second approach, "inpainting," starts from the functional site and fills in addnl. sequence and structure to create a viable protein scaffold in a single forward pass through a specifically trained RoseTTAFold network. We use these two methods to design candidate immunogens, receptor traps, metalloproteins, enzymes, and protein-binding proteins and validate the designs using a combination of in silico and exptl. tests.
- 527Krishna, R.; Wang, J.; Ahern, W.; Sturmfels, P.; Venkatesh, P.; Kalvet, I.; Lee, G. R.; Morey-Burros, F. S.; Anishchenko, I.; Humphreys, I. R. Generalized Biomolecular Modeling and Design with RoseTTAFold All-Atom. Science 2024, DOI: 10.1126/science.adl2528Google ScholarThere is no corresponding record for this reference.
- 528Jumper, J.; Evans, R.; Pritzel, A.; Green, T.; Figurnov, M.; Ronneberger, O.; Tunyasuvunakool, K.; Bates, R.; Žídek, A.; Potapenko, A. Highly Accurate Protein Structure Prediction with AlphaFold. Nature 2021, 596 (7873), 583– 589, DOI: 10.1038/s41586-021-03819-2Google Scholar528Highly accurate protein structure prediction with AlphaFoldJumper, John; Evans, Richard; Pritzel, Alexander; Green, Tim; Figurnov, Michael; Ronneberger, Olaf; Tunyasuvunakool, Kathryn; Bates, Russ; Zidek, Augustin; Potapenko, Anna; Bridgland, Alex; Meyer, Clemens; Kohl, Simon A. A.; Ballard, Andrew J.; Cowie, Andrew; Romera-Paredes, Bernardino; Nikolov, Stanislav; Jain, Rishub; Adler, Jonas; Back, Trevor; Petersen, Stig; Reiman, David; Clancy, Ellen; Zielinski, Michal; Steinegger, Martin; Pacholska, Michalina; Berghammer, Tamas; Bodenstein, Sebastian; Silver, David; Vinyals, Oriol; Senior, Andrew W.; Kavukcuoglu, Koray; Kohli, Pushmeet; Hassabis, DemisNature (London, United Kingdom) (2021), 596 (7873), 583-589CODEN: NATUAS; ISSN:0028-0836. (Nature Portfolio)Proteins are essential to life, and understanding their structure can facilitate a mechanistic understanding of their function. Through an enormous exptl. effort, the structures of around 100,000 unique proteins have been detd., but this represents a small fraction of the billions of known protein sequences. Structural coverage is bottlenecked by the months to years of painstaking effort required to det. a single protein structure. Accurate computational approaches are needed to address this gap and to enable large-scale structural bioinformatics. Predicting the three-dimensional structure that a protein will adopt based solely on its amino acid sequence-the structure prediction component of the 'protein folding problem'-has been an important open research problem for more than 50 years. Despite recent progress, existing methods fall far short of at. accuracy, esp. when no homologous structure is available. Here we provide the first computational method that can regularly predict protein structures with at. accuracy even in cases in which no similar structure is known. We validated an entirely redesigned version of our neural network-based model, AlphaFold, in the challenging 14th Crit. Assessment of protein Structure Prediction (CASP14), demonstrating accuracy competitive with exptl. structures in a majority of cases and greatly outperforming other methods. Underpinning the latest version of AlphaFold is a novel machine learning approach that incorporates phys. and biol. knowledge about protein structure, leveraging multi-sequence alignments, into the design of the deep learning algorithm.
- 529Baumann, T.; Hauf, M.; Richter, F.; Albers, S.; Möglich, A.; Ignatova, Z.; Budisa, N. Computational Aminoacyl-tRNA Synthetase Library Design for Photocaged Tyrosine. Int. J. Mol. Sci. 2019, 20 (9), 2343, DOI: 10.3390/ijms20092343Google ScholarThere is no corresponding record for this reference.
- 530Cervettini, D.; Tang, S.; Fried, S. D.; Willis, J. C. W.; Funke, L. F. H.; Colwell, L. J.; Chin, J. W. Rapid Discovery and Evolution of Orthogonal Aminoacyl-tRNA Synthetase-tRNA Pairs. Nat. Biotechnol. 2020, 38 (8), 989– 999, DOI: 10.1038/s41587-020-0479-2Google Scholar530Rapid discovery and evolution of orthogonal aminoacyl-tRNA synthetase-tRNA pairsCervettini, Daniele; Tang, Shan; Fried, Stephen D.; Willis, Julian C. W.; Funke, Louise F. H.; Colwell, Lucy J.; Chin, Jason W.Nature Biotechnology (2020), 38 (8), 989-999CODEN: NABIF9; ISSN:1087-0156. (Nature Research)Abstr.: A central challenge in expanding the genetic code of cells to incorporate noncanonical amino acids into proteins is the scalable discovery of aminoacyl-tRNA synthetase (aaRS)-tRNA pairs that are orthogonal in their aminoacylation specificity. Here we computationally identify candidate orthogonal tRNAs from millions of sequences and develop a rapid, scalable approach-named tRNA Extension (tREX)-to det. the in vivo aminoacylation status of tRNAs. Using tREX, we test 243 candidate tRNAs in Escherichia coli and identify 71 orthogonal tRNAs, covering 16 isoacceptor classes, and 23 functional orthogonal tRNA-cognate aaRS pairs. We discover five orthogonal pairs, including three highly active amber suppressors, and evolve new amino acid substrate specificities for two pairs. Finally, we use tREX to characterize a matrix of 64 orthogonal synthetase-orthogonal tRNA specificities. This work expands the no. of orthogonal pairs available for genetic code expansion and provides a pipeline for the discovery of addnl. orthogonal pairs and a foundation for encoding the cellular synthesis of noncanonical biopolymers.
- 531Taylor, C. J.; Hardy, F. J.; Burke, A. J.; Bednar, R. M.; Mehl, R. A.; Green, A. P.; Lovelock, S. L. Engineering Mutually Orthogonal PylRS/tRNA Pairs for Dual Encoding of Functional Histidine Analogues. Protein Sci. 2023, 32 (5), e4640 DOI: 10.1002/pro.4640Google ScholarThere is no corresponding record for this reference.
- 532Fredens, J.; Wang, K.; de la Torre, D.; Funke, L. F. H.; Robertson, W. E.; Christova, Y.; Chia, T.; Schmied, W. H.; Dunkelmann, D. L.; Beránek, V. Total Synthesis of Escherichia coli with a Recoded Genome. Nature 2019, 569 (7757), 514– 518, DOI: 10.1038/s41586-019-1192-5Google Scholar532Total synthesis of Escherichia coli with a recoded genomeFredens, Julius; Wang, Kaihang; de la Torre, Daniel; Funke, Louise F. H.; Robertson, Wesley E.; Christova, Yonka; Chia, Tiongsun; Schmied, Wolfgang H.; Dunkelmann, Daniel L.; Beranek, Vaclav; Uttamapinant, Chayasith; Llamazares, Andres Gonzalez; Elliott, Thomas S.; Chin, Jason W.Nature (London, United Kingdom) (2019), 569 (7757), 514-518CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon-out of up to 6 synonyms-to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the no. of codons used to encode the canonical amino acids can be reduced, through the genome-wide substitution of target codons by defined synonyms. We create a variant of Escherichia coli with a four-megabase synthetic genome through a high-fidelity convergent total synthesis. Our synthetic genome implements a defined recoding and refactoring scheme-with simple corrections at just seven positions-to replace every known occurrence of two sense codons and a stop codon in the genome. Thus, we recode 18,214 codons to create an organism with a 61-codon genome; this organism uses 59 codons to encode the 20 amino acids, and enables the deletion of a previously essential tRNA.
- 533Lajoie, M. J.; Rovner, A. J.; Goodman, D. B.; Aerni, H.-R.; Haimovich, A. D.; Kuznetsov, G.; Mercer, J. A.; Wang, H. H.; Carr, P. A.; Mosberg, J. A. Genomically Recoded Organisms Expand Biological Functions. Science 2013, 342 (6156), 357– 360, DOI: 10.1126/science.1241459Google Scholar533Genomically recoded organisms expand biological functionsLajoie, Marc J.; Rovner, Alexis J.; Goodman, Daniel B.; Aerni, Hans-Rudolf; Haimovich, Adrian D.; Kuznetsov, Gleb; Mercer, Jaron A.; Wang, Harris H.; Carr, Peter A.; Mosberg, Joshua A.; Rohland, Nadin; Schultz, Peter G.; Jacobson, Joseph M.; Rinehart, Jesse; Church, George M.; Isaacs, Farren J.Science (Washington, DC, United States) (2013), 342 (6156), 357-360CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)We describe the construction and characterization of a genomically recoded organism (GRO). We replaced all known UAG stop codons in Escherichia coli MG1655 with synonymous UAA codons, which permitted the deletion of release factor 1 and reassignment of UAG translation function. This GRO exhibited improved properties for incorporation of nonstandard amino acids that expand the chem. diversity of proteins in vivo. The GRO also exhibited increased resistance to T7 bacteriophage, demonstrating that new genetic codes could enable increased viral resistance.
- 534Mukai, T.; Hoshi, H.; Ohtake, K.; Takahashi, M.; Yamaguchi, A.; Hayashi, A.; Yokoyama, S.; Sakamoto, K. Highly Reproductive Escherichia coli Cells with No Specific Assignment to the UAG Codon. Sci. Rep. 2015, 5 (1), 9699, DOI: 10.1038/srep09699Google Scholar534Highly reproductive Escherichia coli cells with no specific assignment to the UAG codonMukai, Takahito; Hoshi, Hiroko; Ohtake, Kazumasa; Takahashi, Mihoko; Yamaguchi, Atsushi; Hayashi, Akiko; Yokoyama, Shigeyuki; Sakamoto, KensakuScientific Reports (2015), 5 (), 9699CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)Escherichia coli is a widely used host organism for recombinant technol., and the bacterial incorporation of non-natural amino acids promises the efficient synthesis of proteins with novel structures and properties. In the present study, we developed E. coli strains in which the UAG codon was reserved for non-natural amino acids, without compromising the reproductive strength of the host cells. Ninety-five of the 273 UAG stop codons were replaced synonymously in the genome of E. coli BL21(DE3), by exploiting the oligonucleotide-mediated base-mismatch-repair mechanism. This genomic modification allowed the safe elimination of the UAG-recognizing cellular component (RF-1), thus leaving the remaining 178 UAG codons with no specific mol. recognizing them. The resulting strain B-95.ΔA grew as vigorously as BL21(DE3) in rich medium at 25-42°C, and its deriv. B-95.ΔAΔfabR was better adapted to low temps. and minimal media than B-95.ΔA. UAG was reassigned to synthetic amino acids by expressing the specific pairs of UAG-reading tRNA and aminoacyl-tRNA synthetase. Due to the preserved growth vigor, the B-95.ΔA strains showed superior productivities for hirudin mols. sulfonated on a particular tyrosine residue, and the Fab fragments of Herceptin contg. multiple azido groups.
- 535Wang, T.; Liang, C.; An, Y.; Xiao, S.; Xu, H.; Zheng, M.; Liu, L.; Wang, G.; Nie, L. Engineering the Translational Machinery for Biotechnology Applications. Mol. Biotechnol. 2020, 62 (4), 219– 227, DOI: 10.1007/s12033-020-00246-yGoogle ScholarThere is no corresponding record for this reference.
- 536Zackin, M. T.; Stieglitz, J. T.; Van Deventer, J. A. Genome-Wide Screen for Enhanced Noncanonical Amino Acid Incorporation in Yeast. ACS Synth. Biol. 2022, 11 (11), 3669– 3680, DOI: 10.1021/acssynbio.2c00267Google ScholarThere is no corresponding record for this reference.
- 537Exner, M. P.; Kuenzl, T.; To, T. M. T.; Ouyang, Z.; Schwagerus, S.; Hoesl, M. G.; Hackenberger, C. P. R.; Lensen, M. C.; Panke, S.; Budisa, N. Design of S-Allylcysteine in Situ Production and Incorporation Based on a Novel Pyrrolysyl-tRNA Synthetase Variant. ChemBioChem 2017, 18 (1), 85– 90, DOI: 10.1002/cbic.201600537Google ScholarThere is no corresponding record for this reference.
- 538Kim, S.; Sung, B. H.; Kim, S. C.; Lee, H. S. Genetic Incorporation of L-Dihydroxyphenylalanine (DOPA) Biosynthesized by a Tyrosine Phenol-Lyase. Chem. Commun. 2018, 54 (24), 3002– 3005, DOI: 10.1039/C8CC00281AGoogle ScholarThere is no corresponding record for this reference.
- 539Nojoumi, S.; Ma, Y.; Schwagerus, S.; Hackenberger, C. P. R.; Budisa, N. In-Cell Synthesis of Bioorthogonal Alkene Tag S-Allyl-Homocysteine and Its Coupling with Reprogrammed Translation. Int. J. Mol. Sci. 2019, 20 (9), 2299, DOI: 10.3390/ijms20092299Google ScholarThere is no corresponding record for this reference.
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Abstract
Figure 1
Figure 1. NcAAs discussed in this review. (A) NcAAs incorporated via selective pressure incorporation (SPI), expressed protein ligation (EPL), or solid-phase peptide synthesis (SPPS). (B) NcAAs incorporated by GCE. The orthogonal translation system(s) used to incorporate each ncAA are listed. For several ncAAs, multiple incorporation techniques are discussed in this review, and these are also listed. †DAP is incorporated as a precursor featuring a photocleavable group, which matures to DAP upon irradiation at 365 nm. ‡4-NH2Phe is incorporated as 4-AzPhe, which is then chemically reduced in situ to form 4-NH2Phe.
Figure 2
Figure 2. SPI of ncAAs. SPI employs an auxotrophic expression system to globally replace a target canonical amino acid (cAA) with a close structural analogue. An endogenous aaRS loads its cognate tRNA with the ncAA which is incorporated into proteins. Created with BioRender.com.
Figure 3
Figure 3. Strategies for the generation of ncAA-loaded tRNAs employ either chemoenzymatic methods (top left, PDB: 2C5U (102)) or Flexizymes (bottom left, PDB: 3CUN (103)). These ncAA-tRNAs can then be incorporated into a polypeptide chain using cell-free expression (CFE) systems (right). Created with BioRender.com.
Figure 4
Figure 4. Positive and negative selection processes can be used to engineer orthogonal aaRS-tRNA pairs to improve incorporation efficiency and/or specificity. The engineered aaRS catalyzes an aminoacylation reaction between its cognate tRNA and ncAA, with the ncAA added to the growing polypeptide chain during translation in response to a repurposed codon (e.g., the amber stop codon, UAG). Created with BioRender.com.
Figure 5
Figure 5. DAP incorporation into Valinomycin synthetase. (A) Genetically encoded (2S)-2-amino-3-([(2-[1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl]thio)ethoxy)carbonyl] ncAA is photodeprotected by irradiation at 365 nm to give DAP, which forms stable acyl-enzyme intermediates with an amide bond that is resistant to hydrolysis. (B) The active site of Valinomycin synthetase (protein shown as a gray cartoon, PDB: 6ECE (213)) with a noncanonical DAP nucleophile in position 2463 (atom-colored sticks, brown carbons) bound to a dodecadepsipeptide substrate (atom colored sticks, blue carbons). (C) Large structural differences are observed in the lid region of Valinomycin synthetase when bound to a dodecadepsipeptidyl intermediate (gray cartoon, PDB: 6ECE (213)) in comparison to a tetradepsipeptidyl intermediate (blue cartoon, PDB: 6ECD (213)).
Figure 6
Figure 6. Mechanistic studies on RNRs using ncAAs have shed light on the electron transfer pathway and enabled structural characterization of the active form of the multimer. (A) A cryogenic electron microscopy structure of RNR (PDB: 6W4X (231)) in its active α2β2 form was captured using a 2,3,5-F3Tyr122 mutation. The protein chains are shown as cartoons, and GDP and TPP are shown as red and gray spheres, respectively. (B) The mechanism of RNRs, which catalyze the conversion of nucleoside di- and triphosphates to deoxynucleotides. (236) TR = thioredoxin. (C) DEER experiments provided information on the relative distances between the Tyr122 radical in the unreacted α/β pair and radicals on an N3NDP mechanistic inhibitor or radicals trapped on 3-NH2Tyr.
Figure 7
Figure 7. Mechanistic proposal for the FtmOx1-catalyzed hydrogen atom transfer from Tyr68 to C26•.
Figure 8
Figure 8. 3-ClTyr incorporation into Ketosteroid Isomerase (KSI) to tune the active site electric field. (A) The mechanism of KSI. (B) The active site of KSI (PDB: 5KP1 (254)) with the ncAA 3-ClTyr in the active site, shown with orange carbons. The protein backbone is shown as a gray cartoon. Active site residues and the substrate and transition state analogue equilenin are shown as atom-colored sticks, with gray and blue carbons, respectively. (C) The product analogue 19-nortestosterone used for VSE experiments.
Figure 9
Figure 9. Active site of WT NiSOD (left) and a variant with a secondary amine backbone substitution (right).
Figure 10
Figure 10. Electron donation to the iron center affects ferryl reactivity. (top) Cytochrome P450s are capable of hydrogen atom abstraction by the intermediate Compound I. Increased electron donation through an ncAA selenolate ligand increases the rate compared to WT P450. (bottom) Heme peroxidase compound II is reduced through proton coupled electron transfer. His to MeHis substitution decreases the electron donation to the ferryl intermediate and reduces its proton affinity, slowing the rate of compound II reduction.
Figure 11
Figure 11. Anaerobic X-ray crystal structures of the active sites of Human Cysteine Dioxygenase (CDO, PDB: 6N43 (306)) and CDO Tyr157F2-Tyr (PDB: 6BPR (306)) in complex with the substrate cysteine and NO. CDO and CDO Tyr157F2-Tyr are shown as cartoons in blue and gray, respectively, with key active site residues and the substrate cysteine shown as atom-colored sticks with blue and gray carbon atoms. The noncanonical F2-Tyr157 is shown with orange carbon atoms.
Figure 12
Figure 12. NcAA-mediated noncovalent interactions influence enzyme stability. (A) SPI of 4-R-FPro in KlenTaq DNA polymerase switches many Pro puckers from endo to exo, as illustrated by the substitution of Pro555 (left, gray carbons) to 4-R-FPro555 (right, orange carbons) (PDB: 4DLG, 4DLE (335)). (B) Evolutionary trajectory of TFLeu-incorporating CAT (orange bars) starting from WT CAT (gray bar) against the half-life of enzyme inactivation at 60 °C. (C) Structures of T4 lysozyme with canonical Tyr18 (left, gray carbons) and noncanonical 3-ClTyr18 (right, orange carbons). Glu11 and Gly28 backbone atoms shown (white carbons). Halogen bond between Gly28 backbone oxygen and 3-ClTyr18 chlorine atom indicated with a dashed line (PDB: 1L63, (340) 5V7E (339)).
Figure 13
Figure 13. Covalent cross-links mediated by ncAAs. (A) Cross-links generated between cAAs (black) and ncAAs (orange). Cross-linking bonds shown in gray. Top left: canonical Cys-Cys cross-link. Top right: Cys-SbuTyr cross-link. Middle left: Cys-BpAla cross-link. Middle right: amino group-4-NCSPhe cross-link. Bottom left: Cys-O-2-BeTyr cross-link. Bottom right: Cys-4-CaaPhe cross-link. (B) Structures of Cys-O-2-BeTyr cross-link (left) and Cys-4-CaaPhe cross-link (right) in Mb(H64V,V68A), with Tm increases given by one and two cross-links indicated. ncAAs shown with orange carbons and Cys with white carbons (PDB: 7SPE, 7SPH (351)).
Figure 14
Figure 14. NcAA-mediated enzyme immobilization. (A) Schematic representation of nonspecific enzyme immobilization, mediated by cross-linking at multiple reactive surface residues (gray circles), resulting in multiple enzyme orientations relative to the solid support, as well as enzyme–enzyme cross-linking leading to multilayer immobilization. (B) Schematic representation of site-specific enzyme immobilization, mediated by a ncAA (orange circles) incorporated site specifically, resulting in a monolayer with a single defined enzyme orientation. (C) Immobilization chemistries utilizing ncAAs (orange). From top to bottom: CuAAC, SPAAC, DOPhe–amine coupling, tetrazine-sTCO Diels–Alder cycloaddition, 3-NH2Tyr-acryloyl Diels–Alder cycloaddition, Glaser–Hay alkynyl coupling, and 4-SHPhe-BODIPY coupling.
Figure 15
Figure 15. Introduction of 4-AcPhe into PikC, a CYP450 enzyme, enabled biosynthetic reprogramming through allowing C(sp3)–H oxidation to occur in the absence of an amino-sugar moiety (brown).
Figure 16
Figure 16. Incorporation of ncAAs at various positions within P450BM3 alters the oxidation product distributions for (S)-ibuprofen-OMe and (+)-nootkatone substrates.
Figure 17
Figure 17. Peroxidases with MeHis proximal ligands. (A) An overlay of the crystal structures of APX2 (PDB: 1OAG (382)) and APX2 MeHis163 (PDB: 5L86 (381)). Key active site residues and the heme are shown as atom-colored sticks with gray and blue carbons, respectively. MeHis is shown with brown carbons. (B) TTN achieved by APX2 and APX2 MeHis. (C) The catalytic efficiency toward guaiacol (2-methoxyphenol) oxidation for Mb variants and horseradish peroxidase (HRP). (D) An overlay of the crystal structures of Mb (PDB: 1A6K (383)) and Mb MeHis93 (PDB: 5OJ9 (384)). The protein backbones are shown as cartoons, and key active site residues and the heme are shown as atom-colored sticks with gray and blue carbons, respectively. MeHis93 is shown with brown carbon atoms.
Figure 18
Figure 18. Biocatalytic cyclopropanations by Mb* MeHis93. (A) The bridged ion carbenoid intermediate observed by X-ray crystallography (PDB: 6F17 (386)). A 2FO–FC map contoured at 1.5 σ is shown around the bridged carbenoid intermediate and the iron atom. (B) The cyclopropanation reaction catalyzed by engineered Mbs. (C) The non-native cofactor and MeHis ligand used to expand the scope of biocatalytic cyclopropanations. (388)
Figure 19
Figure 19. Introduction of 4-AzPhe into selected sites of formate dehydrogenase (FDH) and mannitol dehydrogenase (MNDH) created bioorthogonal handles for SPAAC conjugation to either a heterobifunctional linker harboring a tetrazine handle or an alternative linker with a cyclooctene handle (PDB: 3WR5, (407) 1LJ8 (408)). FDH and MNDH are shown as gray and blue cartoons, respectively. The sites of 4-AzPhe incorporation are shown as red spheres.
Figure 20
Figure 20. Introduction of a photocaged ncAA into a DNA polymerase through GCE occludes the active site, preventing the diffusion of nucleotides for extension. Brief irradiation with UV light cleaves the O-NB moiety to reveal the catalytic Tyr and restore polymerase activity. Created with BioRender.com.
Figure 21
Figure 21. Photoresponsive ncAAs used in the allosteric light regulation of ImGPS. AzoPhe undergoes light induced reversible E/Z isomerizations enabling on–off switching of HisH activity.
Figure 22
Figure 22. Introduction of a pair of BpyAlas into Pfu POP (PDB: 5T88 (458)) enabled inhibition of protease activity when incubated in divalent metal salts. Metal binding of the noncanonical ligands holds POP in a closed inactive conformation, which can be released through chelation of metal ions with EDTA addition, thereby allowing reversible allosteric control of biocatalyst activity. Created with BioRender.com.
Figure 23
Figure 23. Catalytic metal-coordinating ncAAs. (A) Crystal structure of dimeric LmrR, with the positions Val15, Met89, and Trp96 in the binding pocket shown with blue carbons (PDB: 3F8B (474)). (B) BpyAla-coordinated Cu(II) complex which activates 1-(1-methyl-1H-imidazol-2-yl)but-2-en-1-one toward nucleophilic attack. (C) Schemes of vinylogous Friedel–Crafts alkylations (top) and α,β-unsaturated 2-acyl pyridine hydrations (bottom) catalyzed by BpyAla-Cu(II) or 3-HqAla-Cu(II) metalloenzymes.
Figure 24
Figure 24. 4-AzPhe-anchored metalloenzymes. (A) BCN-Derivatised dirhodium complex. OAc– = acetate anion. (B) Crystal structure of POP, with positions of 4-AzPhe incorporation (orange spheres) and pore-opening alanine mutations (blue spheres) shown (PDB: 5T88 (479)). (C) Schemes of styrene cyclopropanations (top) and the diazo cross-coupling cascade (bottom) catalyzed by POP variants containing 4-AzPhe-tethered dirhodium complexes.
Figure 25
Figure 25. Nucleophilic catalysis utilizing MeHis. (A) Scheme of ester hydrolysis, showing the reactive covalent intermediate formed between the substrate and MeHis23 (orange). (B) Structure of OE1.3, with MeHis23 (orange carbons) and sites of mutations installed during evolution (blue spheres) shown (PDB: 6Q7Q (486)). (C) Scheme highlighting the proton transfer role of Glu26 (gray) in the evolved MBHase BHMeHis1.8. Intermediates 2 (left) and 3 (right) are shown, covalently bound to MeHis23 (orange).
Figure 26
Figure 26. Nucleophilic catalysis utilizing 4-NH2Phe. (A) Scheme of hydrazone (X = N) and oxime (X = O) formations catalyzed by 4-NH2Phe (orange) incorporated into LmrR, with the covalent adduct formed by the carbonyl substrate and 4-NH2Phe15 shown. (B) Scheme of vinylogous Friedel–Crafts alkylations catalyzed by LmrR_V15_4-NH2Phe_RGN, with the activated imine intermediate formed between 4-NH2Phe15 (orange) and the aldehyde substrate shown. At the end of the reaction time NaBH4 is added to reduce the enzymatic product to the corresponding alcohol (right).
Figure 27
Figure 27. [2 + 2] Photocycloadditions catalyzed by BpAla. (A) Schemes of intramolecular [2 + 2] photocycloadditions of derivatized quinolones (top) and indoles (bottom). X = O or C, n = 1 or 2. (B) Crystal structure of EnT1.3 with product (green carbons) bound between BpAla (orange carbons), Trp244, and His287 (blue carbons) (PDB: 7ZP7 (28)).
Figure 28
Figure 28. Metal-dependent ncAA-incorporating photoenzymes. (A) Chromophore autocatalytically generated in sfYFP and in PSP2, which incorporates BpAla (orange side chain) at position 66. (B) Structure of PSP2, with a chromophore shown (backbone indicated with gray carbons, BpAla side chain with orange carbons). The Cys95 site of nickel–terpyridine complex ligation is shown in dark gray (PDB: 5YR3 (506)). (C) Scheme of dehalogenation reactions catalyzed by BpAla-incorporating PSP2T2 or by BpyAla-incorporating Mb. X = Cl, Br, or I. (D) Structure of Mb incorporating BpyAla (orange carbons) and with an iridium photocatalyst (green carbons) ligated to Cys45 (gray carbons) (PDB: 7YLK (516)).
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- 5Turner, N. J. Directed Evolution Drives the Next Generation of Biocatalysts. Nat. Chem. Biol. 2009, 5, 567– 573, DOI: 10.1038/nchembio.2035Directed evolution drives the next generation of biocatalystsTurner, Nicholas J.Nature Chemical Biology (2009), 5 (8), 567-573CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)A review. Enzymes are increasingly being used as biocatalysts in the generation of products that have until now been derived using traditional chem. processes. Such products range from pharmaceutical and agrochem. building blocks to fine and bulk chems. and, more recently, components of biofuels. For a biocatalyst to be effective in an industrial process, it must be subjected to improvement and optimization, and in this respect the directed evolution of enzymes has emerged as a powerful enabling technol. Directed evolution involves repeated rounds of (1) random gene library generation, (2) expression of genes in a suitable host, and (3) screening of libraries of variant enzymes for the property of interest. Both in vitro screening-based methods and in vivo selection-based methods have been applied to the evolution of enzyme function and properties. Significant developments have occurred recently, particularly with respect to library design, screening methodol., applications in synthetic transformations, and strategies for the generation of new enzyme function.
- 6Dauparas, J.; Anishchenko, I.; Bennett, N.; Bai, H.; Ragotte, R. J.; Milles, L. F.; Wicky, B. I. M.; Courbet, A.; de Haas, R. J.; Bethel, N. Robust Deep Learning-Based Protein Sequence Design Using ProteinMPNN. Science 2022, 378 (6615), 49– 56, DOI: 10.1126/science.add21876Robust deep learning-based protein sequence design using ProteinMPNNDauparas, J.; Anishchenko, I.; Bennett, N.; Bai, H.; Ragotte, R. J.; Milles, L. F.; Wicky, B. I. M.; Courbet, A.; de Haas, R. J.; Bethel, N.; Leung, P. J. Y.; Huddy, T. F.; Pellock, S.; Tischer, D.; Chan, F.; Koepnick, B.; Nguyen, H.; Kang, A.; Sankaran, B.; Bera, A. K.; King, N. P.; Baker, D.Science (Washington, DC, United States) (2022), 378 (6615), 49-56CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Although deep learning has revolutionized protein structure prediction, almost all exptl. characterized de novo protein designs have been generated using phys. based approaches such as Rosetta. Here, we describe a deep learning-based protein sequence design method, ProteinMPNN, that has outstanding performance in both in silico and exptl. tests. On native protein backbones, ProteinMPNN has a sequence recovery of 52.4% compared with 32.9% for Rosetta. The amino acid sequence at different positions can be coupled between single or multiple chains, enabling application to a wide range of current protein design challenges. We demonstrate the broad utility and high accuracy of ProteinMPNN using x-ray crystallog., cryo-electron microscopy, and functional studies by rescuing previously failed designs, which were made using Rosetta or AlphaFold, of protein monomers, cyclic homo-oligomers, tetrahedral nanoparticles, and target-binding proteins.
- 7Watson, J. L.; Juergens, D.; Bennett, N. R.; Trippe, B. L.; Yim, J.; Eisenach, H. E.; Ahern, W.; Borst, A. J.; Ragotte, R. J.; Milles, L. F. De Novo Design of Protein Structure and Function with RFdiffusion. Nature 2023, 620, 1089– 1100, DOI: 10.1038/s41586-023-06415-87De novo design of protein structure and function with RFdiffusionWatson, Joseph L.; Juergens, David; Bennett, Nathaniel R.; Trippe, Brian L.; Yim, Jason; Eisenach, Helen E.; Ahern, Woody; Borst, Andrew J.; Ragotte, Robert J.; Milles, Lukas F.; Wicky, Basile I. M.; Hanikel, Nikita; Pellock, Samuel J.; Courbet, Alexis; Sheffler, William; Wang, Jue; Venkatesh, Preetham; Sappington, Isaac; Torres, Susana Vazquez; Lauko, Anna; De Bortoli, Valentin; Mathieu, Emile; Ovchinnikov, Sergey; Barzilay, Regina; Jaakkola, Tommi S.; DiMaio, Frank; Baek, Minkyung; Baker, DavidNature (London, United Kingdom) (2023), 620 (7976), 1089-1100CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)There has been considerable recent progress in designing new proteins using deep-learning methods1-9. Despite this progress, a general deep-learning framework for protein design that enables soln. of a wide range of design challenges, including de novo binder design and design of higher-order sym. architectures, has yet to be described. Diffusion models10,11 have had considerable success in image and language generative modeling but limited success when applied to protein modeling, probably due to the complexity of protein backbone geometry and sequence-structure relationships. Here we show that by fine-tuning the RoseTTAFold structure prediction network on protein structure denoising tasks, we obtain a generative model of protein backbones that achieves outstanding performance on unconditional and topol.-constrained protein monomer design, protein binder design, sym. oligomer design, enzyme active site scaffolding and sym. motif scaffolding for therapeutic and metal-binding protein design. We demonstrate the power and generality of the method, called RoseTTAFold diffusion (RFdiffusion), by exptl. characterizing the structures and functions of hundreds of designed sym. assemblies, metal-binding proteins and protein binders. The accuracy of RFdiffusion is confirmed by the cryogenic electron microscopy structure of a designed binder in complex with influenza haemagglutinin that is nearly identical to the design model. In a manner analogous to networks that produce images from user-specified inputs, RFdiffusion enables the design of diverse functional proteins from simple mol. specifications.
- 8Sumida, K. H.; Núñez-Franco, R.; Kalvet, I.; Pellock, S. J.; Wicky, B. I. M.; Milles, L. F.; Dauparas, J.; Wang, J.; Kipnis, Y.; Jameson, N. Improving Protein Expression, Stability, and Function with ProteinMPNN. J. Am. Chem. Soc. 2024, 146 (3), 2054– 2061, DOI: 10.1021/jacs.3c10941There is no corresponding record for this reference.
- 9Gargiulo, S.; Soumillion, P. Directed Evolution for Enzyme Development in Biocatalysis. Curr. Opin. Chem. Biol. 2021, 61, 107– 113, DOI: 10.1016/j.cbpa.2020.11.0069Directed evolution for enzyme development in biocatalysisGargiulo, Serena; Soumillion, PatriceCurrent Opinion in Chemical Biology (2021), 61 (), 107-113CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. As an important sector of the chem. industry, biocatalysis requires the continuous development of enzymes with tailor-made activity, selectivity, stability, or tolerance to unnatural environments. This is now routinely achieved by directed evolution based on iterative cycles of genetic diversification and activity screening. Here, we highlight its recent developments. First, the design of "smarter" libraries by focused mutagenesis may be a crucial start-up for a fast and successful outcome. Then library assembly and expression are also key steps that benefits from modern mol. biol. progresses. Finally, various strategies may be considered for library screening depending on the final objective: while low-throughput direct assays have been very successful in generating enzymes for important biocatalytic processes, even in bringing completely new chemistries to the enzyme world, ultrahigh-throughput screening methods are emerging as powerful approaches for engineering the next generation of industrial enzymes.
- 10Planas-Iglesias, J.; Marques, S. M.; Pinto, G. P.; Musil, M.; Stourac, J.; Damborsky, J.; Bednar, D. Computational Design of Enzymes for Biotechnological Applications. Biotechnol. Adv. 2021, 47, 107696, DOI: 10.1016/j.biotechadv.2021.10769610Computational design of enzymes for biotechnological applicationsPlanas-Iglesias, Joan; Marques, Sergio M.; Pinto, Gaspar P.; Musil, Milos; Stourac, Jan; Damborsky, Jiri; Bednar, DavidBiotechnology Advances (2021), 47 (), 107696CODEN: BIADDD; ISSN:0734-9750. (Elsevier Inc.)A review. Enzymes are the natural catalysts that execute biochem. reactions upholding life. Their natural effectiveness has been fine-tuned as a result of millions of years of natural evolution. Such catalytic effectiveness has prompted the use of biocatalysts from multiple sources on different applications, including the industrial prodn. of goods (food and beverages, detergents, textile, and pharmaceutics), environmental protection, and biomedical applications. Natural enzymes often need to be improved by protein engineering to optimize their function in non-native environments. Recent technol. advances have greatly facilitated this process by providing the exptl. approaches of directed evolution or by enabling computer-assisted applications. Directed evolution mimics the natural selection process in a highly accelerated fashion at the expense of arduous lab. work and economic resources. Theor. methods provide predictions and represent an attractive complement to such expts. by waiving their inherent costs. Computational techniques can be used to engineer enzymic reactivity, substrate specificity and ligand binding, access pathways and ligand transport, and global properties like protein stability, soly., and flexibility. Theor. approaches can also identify hotspots on the protein sequence for mutagenesis and predict suitable alternatives for selected positions with expected outcomes. This review covers the latest advances in computational methods for enzyme engineering and presents many successful case studies.
- 11Świderek, K.; Tuñón, I.; Moliner, V.; Bertran, J. Computational Strategies for the Design of New Enzymatic Functions. Arch. Biochem. Biophys. 2015, 582, 68– 79, DOI: 10.1016/j.abb.2015.03.01311Computational strategies for the design of new enzymatic functionsSwiderek, K.; Tunon, I.; Moliner, V.; Bertran, J.Archives of Biochemistry and Biophysics (2015), 582 (), 68-79CODEN: ABBIA4; ISSN:0003-9861. (Elsevier B.V.)A review. In this contribution, recent developments in the design of biocatalysts are reviewed with particular emphasis in the de novo strategy. Studies based on three different reactions, Kemp elimination, Diels-Alder and Retro-Aldolase, are used to illustrate different success achieved during the last years. Finally, a section is devoted to the particular case of designed metalloenzymes. As a general conclusion, the interplay between new and more sophisticated engineering protocols and computational methods, based on mol. dynamics simulations with Quantum Mechanics/Mol. Mechanics potentials and fully flexible models, seems to constitute the bed rock for present and future successful design strategies.
- 12Renata, H.; Wang, Z. J.; Arnold, F. H. Expanding the Enzyme Universe: Accessing Non-Natural Reactions by Mechanism-Guided Directed Evolution. Angew. Chem. Int. Ed. 2015, 54 (11), 3351– 3367, DOI: 10.1002/anie.20140947012Expanding the Enzyme Universe: Accessing Non-Natural Reactions by Mechanism-Guided Directed EvolutionRenata, Hans; Wang, Z. Jane; Arnold, Frances H.Angewandte Chemie, International Edition (2015), 54 (11), 3351-3367CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. High selectivity and exquisite control over the outcome of reactions entice chemists to use biocatalysts in org. synthesis. However, many useful reactions are not accessible because they are not in nature's known repertoire. In this Review, we outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progression has been recapitulated in the lab. starting from extant enzymes. We then examine non-native enzyme activities that have been exploited for chem. synthesis, with an emphasis on reactions that do not have natural counterparts. Non-natural activities can be improved by directed evolution, thus mimicking the process used by nature to create new catalysts. Finally, we describe the discovery of non-native catalytic functions that may provide future opportunities for the expansion of the enzyme universe.
- 13Pagar, A. D.; Patil, M. D.; Flood, D. T.; Yoo, T. H.; Dawson, P. E.; Yun, H. Recent Advances in Biocatalysis with Chemical Modification and Expanded Amino Acid Alphabet. Chem. Rev. 2021, 121 (10), 6173– 6245, DOI: 10.1021/acs.chemrev.0c0120113Recent Advances in Biocatalysis with Chemical Modification and Expanded Amino Acid AlphabetPagar, Amol D.; Patil, Mahesh D.; Flood, Dillon T.; Yoo, Tae Hyeon; Dawson, Philip E.; Yun, HyungdonChemical Reviews (Washington, DC, United States) (2021), 121 (10), 6173-6245CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. The two main strategies for enzyme engineering, directed evolution and rational design, have found widespread applications in improving the intrinsic activities of proteins. Although numerous advances have been achieved using these ground-breaking methods, the limited chem. diversity of the biopolymers, restricted to the 20 canonical amino acids, hampers creation of novel enzymes that Nature has never made thus far. To address this, much research has been devoted to expanding the protein sequence space via chem. modifications and/or incorporation of noncanonical amino acids (ncAAs). This review provides a balanced discussion and crit. evaluation of the applications, recent advances, and tech. breakthroughs in biocatalysis for three approaches: (i) chem. modification of cAAs, (ii) incorporation of ncAAs, and (iii) chem. modification of incorporated ncAAs. Furthermore, the applications of these approaches and the result on the functional properties and mechanistic study of the enzymes are extensively reviewed. We also discuss the design of artificial enzymes and directed evolution strategies for enzymes with ncAAs incorporated. Finally, we discuss the current challenges and future perspectives for biocatalysis using the expanded amino acid alphabet.
- 14Zhao, J.; Burke, A. J.; Green, A. P. Enzymes with Noncanonical Amino Acids. Curr. Opin. Chem. Biol. 2020, 55, 136– 144, DOI: 10.1016/j.cbpa.2020.01.00614Enzymes with noncanonical amino acidsZhao, Jingming; Burke, Ashleigh J.; Green, Anthony P.Current Opinion in Chemical Biology (2020), 55 (), 136-144CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. Enzyme design and engineering strategies rely almost exclusively on nature's alphabet of twenty canonical amino acids. Recent years have seen the emergence of powerful genetic code expansion methods that allow hundreds of structurally diverse amino acids to be installed into proteins in a site-selective manner. Here, we will highlight how the availability of an expanded alphabet of amino acids has opened new avenues in enzyme engineering research. Genetically encoded noncanonical amino acids have provided new tools to probe complex enzyme mechanisms, improve biocatalyst activity and stability, and most ambitiously to design enzymes with new catalytic mechanisms that would be difficult to access within the constraints of the genetic code. We anticipate that the studies highlighted in this article, coupled with the continuing advancements in genetic code expansion technol., will promote the widespread use of noncanonical amino acids in biocatalysis research in the coming years.
- 15Huang, Y.; Liu, T. Therapeutic Applications of Genetic Code Expansion. Synth. Syst. Biotechnol. 2018, 3 (3), 150– 158, DOI: 10.1016/j.synbio.2018.09.00315Therapeutic applications of genetic code expansionHuang Yujia; Liu TaoSynthetic and systems biotechnology (2018), 3 (3), 150-158 ISSN:.In nature, a limited, conservative set of amino acids are utilized to synthesize proteins. Genetic code expansion technique reassigns codons and incorporates noncanonical amino acids (ncAAs) through orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs. The past decade has witnessed the rapid growth in diversity and scope for therapeutic applications of this technology. Here, we provided an update on the recent progress using genetic code expansion in the following areas: antibody-drug conjugates (ADCs), bispecific antibodies (BsAb), immunotherapies, long-lasting protein therapeutics, biosynthesized peptides, engineered viruses and cells, as well as other therapeutic related applications, where the technique was used to elucidate the mechanisms of biotherapeutics and drug targets.
- 16Icking, L.-S.; Riedlberger, A. M.; Krause, F.; Widder, J.; Frederiksen; Anne, S.; Stockert, F.; Spädt, M.; Edel, N.; Armbruster, D.; Forlani, G. iNClusive: a Database Collecting Useful Information on Non-Canonical Amino Acids and Their Incorporation into Proteins for Easier Genetic Code Expansion Implementation. Nucleic Acids Res. 2024, 52, D476, DOI: 10.1093/nar/gkad1090There is no corresponding record for this reference.
- 17Chatterjee, A.; Guo, J.; Lee, H. S.; Schultz, P. G. A Genetically Encoded Fluorescent Probe in Mammalian Cells. J. Am. Chem. Soc. 2013, 135 (34), 12540– 12543, DOI: 10.1021/ja405955317A Genetically Encoded Fluorescent Probe in Mammalian CellsChatterjee, Abhishek; Guo, Jiantao; Lee, Hyun Soo; Schultz, Peter G.Journal of the American Chemical Society (2013), 135 (34), 12540-12543CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Fluorescent reporters are useful in vitro and in vivo probes of protein structure, function, and localization. Here we report that the fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), can be site-specifically incorporated into proteins in mammalian cells in response to the TAG codon with high efficiency using an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair. We further demonstrate that Anap can be used to image the subcellular localization of proteins in live mammalian cells. The small size of Anap, its environment-sensitive fluorescence, and the ability to introduce Anap at specific sites in the proteome by simple mutagenesis make it a unique and valuable tool in eukaryotic cell biol.
- 18Evans, H. T.; Benetatos, J.; van Roijen, M.; Bodea, L. G.; Götz, J. Decreased Synthesis of Ribosomal Proteins in Tauopathy Revealed by Non-Canonical Amino Acid Labelling. EMBO J. 2019, 38 (13), e101174 DOI: 10.15252/embj.2018101174There is no corresponding record for this reference.
- 19Schultz, K. C.; Supekova, L.; Ryu, Y.; Xie, J.; Perera, R.; Schultz, P. G. A Genetically Encoded Infrared Probe. J. Am. Chem. Soc. 2006, 128 (43), 13984– 13985, DOI: 10.1021/ja063669019A Genetically Encoded Infrared ProbeSchultz, Kathryn C.; Supekova, Lubica; Ryu, Youngha; Xie, Jianming; Perera, Roshan; Schultz, Peter G.Journal of the American Chemical Society (2006), 128 (43), 13984-13985CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)An orthogonal tRNA/aminoacyl-tRNA synthetase pair has been evolved that makes it possible to selectively and efficiently incorporate p-cyanophenylalanine (pCNPhe) into proteins in E. coli at sites specified by the amber nonsense codon, TAG. Substitution of pCNPhe for histidine-64 in myoglobin (Mb) affords a sensitive vibrational probe of ligand binding. This methodol. provides a useful IR reporter of protein structure, biomol. interactions, and conformational changes.
- 20Goettig, P.; Koch, N. G.; Budisa, N. Non-Canonical Amino Acids in Analyses of Protease Structure and Function. Int. J. Mol. Sci. 2023, 24 (18), 14035, DOI: 10.3390/ijms241814035There is no corresponding record for this reference.
- 21Tinzl, M.; Hilvert, D. Trapping Transient Protein Species by Genetic Code Expansion. ChemBioChem 2021, 22 (1), 92– 99, DOI: 10.1002/cbic.20200052321Trapping Transient Protein Species by Genetic Code ExpansionTinzl, Matthias; Hilvert, DonaldChemBioChem (2021), 22 (1), 92-99CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Nature employs a limited no. of genetically encoded amino acids for the construction of functional proteins. By engineering components of the cellular translation machinery, however, it is now possible to genetically encode noncanonical building blocks with tailored electronic and structural properties. The ability to incorporate unique chem. functionality into proteins provides a powerful tool to probe mechanism and create novel function. In this minireview, we highlight several recent studies that illustrate how noncanonical amino acids have been used to capture and characterize reactive intermediates, fine-tune the catalytic properties of enzymes, and stabilize short-lived protein-protein complexes.
- 22Birch-Price, Z.; Taylor, C. J.; Ortmayer, M.; Green, A. P. Engineering Enzyme Activity Using an Expanded Amino Acid Alphabet. Protein Eng. Des. Sel. 2023, 36, gzac013, DOI: 10.1093/protein/gzac013There is no corresponding record for this reference.
- 23Giger, S.; Buller, R. Advances in Noncanonical Amino Acid Incorporation for Enzyme Engineering Applications. CHIMIA 2023, 77 (6), 395– 402, DOI: 10.2533/chimia.2023.395There is no corresponding record for this reference.
- 24Hayashi, T.; Hilvert, D.; Green, A. P. Engineered Metalloenzymes with Non-Canonical Coordination Environments. Chem. Eur. J. 2018, 24 (46), 11821– 11830, DOI: 10.1002/chem.201800975There is no corresponding record for this reference.
- 25Lugtenburg, T.; Gran-Scheuch, A.; Drienovská, I. Non-Canonical Amino Acids as a Tool for the Thermal Stabilization of Enzymes. Protein Eng. Des. Sel. 2023, 36, gzad003, DOI: 10.1093/protein/gzad003There is no corresponding record for this reference.
- 26Mirts, E. N.; Bhagi-Damodaran, A.; Lu, Y. Understanding and Modulating Metalloenzymes with Unnatural Amino Acids, Non-Native Metal Ions, and Non-Native Metallocofactors. Acc. Chem. Res. 2019, 52 (4), 935– 944, DOI: 10.1021/acs.accounts.9b0001126Understanding and Modulating Metalloenzymes with Unnatural Amino Acids, Non-Native Metal Ions, and Non-Native MetallocofactorsMirts, Evan N.; Bhagi-Damodaran, Ambika; Lu, YiAccounts of Chemical Research (2019), 52 (4), 935-944CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. Metalloproteins set the gold std. for performing important functions, including catalyzing demanding reactions under mild conditions. Designing artificial metalloenzymes (ArMs) to catalyze abiol. reactions has been a major endeavor for many years, but most ArMs' activities are far below those of native enzymes, making them unsuitable for most pratical applications. A crit. step to advance the field is to fundamentally understand what it takes to not only confer but also fine-tune ArM activities so they match native enzymes. Indeed, only once we can freely modulate ArM activity to rival (or surpass!) natural enzymes can the potential of ArMs be fully realized. A key to unlocking ArM potential is the observation that one metal primary coordination sphere (PCS) can display a range of functions and levels of activity, leading to the realization that secondary coordination sphere (SCS) interactions are critically important. However, SCS interactions are numerous, long-range, and weak, making them very difficult to reproduce in ArMs. Furthermore, natural enzymes are tied to a small set of biol. available functional moieties from canonical amino acids and the physiol. available metal ions and metallocofactors, severely limiting the chem. space available to probe and tune ArMs. In this Account, we summarize our group's use of unnatural amino acids (UAAs) and non-native metal ions and metallocofactors to probe and modulate ArM functions. We incorporated isostructural UAAs in a type 1 copper (T1Cu) protein azurin to provide conclusive evidence that the axial ligand hydrophobicity is a major determinant of T1Cu redunction potential (E'°). We also probed the role of protein backbone interactions that cannot be altered by std. mutagenesis by replacing the peptide bond with an ester linkage. We used insight gained from these studies to tune the E'° of azurin across the entire physiol. range, the broadest range ever achieved in a single metalloprotein. Introducing UAA analogs of Tyr into ArM models of heme-copper oxidase (HCO) revealed a linear relationship between pKa, E'°, and activity. We have also substituted non-native hemes and non-native metal ions for their native equiv. in these models to resolve several issues that were intractable in native HCOs and the closely related nitric oxide reductases (NOR), such as their roles in modulating substrate affinity, ET rate, and activity. We have incorporated abiol. cofactors such as ferrocene and Mn(salen) into azurin and myoglobin, resp., to stabilize these inorg. and organometallic compds. in water, confer abiol. functions, tune their E'° and activity through SCS interactions, and show that the approach to metallocofactor anchoring and orientation can tune enantioselectivity and alter function. Replacing Cu in azurin with non-native Fe or Ni can impart novel activities, such as superoxide redn. and C-C bond formation. While progress has been made, we have identified only a small fraction of the interactions that can be generally applied to ArMs to fine-tune their functions. Because SCS interactions are subtle and heavily interconnected, it has been difficult to characterize their effects quant. It is vital to develop spectroscopic and computational techniques to detect and quantify their effects in both resting states and catalytic intermediates.
- 27Drienovská, I.; Roelfes, G. Expanding the Enzyme Universe with Genetically Encoded Unnatural Amino Acids. Nat. Catal. 2020, 3 (3), 193– 202, DOI: 10.1038/s41929-019-0410-827Expanding the enzyme universe with genetically encoded unnatural amino acidsDrienovska, Ivana; Roelfes, GerardNature Catalysis (2020), 3 (3), 193-202CODEN: NCAACP; ISSN:2520-1158. (Nature Research)A review. The emergence of robust methods to expand the genetic code allows incorporation of non-canonical amino acids into the polypeptide chain of proteins, thus making it possible to introduce unnatural chem. functionalities in enzymes. In this Perspective, we show how this powerful methodol. is used to create enzymes with improved and novel, even new-to-nature, catalytic activities. We provide an overview of the current state of the art, and discuss the potential benefits of developing and using enzymes with genetically encoded non-canonical amino acids compared with enzymes contg. only canonical amino acids.
- 28Trimble, J. S.; Crawshaw, R.; Hardy, F. J.; Levy, C. W.; Brown, M. J. B.; Fuerst, D. E.; Heyes, D. J.; Obexer, R.; Green, A. P. A Designed Photoenzyme for Enantioselective [2 + 2] Cycloadditions. Nature 2022, 611 (7937), 709– 714, DOI: 10.1038/s41586-022-05335-328A designed photoenzyme for enantioselective [2+2] cycloadditionsTrimble, Jonathan S.; Crawshaw, Rebecca; Hardy, Florence J.; Levy, Colin W.; Brown, Murray J. B.; Fuerst, Douglas E.; Heyes, Derren J.; Obexer, Richard; Green, Anthony P.Nature (London, United Kingdom) (2022), 611 (7937), 709-714CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)The ability to program new modes of catalysis into proteins would allow the development of enzyme families with functions beyond those found in nature. To this end, genetic code expansion methodol. holds particular promise, as it allows the site-selective introduction of new functional elements into proteins as noncanonical amino acid side chains1-4. Here we exploit an expanded genetic code to develop a photoenzyme that operates by means of triplet energy transfer (EnT) catalysis, a versatile mode of reactivity in org. synthesis that is not accessible to biocatalysis at present5-12. Installation of a genetically encoded photosensitizer into the beta-propeller scaffold of DA_20_00 (ref. 13) converts a de novo Diels-Alderase into a photoenzyme for [2+2] cycloaddns. (EnT1.0). Subsequent development and implementation of a platform for photoenzyme evolution afforded an efficient and enantioselective enzyme (EnT1.3, up to 99% enantiomeric excess (e.e.)) that can promote intramol. and bimol. cycloaddns., including transformations that have proved challenging to achieve selectively with small-mol. catalysts. EnT1.3 performs >300 turnovers and, in contrast to small-mol. photocatalysts, can operate effectively under aerobic conditions and at ambient temps. An X-ray crystal structure of an EnT1.3-product complex shows how multiple functional components work in synergy to promote efficient and selective photocatalysis. This study opens up a wealth of new excited-state chem. in protein active sites and establishes the framework for developing a new generation of enantioselective photocatalysts.
- 29Boutureira, O.; Bernardes, G. J. L. Advances in Chemical Protein Modification. Chem. Rev. 2015, 115 (5), 2174– 2195, DOI: 10.1021/cr500399p29Advances in Chemical Protein ModificationBoutureira, Omar; Bernardes, Goncalo J. L.Chemical Reviews (Washington, DC, United States) (2015), 115 (5), 2174-2195CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Transition metal-free and -mediated approaches are covered.
- 30Pfleiderer, G. Chemical Modification of Proteins. In Review Articles, including those from an International Conference held in Bielefeld, FR of Germany, June 1–2, 1984 , 2021; Walter de Gruyter GmbH & Co KG, p 207. DOI: 10.1515/9783112393048-011There is no corresponding record for this reference.
- 31Gunnoo, S. B.; Madder, A. Chemical Protein Modification through Cysteine. ChemBioChem 2016, 17 (7), 529– 553, DOI: 10.1002/cbic.20150066731Chemical Protein Modification through CysteineGunnoo, Smita B.; Madder, AnnemiekeChemBioChem (2016), 17 (7), 529-553CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)The modification of proteins with non-protein entities is important for a wealth of applications, and methods for chem. modifying proteins attract considerable attention. Generally, modification is desired at a single site to maintain homogeneity and to minimise loss of function. Though protein modification can be achieved by targeting some natural amino acid side chains, this often leads to ill-defined and randomly modified proteins. Amongst the natural amino acids, cysteine combines advantageous properties contributing to its suitability for site-selective modification, including a unique nucleophilicity, and a low natural abundance-both allowing chemo- and regioselectivity. Native cysteine residues can be targeted, or Cys can be introduced at a desired site in a protein by means of reliable genetic engineering techniques. This review on chem. protein modification through cysteine should appeal to those interested in modifying proteins for a range of applications.
- 32Bischak, C. G.; Longhi, S.; Snead, D. M.; Costanzo, S.; Terrer, E.; Londergan, C. H. Probing Structural Transitions in the Intrinsically Disordered C-Terminal Domain of the Measles Virus Nucleoprotein by Vibrational Spectroscopy of Cyanylated Cysteines. Biophys. J. 2010, 99 (5), 1676– 1683, DOI: 10.1016/j.bpj.2010.06.06032Probing Structural Transitions in the Intrinsically Disordered C-Terminal Domain of the Measles Virus Nucleoprotein by Vibrational Spectroscopy of Cyanylated CysteinesBischak, Connor G.; Longhi, Sonia; Snead, David M.; Costanzo, Stephanie; Terrer, Elodie; Londergan, Casey H.Biophysical Journal (2010), 99 (5), 1676-1683CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)Four single-cysteine variants of the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) were cyanylated at cysteine and their IR spectra in the C≡N stretching region were recorded both in the absence and in the presence of one of the physiol. partners of NTAIL, namely the C-terminal X domain (XD) of the viral phosphoprotein. Consistent with previous studies showing that XD triggers a disorder-to-order transition within NTAIL, the C≡N stretching bands of the IR probe were found to be significantly affected by XD, with this effect being position-dependent. When the cyanylated cysteine side chain is solvent-exposed throughout the structural transition, its changing linewidth reflects a local gain of structure. When the probe becomes partially buried due to binding, its frequency reports on the mean hydrophobicity of the microenvironment surrounding the labeled side chain of the bound form. The probe moiety is small compared to other common covalently attached spectroscopic probes, thereby minimizing possible steric hindrance/perturbation at the binding interface. These results show for the first time to our knowledge the suitability of site-specific cysteine mutagenesis followed by cyanylation and IR spectroscopy to document structural transitions occurring within intrinsically disordered regions, with regions involved in binding and folding being identifiable at the residue level.
- 33Fafarman, A. T.; Webb, L. J.; Chuang, J. I.; Boxer, S. G. Site-Specific Conversion of Cysteine Thiols into Thiocyanate Creates an IR Probe for Electric Fields in Proteins. J. Am. Chem. Soc. 2006, 128 (41), 13356– 13357, DOI: 10.1021/ja065040333Site-Specific Conversion of Cysteine Thiols into Thiocyanate Creates an IR Probe for Electric Fields in ProteinsFafarman, Aaron T.; Webb, Lauren J.; Chuang, Jessica I.; Boxer, Steven G.Journal of the American Chemical Society (2006), 128 (41), 13356-13357CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The nitrile stretching mode of the thiocyanate moiety is a nearly ideal probe for measuring the local elec. field arising from the organized environment of the interior of a protein. Nitriles were introduced into three proteins: RNase S, human aldose reductase (hALR2), and the reaction center (RC) of Rhodobacter capsulatus, through a facile synthetic scheme for the transformation of cysteine residues into thiocyanatoalanine. Vibrational Stark effect spectroscopy and Fourier transform IR spectroscopy on the modified proteins demonstrated that thiocyanate residues are a highly general tool for probing electrostatic fields in proteins.
- 34Weeks, C. L.; Jo, H.; Kier, B.; DeGrado, W. F.; Spiro, T. G. Cysteine-Linked Aromatic Nitriles as UV Resonance Raman Probes of Protein Structure. J. Raman Spectrosc. 2012, 43 (9), 1244– 1249, DOI: 10.1002/jrs.3167There is no corresponding record for this reference.
- 35Boutureira, O.; Bernardes, G. J.; D’Hooge, F.; Davis, B. G. Direct Radiolabelling of Proteins at Cysteine Using [18 F]-Fluorosugars. Chem. Commun. 2011, 47 (36), 10010– 10012, DOI: 10.1039/c1cc13524dThere is no corresponding record for this reference.
- 36Kaiser, E. T.; Lawrence, D. S. Chemical Mutation of Enzyme Active Sites. Science 1984, 226 (4674), 505– 511, DOI: 10.1126/science.6238407There is no corresponding record for this reference.
- 37Levine, H. L.; Kaiser, E. Oxidation of Dihydronicotinamides by Flavopapain. J. Am. Chem. Soc. 1978, 100 (24), 7670– 7677, DOI: 10.1021/ja00492a040There is no corresponding record for this reference.
- 38Levine, H. L.; Nakagawa, Y.; Kaiser, E. Flavopapain: Synthesis and Properties of Semi-Synthetic Enzymes. Biochem. Biophys. Res. Commun. 1977, 76 (1), 64– 70, DOI: 10.1016/0006-291X(77)91668-0There is no corresponding record for this reference.
- 39Mayer, C.; Gillingham, D. G.; Ward, T. R.; Hilvert, D. An Artificial Metalloenzyme for Olefin Metathesis. Chem. Commun. 2011, 47 (44), 12068– 12070, DOI: 10.1039/c1cc15005g39An artificial metalloenzyme for olefin metathesisMayer, Clemens; Gillingham, Dennis G.; Ward, Thomas R.; Hilvert, DonaldChemical Communications (Cambridge, United Kingdom) (2011), 47 (44), 12068-12070CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)A Grubbs-Hoveyda type olefin metathesis catalyst, equipped with an electrophilic bromoacetamide group, was used to modify a cysteine-contg. variant of a small heat shock protein from Methanocaldococcus jannaschii. The resulting artificial metalloenzyme was found to be active under acidic conditions in a benchmark ring closing metathesis reaction.
- 40den Heeten, R.; Muñoz, B. K.; Popa, G.; Laan, W.; Kamer, P. C. J. Synthesis of Hybrid Transition-Metalloproteins via Thiol-Selective Covalent Anchoring of Rh-Phosphine and Ru-Phenanthroline Complexes. Dalton Trans. 2010, 39 (36), 8477– 8483, DOI: 10.1039/c0dt00239aThere is no corresponding record for this reference.
- 41Deuss, P. J.; Popa, G.; Botting, C. H.; Laan, W.; Kamer, P. C. J. Highly Efficient and Site-Selective Phosphane Modification of Proteins through Hydrazone Linkage: Development of Artificial Metalloenzymes. Angew. Chem. Int. Ed. 2010, 49 (31), 5315– 5317, DOI: 10.1002/anie.201002174There is no corresponding record for this reference.
- 42Jarvis, A. G.; Obrecht, L.; Deuss, P. J.; Laan, W.; Gibson, E. K.; Wells, P. P.; Kamer, P. C. J. Enzyme Activity by Design: An Artificial Rhodium Hydroformylase for Linear Aldehydes. Angew. Chem. Int. Ed. 2017, 56 (44), 13596– 13600, DOI: 10.1002/anie.201705753There is no corresponding record for this reference.
- 43Merrifield, R. B. Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J. Am. Chem. Soc. 1963, 85 (14), 2149– 2154, DOI: 10.1021/ja00897a02543Solid phase peptide synthesis. I. The synthesis of a tetrapeptideMerrifield, R. B.Journal of the American Chemical Society (1963), 85 (14), 2149-54CODEN: JACSAT; ISSN:0002-7863.A new approach to the chem. synthesis of polypeptides was investigated. It involved the stepwise addition of protected amino acids to a growing peptide chain which was bound by a covalent bond to a solid resin particle. This provided a procedure whereby reagents anti by-products were removed by filtration, and the recrystn. of intermediates was eliminated. The advantages of the new method were speed and simplicity of operation. The feasibility of the idea was demonstrated by the synthesis of the model tetrapeptide L-leucyl-L-alanylglycyl-L-valine. The peptide was identical with a sample prepd. by the standard p-nitrophenyl ester procedure.
- 44Eckert, D. M.; Malashkevich, V. N.; Hong, L. H.; Carr, P. A.; Kim, P. S. Inhibiting HIV-1 Entry: Discovery of D-Peptide Inhibitors that Target the gp41 Coiled-Coil Pocket. Cell 1999, 99 (1), 103– 115, DOI: 10.1016/S0092-8674(00)80066-544Inhibiting HIV-1 entry: discovery of D-peptide inhibitors that target the gp41 coiled-coil pocketEckert, Debra M.; Malashkevich, Vladimir N.; Hong, Lily H.; Carr, Peter A.; Kim, Peter S.Cell (Cambridge, Massachusetts) (1999), 99 (1), 103-115CODEN: CELLB5; ISSN:0092-8674. (Cell Press)The HIV-1 gp41 protein promotes viral entry by mediating the fusion of viral and cellular membranes. A prominent pocket on the surface of a central trimeric coiled coil within gp41 was previously identified as a potential target for drugs that inhibit HIV-1 entry. The authors designed a peptide, IQN17, which properly presents this pocket. Utilizing IQN17 and mirror-image phage display, the authors identified cyclic, D-peptide inhibitors of HIV-1 infection that share a sequence motif. A 1.5 Å cocrystal structure of IQN17 in complex with a D-peptide, and NMR studies, show that conserved residues of these inhibitors make intimate contact with the gp41 pocket. The authors studies validate the pocket per se as a target for drug development. IQN17 and these D-peptide inhibitors are likely to be useful for development and identification of a new class of orally bioavailable anti-HIV drugs.
- 45Kent, S.; Sohma, Y.; Liu, S.; Bang, D.; Pentelute, B.; Mandal, K. Through The Looking Glass - a New World of Proteins Enabled by Chemical Synthesis. J. Pept. Sci. 2012, 18 (7), 428– 436, DOI: 10.1002/psc.2421There is no corresponding record for this reference.
- 46Schumacher, T. N. M.; Mayr, L. M.; Minor, D. L.; Milhollen, M. A.; Burgess, M. W.; Kim, P. S. Identification of D-Peptide Ligands Through Mirror-Image Phage Display. Science 1996, 271 (5257), 1854– 1857, DOI: 10.1126/science.271.5257.185446Identification of D-peptide ligands through mirror-image phage displaySchumacher, Ton N. M.; Mayr, Lorenz M.; Minor, Daniel L., Jr.; Milhollen, Michael A.; Burgess, Michael W.; Kim, Peter S.Science (Washington, D. C.) (1996), 271 (5257), 1854-7CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Genetically encoded libraries of peptides and oligonucleotides are well suited for the identification of ligands for many macromols. A major drawback of these techniques is that the resultant ligands are subject to degrdn. by naturally occurring enzymes. Here, a method is described that uses a biol. encoded library for the identification of D-peptide ligands, which should be resistant to proteolytic degrdn. In this approach, a protein is synthesized in the D-amino acid configuration and used to select peptides from a phage display library expressing random L-amino acid peptides. For reasons of sym., the mirror images of these phage-displayed peptides interact with the target protein of the natural handedness. The value of this approach was demonstrated by the identification of a cyclic D-peptide partially overlaps the site for the physiol. ligands of this domain.
- 47Pech, A.; Achenbach, J.; Jahnz, M.; Schülzchen, S.; Jarosch, F.; Bordusa, F.; Klussmann, S. A Thermostable D-Polymerase for Mirror-Image PCR. Nucleic Acids Res. 2017, 45 (7), 3997– 4005, DOI: 10.1093/nar/gkx07947A thermostable D-polymerase for mirror-image PCRPech, Andreas; Achenbach, John; Jahnz, Michael; Schulzchen, Simone; Jarosch, Florian; Bordusa, Frank; Klussmann, SvenNucleic Acids Research (2017), 45 (7), 3997-4005CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)Biol. evolution resulted in a homochiral world in which nucleic acids consist exclusively of Dnucleotides and proteins made by ribosomal translation of L-amino acids. From the perspective of synthetic biol., however, particularly anabolic enzymes that could build the mirror-image counterparts of biol. macromols. such as L-DNA or L-RNA are lacking. Based on a convergent synthesis strategy, we have chem. produced and characterized a thermostable mirror-image polymerase that efficiently replicates and amplifies mirror-image (L)-DNA. This artificial enzyme, dubbed D-Dpo4-3C, is a mutant of Sulfolobus solfataricus DNA polymerase IV consisting of 352 D-amino acids. D-Dpo4-3C was reliably deployed in classical polymerase chain reactions (PCR) and it was used to assemble a first mirror-image gene coding for the protein Sso7d. We believe that this D-polymerase provides a valuable tool to further investigate the mysteries of biol. (homo) chirali ty and to pave the way for potential novel life forms running on a mirror-image genome.
- 48Jackson, D. Y.; Burnier, J.; Quan, C.; Stanley, M.; Tom, J.; Wells, J. A. A Designed Peptide Ligase for Total Synthesis of Ribonuclease A with Unnatural Catalytic Residues. Science 1994, 266 (5183), 243– 247, DOI: 10.1126/science.7939659There is no corresponding record for this reference.
- 49Kaiser, E. Synthetic Approaches to Biologically Active Peptides and Proteins Including Enzymes. Acc. Chem. Res. 1989, 22 (2), 47– 54, DOI: 10.1021/ar00158a001There is no corresponding record for this reference.
- 50Schnölzer, M.; Kent, S. B. Constructing Proteins by Dovetailing Unprotected Synthetic Peptides: Backbone-Engineered HIV Protease. Science 1992, 256 (5054), 221– 225, DOI: 10.1126/science.156606950Constructing proteins by dovetailing unprotected synthetic peptides: backbone-engineered HIV proteaseSchnolzer M; Kent S BScience (New York, N.Y.) (1992), 256 (5054), 221-5 ISSN:0036-8075.Backbone-engineered HIV-1 protease was prepared by a total chemical synthesis approach that combines the act of joining two peptides with the generation of an analog structure. Unprotected synthetic peptide segments corresponding to the two halves of the HIV-1 protease monomer polypeptide chain were joined cleanly and in high yield through unique mutually reactive functional groups, one on each segment. Ligation was performed in 6 molar guanidine hydrochloride, thus circumventing limited solubility of protected peptide segments, the principal problem of the classical approach to the chemical synthesis of proteins. The resulting fully active HIV-1 protease analog contained a thioester replacement for the natural peptide bond between Gly51-Gly52 in each of the two active site flaps, a region known to be highly sensitive to mutational changes of amino acid side chains.
- 51Bode, J. W. Chemical Protein Synthesis with the α-Ketoacid-Hydroxylamine Ligation. Acc. Chem. Res. 2017, 50 (9), 2104– 2115, DOI: 10.1021/acs.accounts.7b0027751Chemical Protein Synthesis with the α-Ketoacid-Hydroxylamine LigationBode, Jeffrey W.Accounts of Chemical Research (2017), 50 (9), 2104-2115CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. The coupling of an α-ketoacid and a hydroxylamine (KAHA ligation) affords amide bonds under aq., acidic conditions without the need for protecting groups or coupling agents. Translating this finding into a general approach to chem. protein synthesis required the identification of methods to incorporate the key functional groups into unprotected peptide segments-ideally using well-established Fmoc solid-phase peptide synthesis protocols. A decade of effort has now led to robust, convenient methods for prepg. peptides bearing free or masked C-terminal α-ketoacids and N-terminal hydroxylamines. The facile synthesis of the segments and the aq., acidic conditions of the KAHA ligation make it ideal for the construction of small proteins (up to 200 residues), including SUMO and related modifier proteins, betatrophin and other protein hormones, nitrophorin 4, S100A4, and the cyclic protein AS-48.Key to the successful development of this protein synthesis platform was the identification and gram-scale synthesis of (S)-5-oxaproline. This hydroxylamine monomer is completely stable toward std. methods and practices of solid-phase peptide synthesis while still performing very well in the KAHA ligation. This reaction partner-in contrast to all others examd.-affords esters rather than amides as the primary ligation product. The resulting depsipeptides often offer superior soly. and handling and have been key in the chem. synthesis of hydrophobic and amphiphilic proteins. Upon facile O-to-N acyl shift, peptides bearing a noncanonical homoserine residue at the ligation site are formed. With proper choice of the ligation site, the incorporation of this unnatural amino acid does not appear to affect the structure or biol. activity of the protein targets. The development of the chem. methods for prepg. and masking peptide α-ketoacids and hydroxyalmines, the prepn. of several protein targets by convergent ligation strategies, and the synthesis of new hydroxylamine monomers affording either natural or unnatural residues at the ligation site are discussed. By operation under acidic conditions and with a distinct preference for the ligation site, these efforts establish KAHA ligation as a complementary method to the venerable native chem. ligation (NCL) for chem. protein synthesis. This Account documents both the state of the KAHA ligation and the challenges in identifying, inventing, and optimizing new reactions and building blocks needed to interface KAHA ligation with Fmoc solid-phase peptide chem. With these challenges largely addressed, peptide segments ready for ligation are formed directly upon resin cleavage, facilitating rapid assembly of four to five segments into proteins. This work sets the stage for applications of the KAHA ligation to chem. biol. and protein therapeutics.
- 52Liu, H.; Li, X. Serine/Threonine Ligation: Origin, Mechanistic Aspects, and Applications. Acc. Chem. Res. 2018, 51 (7), 1643– 1655, DOI: 10.1021/acs.accounts.8b0015152Serine/Threonine Ligation: Origin, Mechanistic Aspects, and ApplicationsLiu, Han; Li, XuechenAccounts of Chemical Research (2018), 51 (7), 1643-1655CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)Review. Synthetic proteins are expected to go beyond the boundary of recombinant DNA expression systems, by being flexibly installed with the site-specific natural or unnatural modification structures along synthesis. To enable protein chem. synthesis, peptide ligations provide effective strategies to assemble short peptide fragments obtained from solid phase peptide synthesis (SPPS) into long peptides and proteins. In this regard, chemoselective peptide ligation represents a simple but powerful transformation realizing selective amide formation between the C-terminus and N-terminus of two side-chain unprotected peptide fragments. These reactions are highly chemo- and regioselective to tolerate the side-chain functionalities present on the unprotected peptides, highly reactive to work with mM or sub-mM concn. of the substrates, and operationally simple with mild conditions and accessible building blocks. This Account focuses on our work in the development of serine/threonine ligation (STL), which originates from a chemoselective reaction between an unprotected peptide with the C-terminal salicylaldehyde ester and another unprotected peptide with N-terminal serine or threonine residues. Mechanistically, STL involves imine capture, 5-endo-trig ring-chain tautomerization, O-to-N [1,5] acyl transfer to afford the N,O-benzylidene acetal linked peptide, followed by acidolysis to regenerate the Xaa-Ser/Thr linkage (Xaa is the amino acid) at the ligation site. The high abundance of serine and threonine residues (12.7%) in naturally occurring proteins and good compatibility of STL with variable C-terminal residues provide multiple choices for ligation sites. The requisite peptide C-terminal salicylaldehyde (SAL) esters can be prepd. from the peptide fragments obtained from both Fmoc-SPPS and Boc-SPPS through four available methods (safety catch strategy based on phenolysis, direct coupling, ozonolysis and n+1 strategy). In the synthesis of proteins (e.g., ACYP enzyme, MUC1 glycopeptide 40-mer to 80-mer, interleukin 25 and HMGA1a with variable post-translational modification patterns), both C-to-N and N-to-C sequential STL strategies have been developed, through selection of temporal N-terminal protecting groups and proper design of the switch-on/off C-terminal SAL ester surrogate, resp. In the synthesis of cyclic peptide natural products (e.g., daptomycin, teixobactin, cyclomontanin B, yunnanin C) and their analogs, the intramol. head-to-tail STL has been implemented on linear peptide SAL ester precursors contg. four to ten amino acid residues, with good efficiency and minimized oligomerization. As a thiol-independent chemoselective ligation complementary to native chem. ligation (NCL), STL provides an alternative tool to chem. synthesize homogeneous proteins with site specific and structure defined modifications and cyclic peptide natural products, which lays foundation for chem. biol. and medicinal study on those mols. with biol. importance and therapeutic potentials.
- 53Muir, T. W. Semisynthesis of Proteins by Expressed Protein Ligation. Annu. Rev. Biochem. 2003, 72 (1), 249– 289, DOI: 10.1146/annurev.biochem.72.121801.161900There is no corresponding record for this reference.
- 54Antos, J. M.; Truttmann, M. C.; Ploegh, H. L. Recent Advances in Sortase-Catalyzed Ligation Methodology. Curr. Opin. Struct. Biol. 2016, 38, 111– 118, DOI: 10.1016/j.sbi.2016.05.02154Recent advances in sortase-catalyzed ligation methodologyAntos, John M.; Truttmann, Matthias C.; Ploegh, Hidde L.Current Opinion in Structural Biology (2016), 38 (), 111-118CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. The transpeptidation reaction catalyzed by bacterial sortases continues to see increasing use in the construction of novel protein derivs. In addn. to growth in the no. of applications that rely on sortase, this field has also seen methodol. improvements that enhance reaction performance and scope. In this opinion, we present an overview of key developments in the practise and implementation of sortase-based strategies, including applications relevant to structural biol. Topics include the use of engineered sortases to increase reaction rates, the use of redesigned acyl donors and acceptors to mitigate reaction reversibility, and strategies for expanding the range of substrates that are compatible with a sortase-based approach.
- 55Chen, S.-Y.; Cressman, S.; Mao, F.; Shao, H.; Low, D. W.; Beilan, H. S.; Cagle, E. N.; Carnevali, M.; Gueriguian, V.; Keogh, P. J. Synthetic Erythropoietic Proteins: Tuning Biological Performance by Site-Specific Polymer Attachment. Chem. Biol. 2005, 12 (3), 371– 383, DOI: 10.1016/j.chembiol.2005.01.01755Synthetic erythropoietic proteins: tuning biological performance by site-specific polymer attachmentChen, Shiah-Yun; Cressman, Sonya; Mao, Feng; Shao, Haiyan; Low, Donald W.; Beilan, Hal S.; Cagle, E. Neil; Carnevali, Maia; Gueriguian, Vincent; Keogh, Peter J.; Porter, Heather; Stratton, Stephen M.; Wiedeke, M. Con; Savatski, Laura; Adamson, John W.; Bozzini, Carlos E.; Kung, Ada; Kent, Stephen B. H.; Bradburne, James A.; Kochendoerfer, Gerd G.Chemistry & Biology (2005), 12 (3), 371-383CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)Chem. synthesis in combination with precision polymer modification allows the systematic exploration of the effect of protein properties, such as charge and hydrodynamic radius, on potency using defined, homogeneous conjugates. A series of polymer-modified synthetic erythropoiesis proteins were constructed that had a polypeptide chain similar to the amino acid sequence of human erythropoietin but differed significantly in the no. and type of attached polymers. The analogs differed in charge from +5 to -26 at neutral pH and varied in mol. wt. from 30 to 54 kDa. All were active in an in vitro cell proliferation assay. However, in vivo potency was found to be strongly dependent on overall charge and size. The trends obsd. in this study may serve as starting points for the construction of more potent synthetic EPO analogs in the future.
- 56Kochendoerfer, G. G.; Chen, S.-Y.; Mao, F.; Cressman, S.; Traviglia, S.; Shao, H.; Hunter, C. L.; Low, D. W.; Cagle, E. N.; Carnevali, M. Design and Chemical Synthesis of a Homogeneous Polymer-Modified Erythropoiesis Protein. Science 2003, 299 (5608), 884– 887, DOI: 10.1126/science.107908556Design and Chemical Synthesis of a Homogeneous Polymer-Modified Erythropoiesis ProteinKochendoerfer, Gerd G.; Chen, Shiah-Yun; Mao, Feng; Cressman, Sonya; Traviglia, Stacey; Shao, Haiyan; Hunter, Christie L.; Low, Donald W.; Cagle, E. Neil; Carnevali, Maia; Gueriguian, Vincent; Keogh, Peter J.; Porter, Heather; Stratton, Stephen M.; Wiedeke, M. Con; Wilken, Jill; Tang, Jie; Levy, Jay J.; Miranda, Les P.; Crnogorac, Milan M.; Kalbag, Suresh; Botti, Paolo; Schindler-Horvat, Janice; Savatski, Laura; Adamson, John W.; Kung, Ada; Kent, Stephen B. H.; Bradburne, James A.Science (Washington, DC, United States) (2003), 299 (5608), 884-887CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The authors report the design and total chem. synthesis of "synthetic erythropoiesis protein" (SEP), a 51-kilodalton protein-polymer construct consisting of a 166-amino-acid polypeptide chain and two covalently attached, branched, and monodisperse polymer moieties that are neg. charged. The ability to control the chem. allowed the authors to synthesize a macromol. of precisely defined covalent structure. SEP was homogeneous as shown by high-resoln. anal. techniques, with a mass of 50,825 ±10 daltons by electrospray mass spectrometry, and with a pI of 5.0. In cell and animal assays for erythropoiesis, SEP displayed potent biol. activity and had significantly prolonged duration of action in vivo. These chem. methods are a powerful tool in the rational design of protein constructs with potential therapeutic applications.
- 57Kent, S. B. H. Bringing the Science of Proteins into the Realm of Organic Chemistry: Total Chemical Synthesis of SEP (Synthetic Erythropoiesis Protein). Angew. Chem. Int. Ed. 2013, 52 (46), 11988– 11996, DOI: 10.1002/anie.201304116There is no corresponding record for this reference.
- 58Liu, S.; Pentelute, B. L.; Kent, S. B. H. Convergent Chemical Synthesis of [Lysine24, 38, 83] Human Erythropoietin. Angew. Chem. Int. Ed. 2012, 51 (4), 993– 999, DOI: 10.1002/anie.201106060There is no corresponding record for this reference.
- 59Baca, M.; Kent, S. B. Catalytic Contribution of Flap-Substrate Hydrogen Bonds in ″HIV-1 Protease″ Explored by Chemical Synthesis. Proc. Natl. Acad. Sci. U.S.A. 1993, 90 (24), 11638– 11642, DOI: 10.1073/pnas.90.24.11638There is no corresponding record for this reference.
- 60Beadle, J. D.; Knuhtsen, A.; Hoose, A.; Raubo, P.; Jamieson, A. G.; Shipman, M. Solid-Phase Synthesis of Oxetane Modified Peptides. Org. Lett. 2017, 19 (12), 3303– 3306, DOI: 10.1021/acs.orglett.7b01466There is no corresponding record for this reference.
- 61Abdildinova, A.; Kurth, M. J.; Gong, Y.-D. Solid-Phase Synthesis of Peptidomimetics with Peptide Backbone Modifications. Asian J. Org. Chem. 2021, 10 (9), 2300– 2317, DOI: 10.1002/ajoc.20210026461Solid-phase Synthesis of Peptidomimetics with Peptide Backbone ModificationsAbdildinova, Aizhan; Kurth, Mark J.; Gong, Young-DaeAsian Journal of Organic Chemistry (2021), 10 (9), 2300-2317CODEN: AJOCC7; ISSN:2193-5807. (Wiley-VCH Verlag GmbH & Co. KGaA)Peptidomimetics are a class of compds. with promising pharmacol. properties. Peptidomimetics reduce limitations of peptides including low bioavailability, poor stability, and poor cell-permeability. Peptide backbone modifications are a frequently used manipulation to reach desirable properties of the peptide mols. The development of accessible synthetic methodologies plays an important role in peptidomimetic progress. Synthesis of peptidomimetics proceeds via soln. and solid-phase synthesis strategies. Solid-phase org. synthesis serves as a powerful tool for the prepn. of peptidomimetic mols., thus, numerous strategies have been developed over the years. In this review, we discuss solid-phase synthetic approaches for peptide backbone modifications that were presented in the last two decades.
- 62Flavell, R. R.; Muir, T. W. Expressed Protein Ligation (EPL) in the Study of Signal Transduction, Ion Conduction, And Chromatin Biology. Acc. Chem. Res. 2009, 42 (1), 107– 116, DOI: 10.1021/ar800129c62Expressed protein ligation (EPL) in the study of signal transduction, ion conduction, and chromatin biologyFlavell, Robert R.; Muir, Tom W.Accounts of Chemical Research (2009), 42 (1), 107-116CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. EPL is a semisynthetic technique in which a recombinant protein thioester, generated by thiolysis of an intein fusion protein, is reacted with a synthetic or recombinant peptide with an N-terminal cysteine to produce a native peptide bond. This method has been used extensively for the incorporation of biophys. probes, unnatural amino acids, and post-translational modifications in proteins. In the 10 years since this technique was developed, the applications of EPL to studying protein structure and function have grown ever more sophisticated. In this account, we review the use of EPL in selected systems in which substantial mechanistic insights have recently been gained through the use of the semisynthetic protein derivs. EPL has been used in many studies to unravel the complexity of signaling networks and subcellular trafficking. Herein, we highlight this application to 2 different systems. First, we describe how phosphorylated or otherwise modified proteins in the TGF-β signaling network were prepd. and how they were applied to understanding the complexities of this pathway, from receptor activation to nuclear import. Second, Rab-GTPases are multiply modified with lipid derivs., and EPL-based techniques were used to incorporate these modifications, allowing for the elucidation of the biophys. basis of membrane assocn. and dissocn. We also review the use of EPL to understand the biol. of 2 other systems, the potassium channel KcsA and histones. EPL was used to incorporate D-alanine and an amide-to-ester backbone modification in the selectivity filter of the KcsA potassium channel, providing insight into the mechanism of selectivity in ion conduction. In the case of histones, which are among the most heavily post-translationally modified proteins, the modifications play a key role in the regulation of gene transcription and chromatin structure. We describe how native chem. ligation and EPL were used to generate acetylated, phosphorylated, methylated, and ubiquitylated histones and how these modified histones were used to interrogate chromatin biol. Collectively, these studies demonstrate the utility of EPL in protein science. These techniques and concepts are applicable to many other systems, and ongoing advances promise to extend this semisynthetic technique to increasingly complex biol. problems.
- 63Valiyaveetil, F. I.; Leonetti, M.; Muir, T. W.; MacKinnon, R. Ion Selectivity in a Semisynthetic K+ Channel Locked in the Conductive Conformation. Science 2006, 314 (5801), 1004– 1007, DOI: 10.1126/science.1133415There is no corresponding record for this reference.
- 64Vázquez, M. E.; Nitz, M.; Stehn, J.; Yaffe, M. B.; Imperiali, B. Fluorescent Caged Phosphoserine Peptides as Probes to Investigate Phosphorylation-Dependent Protein Associations. J. Am. Chem. Soc. 2003, 125 (34), 10150– 10151, DOI: 10.1021/ja035184764Fluorescent caged phosphoserine peptides as probes to investigate phosphorylation-dependent protein associationsVazquez, M. Eugenio; Nitz, Mark; Stehn, Justine; Yaffe, Michael B.; Imperiali, BarbaraJournal of the American Chemical Society (2003), 125 (34), 10150-10151CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The development of chem. probes for the investigation of the complex phosphorylation signaling cascades that regulate biol. events is crucial to understanding these processes. We describe herein a bifunctional probe that enables spatial and temporal release of a biol. active ligand while allowing simultaneous monitoring of its binding to the protein of interest. Substitution of Tyr(-2) for the environmentally sensitive fluorescent amino acid DANA in the sequence RLYRpSLPA which is known to bind the 14-3-3 protein does not adversely affect binding affinity and allows monitoring of the binding process. The binding of the peptide to 14-3-3 places the fluorescent reporter unit into a hydrophobic pocket, which changes the fluorescent max. emission intensity and wavelength. At the same time, the newly developed photolabile 1-(2-nitrophenyl)ethyl-caged phosphoserine allows control of the release of the biol. active ligand through unmasking of the key phosphoserine functionality upon UV irradn.
- 65Müller, M. M.; Kries, H.; Csuhai, E.; Kast, P.; Hilvert, D. Design, Selection, and Characterization of a Split Chorismate Mutase. Protein Sci. 2010, 19 (5), 1000– 1010, DOI: 10.1002/pro.377There is no corresponding record for this reference.
- 66Choi, Y.; Shin, S. H.; Jung, H.; Kwon, O.; Seo, J. K.; Kee, J.-M. Specific Fluorescent Probe for Protein Histidine Phosphatase Activity. ACS sensors 2019, 4 (4), 1055– 1062, DOI: 10.1021/acssensors.9b00242There is no corresponding record for this reference.
- 67Sainlos, M.; Imperiali, B. Tools For Investigating Peptide-Protein Interactions: Peptide Incorporation of Environment-Sensitive Fluorophores Through SPPS-Based ’Building Block’ Approach. Nat. Protoc. 2007, 2 (12), 3210– 3218, DOI: 10.1038/nprot.2007.443There is no corresponding record for this reference.
- 68Wu, Y.; Tam, W.-S.; Chau, H.-F.; Kaur, S.; Thor, W.; Aik, W. S.; Chan, W.-L.; Zweckstetter, M.; Wong, K.-L. Solid-Phase Fluorescent BODIPY-Peptide Synthesis via in situ Dipyrrin Construction. Chem. Sci. 2020, 11 (41), 11266– 11273, DOI: 10.1039/D0SC04849FThere is no corresponding record for this reference.
- 69Kienhöfer, A.; Kast, P.; Hilvert, D. Selective Stabilization of the Chorismate Mutase Transition State by a Positively Charged Hydrogen Bond Donor. J. Am. Chem. Soc. 2003, 125 (11), 3206– 3207, DOI: 10.1021/ja034199269Selective stabilization of the chorismate mutase transition state by a positively charged hydrogen bond donorKienhofer Alexander; Kast Peter; Hilvert DonaldJournal of the American Chemical Society (2003), 125 (11), 3206-7 ISSN:0002-7863.Citrulline was incorporated via chemical semisynthesis at position 90 in the active site of the AroH chorismate mutase from Bacillus subtilis. The wild-type arginine at this position makes hydrogen-bonding interactions with the ether oxygen of chorismate. Replacement of the positively charged guanidinium group with the isosteric but neutral urea has a dramatic effect on the ability of the enzyme to convert chorismate into prephenate. The Arg90Cit variant exhibits a >104-fold decrease in the catalytic rate constant kcat with a 2.7-fold increase in the Michaelis constant Km. In contrast, its affinity for a conformationally constrained inhibitor molecule that effectively mimics the geometry but not the dissociative character of the transition state is only reduced by a factor of approximately 6. These results show that an active site merely complementary to the reactive conformation of chorismate is insufficient for catalysis of the mutase reaction. Instead, electrostatic stabilization of the polarized transition state by provision of a cationic hydrogen bond donor proximal to the oxygen in the breaking C-O bond is essential for high catalytic efficiency.
- 70Roy, R. S.; Imperiali, B. Pyridoxamine-Amino Acid Chimeras in Semisynthetic Aminotransferase Mimics. Protein eng. 1997, 10 (6), 691– 698, DOI: 10.1093/protein/10.6.691There is no corresponding record for this reference.
- 71Lopez, G.; Anderson, J. C. Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a BL21 (DE3) Biosafety Strain. ACS Synth. Biol. 2015, 4 (12), 1279– 1286, DOI: 10.1021/acssynbio.5b00085There is no corresponding record for this reference.
- 72Budisa, N. Amino Acids and Codons - Code Organization and Protein Structure. Engineering the Genetic Code 2005, 66– 89, DOI: 10.1002/3527607188.ch4There is no corresponding record for this reference.
- 73Budisa, N.; Steipe, B.; Demange, P.; Eckerskorn, C.; Kellermann, J.; Huber, R. High-Level Biosynthetic Substitution of Methionine in Proteins by Its Analogs 2-Aminohexanoic Acid, Selenomethionine, Telluromethionine and Ethionine in Escherichia coli. Eur. J. Biochem. 1995, 230 (2), 788– 796, DOI: 10.1111/j.1432-1033.1995.0788h.xThere is no corresponding record for this reference.
- 74Cowie, D. B.; Cohen, G. N. Biosynthesis by Escherichia coli of Active Altered Proteins Containing Selenium Instead of Sulfur. Biochim. Biophys. Acta 1957, 26 (2), 252– 261, DOI: 10.1016/0006-3002(57)90003-3There is no corresponding record for this reference.
- 75Wong, J. Membership Mutation of the Genetic Code: Loss of Fitness by Tryptophan. Proc. Natl. Acad. Sci. U.S.A. 1983, 80 (20), 6303– 6306, DOI: 10.1073/pnas.80.20.630375Membership mutation of the genetic code: Loss of fitness by tryptophanWong, J. Tze FeiProceedings of the National Academy of Sciences of the United States of America (1983), 80 (20), 6303-6CODEN: PNASA6; ISSN:0027-8424.Bacillus subtilis Strain QB928, a tryptophan auxotroph, was serially mutated to yield strain HR15. For QB928, tryptophan functioned as a competent amino acid and 4-fluorotryptophan as merely an inferior analog. For HR15, these roles were reversed. The tryptophan/4-fluorotryptophan growth ratio decreased by a factor of 2 × 104 in the transition from QB928 to HR15.
- 76Dóring, V.; Mootz, H. D.; Nangle, L. A.; Hendrickson, T. L.; de Crécy-Lagard, V.; Schimmel, P.; Marliere, P. Enlarging the Amino Acid Set of Escherichia coli by Infiltration of the Valine Coding Pathway. Science 2001, 292 (5516), 501– 504, DOI: 10.1126/science.1057718There is no corresponding record for this reference.
- 77Datta, D.; Wang, P.; Carrico, I. S.; Mayo, S. L.; Tirrell, D. A. A Designed Phenylalanyl-tRNA Synthetase Variant Allows Efficient in Vivo Incorporation of Aryl Ketone Functionality into Proteins. J. Am. Chem. Soc. 2002, 124 (20), 5652– 5653, DOI: 10.1021/ja017709677A Designed Phenylalanyl-tRNA Synthetase Variant Allows Efficient in Vivo Incorporation of Aryl Ketone Functionality into ProteinsDatta, Deepshikha; Wang, Pin; Carrico, Isaac S.; Mayo, Stephen L.; Tirrell, David A.Journal of the American Chemical Society (2002), 124 (20), 5652-5653CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Incorporation of non-natural amino acids into proteins in vivo expands the scope of protein synthesis and design. P-Acetylphenylalanine was incorporated into recombinant dihydrofolate reductase (DHFR) in Escherichia coli via a computationally designed mutant form of the phenylalanyl-tRNA synthetase of the host. DHFR outfitted with ketone functionality can be chemoselectively ligated with hydrazide reagents under mild conditions.
- 78Sharma, N.; Furter, R.; Kast, P.; Tirrell, D. A. Efficient Introduction of Aryl Bromide Functionality into Proteins in vivo. FEBS Lett. 2000, 467 (1), 37– 40, DOI: 10.1016/S0014-5793(00)01120-078Efficient introduction of aryl bromide functionality into proteins in vivoSharma, N.; Furter, R.; Kast, P.; Tirrell, D. A.FEBS Letters (2000), 467 (1), 37-40CODEN: FEBLAL; ISSN:0014-5793. (Elsevier Science B.V.)Artificial proteins can be engineered to exhibit interesting solid state, liq. crystal or interfacial properties and may ultimately serve as important alternatives to conventional polymeric materials. The utility of protein-based materials is limited, however, by the availability of just the 20 amino acids that are normally recognized and utilized by biol. systems; many desirable functional groups cannot be incorporated directly into proteins by biosynthetic means. In this study, we incorporate para-bromophenylalanine (p-Br-phe) into a model target protein, mouse dihydrofolate reductase (DHFR), by using a bacterial phenylalanyl-tRNA synthetase (PheRS) variant with relaxed substrate specificity. Coexpression of the mutant PheRS and DHFR in a phenylalanine auxotrophic Escherichia coli host strain grown in p-Br-phe-supplemented minimal medium resulted in 88% replacement of phenylalanine residues by p-Br-phe; variation in the relative amts. of phe and p-Br-phe in the medium allows control of the degree of substitution by the analog. Protein expression yields of 20-25 mg/l were obtained from cultures supplemented with p-Br-phe; this corresponds to about two-thirds of the expression levels characteristic of cultures supplemented with phe. The aryl bromide function is stable under the conditions used to purify DHFR and creates new opportunities for post-translational derivatization of brominated proteins via metal-catalyzed coupling reactions. In addn., bromination may be useful in X-ray studies of proteins via the multiwavelength anomalous diffraction (MAD) technique.
- 79Kiick, K. L.; van Hest, J. C.; Tirrell, D. A. Expanding the Scope of Protein Biosynthesis by Altering the Methionyl-tRNA Synthetase Activity of a Bacterial Expression Host. Angew. Chem. Int. Ed. 2000, 39 (12), 2148– 2152, DOI: 10.1002/1521-3773(20000616)39:12<2148::AID-ANIE2148>3.0.CO;2-7There is no corresponding record for this reference.
- 80Hendrickson, W. A.; Horton, J. R.; LeMaster, D. M. Selenomethionyl Proteins Produced for Analysis by Multiwavelength Anomalous Diffraction (MAD): a Vehicle for Direct Determination of Three-Dimensional Structure. EMBO J. 1990, 9 (5), 1665– 1672, DOI: 10.1002/j.1460-2075.1990.tb08287.x80Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structureHendrickson, Wayne A.; Horton, John R.; LeMaster, David M.EMBO Journal (1990), 9 (5), 1665-72CODEN: EMJODG; ISSN:0261-4189.An expression system has been established for the incorporation of selenomethionine into recombinant proteins produced from plasmids in Escherichia coli. Replacement of methionine by selenomethionine is demonstrated at the level of 100% for both T4 and E. coli thioredoxins. The natural recombinant proteins and the selenomethionyl variants of both thioredoxins crystallize isomorphously. Anomalous scattering factors were deduced from synchrotron x-ray absorption measurements of crystals of the selenomethionyl proteins. Taken with ref. to experience in the structural anal. of selenobiotinyl streptavidin by the method of MAD, these data indicate that recombinant selenomethionyl proteins analyzed by MAD phasing offer a rather general means for the elucidation of at. structures.
- 81Boles, J. O.; Lewinski, K.; Kunkle, M.; Odom, J. D.; Dunlap, R. B.; Lebioda, L.; Hatada, M. Bio-Incorporation of Telluromethionine into Buried Residues of Dihydrofolate Reductase. Nat. Struct. Biol. 1994, 1 (5), 283– 284, DOI: 10.1038/nsb0594-283There is no corresponding record for this reference.
- 82Strub, M. P.; Hoh, F.; Sanchez, J. F.; Strub, J. M.; Böck, A.; Aumelas, A.; Dumas, C. Selenomethionine and Selenocysteine Double Labeling Strategy for Crystallographic Phasing. Structure 2003, 11 (11), 1359– 1367, DOI: 10.1016/j.str.2003.09.014There is no corresponding record for this reference.
- 83Budisa, N.; Karnbrock, W.; Steinbacher, S.; Humm, A.; Prade, L.; Neuefeind, T.; Moroder, L.; Huber, R. Bioincorporation of Telluromethionine into Proteins: A Promising New Approach for X-Ray Structure Analysis of Proteins. J. Mol. Biol. 1997, 270 (4), 616– 623, DOI: 10.1006/jmbi.1997.1132There is no corresponding record for this reference.
- 84Bae, J. H.; Alefelder, S.; Kaiser, J. T.; Friedrich, R.; Moroder, L.; Huber, R.; Budisa, N. Incorporation of β-Selenolo[3,2-b]Pyrrolyl-Alanine into Proteins for Phase Determination in Protein X-Ray Crystallography. J. Mol. Biol. 2001, 309 (4), 925– 936, DOI: 10.1006/jmbi.2001.4699There is no corresponding record for this reference.
- 85Minks, C.; Huber, R.; Moroder, L.; Budisa, N. Atomic Mutations at the Single Tryptophan Residue of Human Recombinant Annexin V: Effects on Structure, Stability, and Activity. Biochemistry 1999, 38 (33), 10649– 10659, DOI: 10.1021/bi990580g85Atomic mutations at the single tryptophan residue of human recombinant annexin V: Effects on structure, stability, and activityMinks, Caroline; Huber, Robert; Moroder, Luis; Budisa, NediljkoBiochemistry (1999), 38 (33), 10649-10659CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The single tryptophan residue (Trp187) of human recombinant annexin V, contg. 320 residues and 5328 atoms, was replaced with three different isosteric analogs where hydrogen atoms at positions 4, 5, and 6 in the indole ring were exchanged with fluorine. Such single atom exchanges of H → F represent at. mutations that result in slightly increased covalent bond lengths and inverted polarities in the residue side-chain structure. These minimal changes in the local geometry do not affect the secondary and tertiary structures of the mutants, which were identical to those of wild-type protein in the crystal form. But the mutants exhibit significant differences in stability, folding cooperativity, biol. activity, and fluorescence properties if compared to the wild-type protein. These rather large global effects, resulting from the minimal local changes, have to be attributed either to the relatively strong changes in polar interactions of the indole ring or to differences in the van der Waals radii or to a combination of both facts. The changes in local geometry that are below resoln. of protein X-ray crystallog. studies are probably of secondary importance in comparison to the strong electronegativity introduced by the fluorine atom. Correspondingly, these types of mutations provide an interesting approach to study cooperative functions of integrated residues and modulation of particular physicochem. properties, in the present case of electronegativity, in a uniquely structured and hierarchically organized protein mol.
- 86Renner, C.; Alefelder, S.; Bae, J. H.; Budisa, N.; Huber, R.; Moroder, L. Fluoroprolines as Tools for Protein Design and Engineering. Angew. Chem. Int. Ed. 2001, 40 (5), 923– 925, DOI: 10.1002/1521-3773(20010302)40:5<923::AID-ANIE923>3.0.CO;2-#86Fluoroprolines as tools for protein design and engineeringRenner, Christian; Alefelder, Stefan; Bae, Jae H.; Budisa, Nediljko; Huber, Robert; Moroder, LuisAngewandte Chemie, International Edition (2001), 40 (5), 923-925CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)The preference of the peptidyl-fluoroproline amide bond for the cis or trans conformation in the model compds. N-acetyl-4-fluoroproline Me esters fully correlates with the thermostability of the related mutants of the model protein barstar. Thus, the (4S)-L-FPro mutants show a higher and the(4R)-L-FPro mutants a lower thermal stability than barstar.
- 87Tang, Y.; Ghirlanda, G.; Petka, W. A.; Nakajima, T.; DeGrado, W. F.; Tirrell, D. A. Fluorinated Coiled-Coil Proteins Prepared in Vivo Display Enhanced Thermal and Chemical Stability. Angew. Chem. Int. Ed. 2001, 40 (8), 1494– 1496, DOI: 10.1002/1521-3773(20010417)40:8<1494::AID-ANIE1494>3.0.CO;2-X87Fluorinated coiled-coil proteins prepared in vivo display enhanced thermal and chemical stabilityTang, Yi; Ghirlanda, Giovanna; Petka, Wendy A.; Nakajima, Tadashi; DeGrado, William F.; Tirrell, David A.Angewandte Chemie, International Edition (2001), 40 (8), 1494-1496CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)In this article the authors present a general approach to the stabilization of leucine-zipper peptides and coiled-coil proteins by incorporation of the hyperhydrophobic leucine isostere trifluoroleucine.
- 88Tang, Y.; Tirrell, D. A. Biosynthesis of a Highly Stable Coiled-Coil Protein Containing Hexafluoroleucine in an Engineered Bacterial Host. J. Am. Chem. Soc. 2001, 123 (44), 11089– 11090, DOI: 10.1021/ja016652k88Biosynthesis of a highly stable coiled-coil protein containing hexafluoroleucine in an engineered bacterial hostTang, Yi; Tirrell, David A.Journal of the American Chemical Society (2001), 123 (44), 11089-11090CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Modification of leucyl-tRNA synthetase (LeuRS) of the host is reported to allow efficient incorporation of hexafluoroleucine (I) into recombinant proteins prepd. in Escherichia coli. The E. coli leuS gene and its endogenous promoter were amplified from genomic DNA and ligated into the expression vector pQEA1 to yield pA1EL; the LeuRS activity of the new strains was approx. 8-fold higher than that of pQEA1-carrying strains and LeuRS was overexpressed. Only the strain carrying pQEA1 supported protein synthesis with I. The compn. and phys. properties of the I-contg. recombinant protein were detd. following affinity chromatog. The yield was 8 mg/L with 74% replacement of leucine by I. The secondary structure was >90% α-helical, and the predominant structure was dimeric. The free energy of unfolding was elevated 3.7 kcal over the leucine-contg. protein and the protein showed remarkable resistance to urea denaturation.
- 89Wang, P.; Tang, Y.; Tirrell, D. A. Incorporation of Trifluoroisoleucine into Proteins in Vivo. J. Am. Chem. Soc. 2003, 125 (23), 6900– 6906, DOI: 10.1021/ja029828789Incorporation of Trifluoroisoleucine into Proteins in VivoWang, Pin; Tang, Yi; Tirrell, David A.Journal of the American Chemical Society (2003), 125 (23), 6900-6906CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Two fluorinated derivs. of isoleucine: D,L-2-amino-3-trifluoromethyl pentanoic acid (3TFI, 2) and D,L-2-amino-5,5,5-trifluoro-3-Me pentanoic acid (5TFI, 3) were prepd. 5TFI was incorporated into a model target protein, murine dihydrofolate reductase (mDHFR), in an isoleucine auxotrophic Escherichia coli host strain suspended in 5TFI-supplemented minimal medium depleted of isoleucine. Incorporation of 5TFI was confirmed by tryptic peptide anal. and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) of the protein product. Amino acid anal. showed that more than 93% of the encoded isoleucine residues were replaced by 5TFI. Measurement of the rate of activation of 5TFI by the E. coli isoleucyl-tRNA synthetase (IleRS) yielded a specificity const. (kcat/Km) 134-fold lower than that for isoleucine. 5TFI was successfully introduced into the cytokine murine interleukin-2 (mIL-2) at the encoded isoleucine positions. The concn. of fluorinated protein that elicits 50% of the maximal proliferative response is 3.87 ng/mL, about 30% higher than that of wild-type mIL-2 (EC50 = 2.70 ng/mL). The maximal responses are equiv. for the fluorinated and wild-type cytokines, indicating that fluorinated proteins can fold into stable and functional structures. 3TFI yielded no evidence for in vivo incorporation into recombinant proteins, and no evidence for activation by IleRS in vitro.
- 90Montclare, J. K.; Tirrell, D. A. Evolving Proteins of Novel Composition. Angew. Chem. Int. Ed. 2006, 45 (27), 4518– 4521, DOI: 10.1002/anie.20060008890Evolving proteins of novel compositionMontclare, Jin Kim; Tirrell, David A.Angewandte Chemie, International Edition (2006), 45 (27), 4518-4521CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Changing its nature: Global incorporation of noncanonical amino acids can alter the behavior of proteins in useful ways. In some cases, however, replacement of natural amino acids by noncanonical analogs can cause loss of protein stability. After several generations, functional proteins of non-natural compn. were prepd. through residue-specific incorporation combined with directed evolution.
- 91Hyun Bae, J.; Rubini, M.; Jung, G.; Wiegand, G.; Seifert, M. H.J.; Azim, M.K.; Kim, J.-S.; Zumbusch, A.; Holak, T. A.; Moroder, L.; Huber, R.; Budisa, N. Expansion of the Genetic Code Enables Design of a Novel “Gold” Class of Green Fluorescent Proteins. J. Mol. Biol. 2003, 328 (5), 1071– 1081, DOI: 10.1016/S0022-2836(03)00364-4There is no corresponding record for this reference.
- 92Budisa, N.; Rubini, M.; Bae, J. H.; Weyher, E.; Wenger, W.; Golbik, R.; Huber, R.; Moroder, L. Global Replacement of Tryptophan with Aminotryptophans Generates Non-Invasive Protein-Based Optical pH Sensors. Angew. Chem. Int. Ed. 2002, 41 (21), 4066– 4069, DOI: 10.1002/1521-3773(20021104)41:21<4066::AID-ANIE4066>3.0.CO;2-6There is no corresponding record for this reference.
- 93Duewel, H. S.; Daub, E.; Robinson, V.; Honek, J. F. Elucidation of Solvent Exposure, Side-Chain Reactivity, and Steric Demands of the Trifluoromethionine Residue in a Recombinant Protein. Biochemistry 2001, 40 (44), 13167– 13176, DOI: 10.1021/bi011381b93Elucidation of solvent exposure, side-chain reactivity, and steric demands of the trifluoromethionine residue in a recombinant proteinDuewel, Henry S.; Daub, Elisabeth; Robinson, Valerie; Honek, John F.Biochemistry (2001), 40 (44), 13167-13176CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)When incorporated into proteins, fluorinated amino acids have been utilized as 19F NMR probes of protein structure and protein-ligand interactions, and as subtle structural replacements for their parent amino acids which is not possible using the std. 20-amino acid repertoire. Recent investigations have shown the ability of various fluorinated methionines, such as difluoromethionine (DFM) and trifluoromethionine (TFM), to be bioincorporated into recombinant proteins and to be extremely useful as 19F NMR biophys. probes. Interestingly, in the case of the bacteriophage lambda lysozyme (LaL) which contains only three Met residues (at positions 1, 14, and 107), four 19F NMR resonances are obsd. when TFM is incorporated into LaL. To elucidate the underlying structural reasons for this anomalous observation and to more fully explore the effect of TFM on protein structure, site-directed mutagenesis was used to assign each 19F NMR resonance. Incorporation of TFM into the M14L mutant resulted in the collapse of the two 19F resonances assocd. with TFM at position 107 into a single resonance, suggesting that when position 14 in wild-type protein contains TFM, a subtle but different environment exists for the methionine at position 107. In addn., 19F and [1H-13C]-HMQC NMR expts. have been utilized with paramagnetic line broadening and K2PtCl4 reactivity expts. to obtain information about the probable spatial position of each Met in the protein. These results are compared with the recently detd. crystal structure of LaL and allow for a more detailed structural explanation for the effect of fluorination on protein structure.
- 94Seifert, M. H. J.; Ksiazek, D.; Azim, M. K.; Smialowski, P.; Budisa, N.; Holak, T. A. Slow Exchange in the Chromophore of a Green Fluorescent Protein Variant. J. Am. Chem. Soc. 2002, 124 (27), 7932– 7942, DOI: 10.1021/ja025772594Slow exchange in the chromophore of a green fluorescent protein variantSeifert, Markus H. J.; Ksiazek, Dorota; Azim, M. Kamran; Smialowski, Pawel; Budisa, Nediljko; Holak, Tad A.Journal of the American Chemical Society (2002), 124 (27), 7932-7942CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Green fluorescent protein and its mutants have become valuable tools in mol. biol. They also provide systems rich in photophys. and photochem. phenomena of which an understanding is important for the development of new and optimized variants of GFP. Surprisingly, not a single NMR study has been reported on GFPs until now, possibly because of their high tendency to aggregate. ABS Here, we report the 19F NMR studies on mutants of the green fluorescent protein (GFP) and cyan fluorescent protein (CFP) labeled with fluorinated tryptophans that enabled the detection of slow mol. motions in these proteins. The concerted use of dynamic NMR and 19F relaxation measurements, supported by temp., concn.- and folding-dependent expts. provides direct evidence for the existence of a slow exchange process between two different conformational states of CFP. 19F NMR relaxation and line shape anal. indicate that the time scale of exchange between these states is in the range of 1.2-1.4 ms. Thermodn. anal. revealed a difference in enthalpy ΔH0 = (18.2±3.8) kJ/mol and entropy TΔS0 = (19.6±1.2) kJ/mol at T = 303 K for the two states involved in the exchange process, indicating an entropy-enthalpy compensation. The free energy of activation was estd. to be approx. 60 kJ/mol. Exchange between two conformations, either of the chromophore itself or more likely of the closely related histidine 148, is suggested to be the structural process underlying the conformational mobility of GFPs. The possibility to generate a series of single-atom exchanges ("at. mutations") like H → F in this study offers a useful approach for characterizing and quantifying dynamic processes in proteins by NMR.
- 95Bann, J. G.; Pinkner, J.; Hultgren, S. J.; Frieden, C. Real-Time and Equilibrium 19F-NMR Studies Reveal the Role of Domain-Domain Interactions in the Folding of the Chaperone PapD. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (2), 709– 714, DOI: 10.1073/pnas.022649599There is no corresponding record for this reference.
- 96Deming, T. J.; Fournier, M. J.; Mason, T. L.; Tirrell, D. A. Biosynthetic Incorporation and Chemical Modification of Alkene Functionality in Genetically Engineered Polymers. Journal of Macromolecular Science, Part A 1997, 34 (10), 2143– 2150, DOI: 10.1080/10601329708010331There is no corresponding record for this reference.
- 97Kiick, K. L.; Saxon, E.; Tirrell, D. A.; Bertozzi, C. R. Incorporation of Azides into Recombinant Proteins for Chemoselective Modification by the Staudinger Ligation. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (1), 19– 24, DOI: 10.1073/pnas.01258329997Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligationKiick, Kristi L.; Saxon, Eliana; Tirrell, David A.; Bertozzi, Carolyn R.Proceedings of the National Academy of Sciences of the United States of America (2002), 99 (1), 19-24CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The introduction of chem. unique groups into proteins by means of non-natural amino acids has numerous applications in protein engineering and functional studies. One method to achieve this involves the utilization of a non-natural amino acid by the cell's native translational app. Here we demonstrate that a methionine surrogate, azidohomoalanine, is activated by the methionyl-tRNA synthetase of Escherichia coli and replaces methionine in proteins expressed in methionine-depleted bacterial cultures. We further show that proteins contg. azidohomoalanine can be selectively modified in the presence of other cellular proteins by means of Staudinger ligation with triarylphosphine reagents. Incorporation of azide-functionalized amino acids into proteins in vivo provides opportunities for protein modification under native conditions and selective labeling of proteins in the intracellular environment.
- 98Kothakota, S.; Mason, T. L.; Tirrell, D. A.; Fournier, M. J. Biosynthesis of a Periodic Protein Containing 3-Thienylalanine: A Step Toward Genetically Engineered Conducting Polymers. J. Am. Chem. Soc. 1995, 117 (1), 536– 537, DOI: 10.1021/ja00106a06498Biosynthesis of a Periodic Protein Containing 3-Thienylalanine: A Step Toward Genetically Engineered Conducting PolymersKothakota, Srinivas; Mason, Thomas L.; Tirrell, David A.; Fournier, Maurille J.Journal of the American Chemical Society (1995), 117 (1), 536-7CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The authors present evidence for the use of 3-thienylalanine (3-TA), a phenylalanine analog, by the Escherichia coli biosynthetic machinery. The analog was incorporated into a periodic polymer of sequence [(GlyAla)3GlyPhe]13, in place of phenylalanine. The extent of substitution was at least 80%, as shown by UV spectroscopy, NMR spectroscopy and amino acid anal. No evidence for modification of the analog was found. The 3-alkylthiophene side chain of 3-TA should be susceptible to oxidative crosslinking or grafting of conventional 3-alkylthiophene monomers, opening a route to genetically engineered polymeric materials with useful properties.
- 99Link, A. J.; Tirrell, D. A. Cell Surface Labeling of Escherichia coli via Copper(I)-Catalyzed [3 + 2] Cycloaddition. J. Am. Chem. Soc. 2003, 125 (37), 11164– 11165, DOI: 10.1021/ja036765z99Cell surface labeling of Escherichia coli via copper(I)-catalyzed [3+2] cycloadditionLink, A. James; Tirrell, David A.Journal of the American Chemical Society (2003), 125 (37), 11164-11165CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Labeling of the cell surface of Escherichia coli was accomplished by expression of a recombinant outer membrane protein, OmpC, in the presence of the unnatural amino acid azidohomoalanine, which acts as a methionine surrogate. The surface-exposed azide moieties of whole cells were biotinylated via Cu(1)-catalyzed [3+2] azide-alkyne cycloaddn. The specificity of labeling of both wild-type OmpC and a mutant contg. addnl. methionine sites for azidohomoalanine incorporation was confirmed by Western blotting. Flow cytometry was performed to examine the specificity of the labeling. Cells that express the mutant form of OmpC in the presence of azidohomoalanine, which were biotinylated and stained with fluorescent avidin, exhibit a mean fluorescence 10-fold higher than the background. Incorporation of an unnatural amino acid can thus be detd. on a single-cell basis.
- 100van Hest, J. C. M.; Kiick, K. L.; Tirrell, D. A. Efficient Incorporation of Unsaturated Methionine Analogues into Proteins in Vivo. J. Am. Chem. Soc. 2000, 122 (7), 1282– 1288, DOI: 10.1021/ja992749j100Efficient incorporation of unsaturated methionine analogues into proteins in vivoVan Hest, Jan C. M.; Kiick, Kristi L.; Tirrell, David A.Journal of the American Chemical Society (2000), 122 (7), 1282-1288CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A set of eight methionine analogs was assayed for translational activity in Escherichia coli. Norvaline and norleucine, which are com. available, were assayed along with 2-amino-5-hexenoic acid (I), 2-amino-5-hexynoic acid (II), cis-2-amino-4-hexenoic acid, trans-2-amino-4-hexenoic acid, 6,6,6-trifluoro-2-aminohexanoic acid, and 2-aminoheptanoic acid, each of which was prepd. by alkylation of di-Et acetamidomalonate with the appropriate tosylate, followed by hydrolysis. The E. coli methionine auxotroph CAG18491, transformed with plasmids pREP4 and pQE15, was used as the expression host, and translational activity was assayed by detn. of the capacity of the analog to support synthesis of the test protein dihydrofolate reductase (DHFR) in the absence of added methionine. The importance of amino acid side chain length was illustrated by the fact that neither norvaline nor 2-aminoheptanoic acid showed translational activity, in contrast to norleucine, which does support protein synthesis under the assay conditions. The internal alkene functions of cis-2-amino-4-hexenoic acid trans-2-amino-4-hexenoic acid prevented incorporation of these analogs into test protein, and the fluorinated analog 6,6,6-trifluoro-2-aminohexanoic acid yielded no evidence of translational activity. The terminally unsatd. compds. I and II, however, proved to be excellent methionine surrogates: 1H NMR spectroscopy, amino acid anal., and N-terminal sequencing indicated ∼85% substitution of methionine by I, while II showed 90-100% replacement. Both analogs also function efficiently in the initiation step of protein synthesis, as shown by their near-quant. occupancy of the N-terminal amino acid site in DHFR. Enzyme kinetics assays were conducted to det. the rate of activation of each of the methionine analogs by methionyl tRNA synthetase (MetRS); results of the in vitro assays corroborate the in vivo incorporation results, suggesting that success or failure of analog incorporation in vivo is controlled by MetRS.
- 101van Hest, J. C. M.; Tirrell, D. A. Efficient introduction of alkene functionality into proteins in vivo. FEBS Lett. 1998, 428 (1–2), 68– 70, DOI: 10.1016/S0014-5793(98)00489-XThere is no corresponding record for this reference.
- 102Omari, K. E.; Ren, J.; Bird, L. E.; Bona, M. K.; Klarmann, G.; LeGrice, S. F. J.; Stammers, D. K. Molecular Architecture and Ligand Recognition Determinants for T4 RNA Ligase. J. Biol. Chem. 2006, 281 (3), 1573– 1579, DOI: 10.1074/jbc.M509658200There is no corresponding record for this reference.
- 103Xiao, H.; Murakami, H.; Suga, H.; Ferré-D’Amaré, A. R. Structural Basis of Specific tRNA Aminoacylation by a Small in vitro Selected Ribozyme. Nature 2008, 454 (7202), 358– 361, DOI: 10.1038/nature07033103Structural basis of specific tRNA aminoacylation by a small in vitro selected ribozymeXiao, Hong; Murakami, Hiroshi; Suga, Hiroaki; Ferre-D'Amare, Adrian R.Nature (London, United Kingdom) (2008), 454 (7202), 358-361CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)In modern organisms, protein enzymes are solely responsible for the aminoacylation of tRNA. However, the evolution of protein synthesis in the RNA world required RNAs capable of catalyzing this reaction. Ribozymes that aminoacylate RNA by using activated amino acids have been discovered through selection in vitro. Flexizyme is a 45-nucleotide ribozyme capable of charging tRNA in trans with various activated L-phenylalanine derivs. In addn. to a more than 105 rate enhancement and more than 104-fold discrimination against some non-cognate amino acids, this ribozyme achieves good regioselectivity: of all the hydroxyl groups of a tRNA, it exclusively aminoacylates the terminal 3'-OH. Here we report the 2.8-Å resoln. structure of flexizyme fused to a substrate RNA. Together with randomization of ribozyme core residues and reselection, this structure shows that very few nucleotides are needed for the aminoacylation of specific tRNAs. Although it primarily recognizes tRNA through base-pairing with the CCA terminus of the tRNA mol., flexizyme makes numerous local interactions to position the acceptor end of tRNA precisely. A comparison of two crystallog. independent flexizyme conformations, only one of which appears capable of binding activated phenylalanine, suggests that this ribozyme may achieve enhanced specificity by coupling active-site folding to tRNA docking. Such a mechanism would be reminiscent of the mutually induced fit of tRNA and protein employed by some aminoacyl-tRNA synthetases to increase specificity.
- 104Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. A General Method for Site-specific Incorporation of Unnatural Amino Acids into Proteins. Science 1989, 244 (4901), 182– 188, DOI: 10.1126/science.2649980104A general method for site-specific incorporation of unnatural amino acids into proteinsNoren, Christopher J.; Anthony-Cahill, Spencer J.; Griffith, Michael C.; Schultz, Peter G.Science (Washington, DC, United States) (1989), 244 (4901), 182-8CODEN: SCIEAS; ISSN:0036-8075.A new method has been developed that makes it possible to site-specifically incorporate unnatural amino acids into proteins. Synthetic amino acids were incorporated into the enzyme β-lactamase by the use of a chem. acylated suppressor tRNA that inserted the amino acid in response to a stop codon substituted for the codon encoding residue of interest. Peptide mapping localized the inserted amino acid to a single peptide, and enough enzyme could be generated for purifn. to homogeneity. The catalytic properties of several mutants at the conserved Phe66 were characterized. The ability to selectively replace amino acids in a protein with a wide variety of structural and electronic variants should provide a more detailed understanding of protein structure and function.
- 105Goto, Y.; Katoh, T.; Suga, H. Flexizymes for Genetic Code Reprogramming. Nat. Protoc. 2011, 6 (6), 779– 790, DOI: 10.1038/nprot.2011.331105Flexizymes for genetic code reprogrammingGoto, Yuki; Katoh, Takayuki; Suga, HiroakiNature Protocols (2011), 6 (6), 779-790CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Genetic code reprogramming is a method for the reassignment of arbitrary codons from proteinogenic amino acids to nonproteinogenic ones; thus, specific sequences of nonstandard peptides can be ribosomally expressed according to their mRNA templates. Here we describe a protocol that facilitates genetic code reprogramming using flexizymes integrated with a custom-made in vitro translation app., referred to as the flexible in vitro translation (FIT) system. Flexizymes are flexible tRNA acylation ribozymes that enable the prepn. of a diverse array of nonproteinogenic acyl-tRNAs. These acyl-tRNAs read vacant codons created in the FIT system, yielding the desired nonstandard peptides with diverse exotic structures, such as N-Me amino acids, D-amino acids and physiol. stable macrocyclic scaffolds. The facility of the protocol allows a wide variety of applications in the synthesis of new classes of nonstandard peptides with biol. functions. Prepn. of flexizymes and tRNA used for genetic code reprogramming, optimization of flexizyme reaction conditions and expression of nonstandard peptides using the FIT system can be completed by one person in ∼1 wk. However, once the flexizymes and tRNAs are in hand and reaction conditions are fixed, synthesis of acyl-tRNAs and peptide expression is generally completed in 1 d, and alteration of a peptide sequence can be achieved by simply changing the corresponding mRNA template.
- 106Shimizu, Y.; Inoue, A.; Tomari, Y.; Suzuki, T.; Yokogawa, T.; Nishikawa, K.; Ueda, T. Cell-Free Translation Reconstituted with Purified Components. Nat. Biotechnol. 2001, 19 (8), 751– 755, DOI: 10.1038/90802106Cell-free translation reconstituted with purified componentsShimizu, Yoshihiro; Inoue, Akio; Tomari, Yukihide; Suzuki, Tsutomu; Yokogawa, Takashi; Nishikawa, Kazuya; Ueda, TakuyaNature Biotechnology (2001), 19 (8), 751-755CODEN: NABIF9; ISSN:1087-0156. (Nature America Inc.)We have developed a protein-synthesizing system reconstituted from recombinant tagged protein factors purified to homogeneity. The system was able to produce protein at a rate of about 160 μg/mL/h in a batch mode without the need for any supplementary app. The protein products were easily purified within 1 h using affinity chromatog. to remove the tagged protein factors. Moreover, omission of a release factor allowed efficient incorporation of an unnatural amino acid using suppressor tRNA.
- 107Chapeville, F.; Lipmann, F.; Ehrenstein, G. v.; Weisblum, B.; Ray, W. J.; Benzer, S. On the Role of Soluble Ribonucleic Acid in Coding for Amino Acids. Proc. Natl. Acad. Sci. U.S.A. 1962, 48 (6), 1086– 1092, DOI: 10.1073/pnas.48.6.1086There is no corresponding record for this reference.
- 108Fahnestock, S.; Rich, A. Ribosome-Catalyzed Polyester Formation. Science 1971, 173 (3994), 340– 343, DOI: 10.1126/science.173.3994.340108Ribosome-catalyzed polyester formationFahnestock, Stephen; Rich, AlexanderScience (Washington, DC, United States) (1971), 173 (3994), 340-3CODEN: SCIEAS; ISSN:0036-8075.The deamination of phenylalanyl-tRNA with HNO2 gave the α-hydroxyacyl analog phenyllactyl-tRNA, which incubated in a protein-synthesizing system directed by polyuridylic acid yielded an acid-precipitable, alkali-labile phenyllactic acid polyester. Similarities with polyphenylalanine formation suggested the existence of the same ribosomal mechanism. The polymer consisted 70-80% of phenyllactic acid residues, the remaining residues being probably phenylalanine.
- 109Hecht, S. M.; Alford, B. L.; Kuroda, Y.; Kitano, S. “Chemical Aminoacylation” of tRNA’s. J. Biol. Chem. 1978, 253 (13), 4517– 4520, DOI: 10.1016/S0021-9258(17)30417-9There is no corresponding record for this reference.
- 110Katoh, T.; Goto, Y.; Passioura, T.; Suga, H. Development of Flexizyme Aminoacylation Ribozymes and Their Applications. Ribozymes 2021, 519– 543, DOI: 10.1002/9783527814527.ch20There is no corresponding record for this reference.
- 111Morimoto, J.; Hayashi, Y.; Iwasaki, K.; Suga, H. Flexizymes: Their Evolutionary History and the Origin of Catalytic Function. Acc. Chem. Res. 2011, 44 (12), 1359– 1368, DOI: 10.1021/ar2000953111Flexizymes: Their Evolutionary History and the Origin of Catalytic FunctionMorimoto, Jumpei; Hayashi, Yuuki; Iwasaki, Kazuhiro; Suga, HiroakiAccounts of Chemical Research (2011), 44 (12), 1359-1368CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. TRNA is an essential component of the cell's translation app. These RNA strands contain the anticodon for a given amino acid, and when "charged" with that amino acid are termed aminoacyl-tRNA. Aminoacylation, which occurs exclusively at one of the 3'-terminal hydroxyl groups of tRNA, is catalyzed by a family of enzymes called aminoacyl-tRNA synthetases (ARSs). In a primitive translation system, before the advent of sophisticated protein-based enzymes, this chem. event could conceivably have been catalyzed solely by RNA enzymes. Given the evolutionary implications, our group attempted in vitro selection of artificial ARS-like ribozymes, successfully uncovering a functional ribozyme (r24) from an RNA pool of random sequences attached to the 5'-leader region of tRNA. This ribozyme preferentially charges arom. amino acids (such as phenylalanine) activated with cyanomethyl ester (CME) onto specific kinds of tRNA. During the course of our studies, we became interested in developing a versatile, rather than a specific, aminoacylation catalyst. Such a ribozyme could facilitate the prepn. of intentionally misacylated tRNAs and thus serve a convenient tool for manipulating the genetic code. On the basis of biochem. studies of r24, we constructed a truncated version of r24 (r24mini) that was 57 nucleotides long. This r24mini was then further shortened to 45 nucleotides. This ribozyme could charge various tRNAs through very simple three-base-pair interactions between the ribozyme's 3'-end and the tRNA's 3'-end. We termed this ribozyme a "flexizyme" (Fx3 for this particular construct) owing to its flexibility in addressing tRNAs. To devise an even more flexible tool for tRNA acylation, we attempted to eliminate the amino acid specificity from Fx3. This attempt yielded an Fx3 variant, termed dFx, which accepts amino acid substrates having 3,5-dinitrobenzyl ester instead of CME as a leaving group. Similar selection attempts with the original phenylalanine-CME and a substrate activated by (2-aminoethyl)amidocarboxybenzyl thioester yielded the variants eFx and aFx (e and a denote enhanced and amino, resp.). In this Account, we describe the history and development of these flexizymes and their appropriate substrates, which provide a versatile and easy-to-use tRNA acylation system. Their use permits the synthesis of a wide array of acyl-tRNAs charged with artificial amino and hydroxy acids. In parallel with these efforts, we initiated a crystn. study of Fx3 covalently conjugated to a microhelix RNA, which is an analog of tRNA. The X-ray crystal structure, solved as a co-complex with phenylalanine Et ester and U1A-binding protein, revealed the structural basis of this enzyme. Most importantly, many biochem. observations were consistent with the crystal structure. Along with the predicted three regular-helix regions, however, the flexizyme has a unique irregular helix that was unexpected. This irregular helix constitutes a recognition pocket for the arom. ring of the amino acid side chain and precisely brings the carbonyl group to the 3'-hydroxyl group of the tRNA 3'-end. This study has clearly defined the mol. interactions between Fx3, tRNA, and the amino acid substrate, revealing the fundamental basis of this unique catalytic system.
- 112Murakami, H.; Ohta, A.; Ashigai, H.; Suga, H. A Highly Flexible tRNA Acylation Method for Non-Natural Polypeptide Synthesis. Nat. Methods 2006, 3 (5), 357– 359, DOI: 10.1038/nmeth877112A highly flexible tRNA acylation method for non-natural polypeptide synthesisMurakami, Hiroshi; Ohta, Atsushi; Ashigai, Hiroshi; Suga, HiroakiNature Methods (2006), 3 (5), 357-359CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Here the authors describe a de novo tRNA acylation system, the flexizyme (Fx) system, for the prepn. of acyl tRNAs with nearly unlimited selection of amino and hydroxy acids and tRNAs. The combination of the Fx system with an appropriate cell-free translation system allows the authors to readily perform mRNA-encoded synthesis of proteins and short polypeptides involving multiple nonnatural amino acids.
- 113Katoh, T.; Suga, H. In Vitro Genetic Code Reprogramming for the Expansion of Usable Noncanonical Amino Acids. Annu. Rev. Biochem. 2022, 91 (1), 221– 243, DOI: 10.1146/annurev-biochem-040320-103817There is no corresponding record for this reference.
- 114Goto, Y.; Suga, H. The RaPID Platform for the Discovery of Pseudo-Natural Macrocyclic Peptides. Acc. Chem. Res. 2021, 54 (18), 3604– 3617, DOI: 10.1021/acs.accounts.1c00391114The RaPID Platform for the Discovery of Pseudo-Natural Macrocyclic PeptidesGoto, Yuki; Suga, HiroakiAccounts of Chemical Research (2021), 54 (18), 3604-3617CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. Conspectus: Although macrocyclic peptides bearing exotic building blocks have proven their utility as pharmaceuticals, the sources of macrocyclic peptide drugs have been largely limited to mimetics of native peptides or natural product peptides. However, the recent emergence of technologies for discovering de novo bioactive peptides has led to their reconceptualization as a promising therapeutic modality. For the construction and screening of libraries of such macrocyclic peptides, our group has devised a platform to conduct affinity-based selection of massive libraries (>1012 unique sequences) of in vitro expressed macrocyclic peptides, which is referred to as the random nonstandard peptides integrated discovery (RaPID) system. The RaPID system integrates genetic code reprogramming using the FIT (flexible in vitro translation) system, which is largely facilitated by flexizymes (flexible tRNA-aminoacylating ribozymes), with mRNA display technol. We have demonstrated that the RaPID system enables rapid discovery of various de novo pseudo-natural peptide ligands for protein targets of interest. Many examples discussed in this Account prove that thioether-closed macrocyclic peptides (teMPs) obtained by the RaPID system generally exhibit remarkably high affinity and specificity, thereby potently inhibiting or activating a specific function(s) of the target. Moreover, such teMPs are used for a wide range of biochem. applications, for example, as crystn. chaperones for intractable transmembrane proteins and for in vivo recognition of specific cell types. Furthermore, recent studies demonstrate that some teMPs exhibit pharmacol. activities in animal models and that even intracellular proteins can be inhibited by teMPs, illustrating the potential of this class of peptides as drug leads. Besides the ring-closing thioether linkage in the teMPs, genetic code reprogramming by the FIT system allows for incorporation of a variety of other exotic building blocks. For instance, diverse nonproteinogenic amino acids, hydroxy acids (ester linkage), amino carbothioic acid (thioamide linkage), and abiotic foldamer units have been successfully incorporated into ribosomally synthesized peptides. Despite such enormous successes in the conventional FIT system, multiple or consecutive incorporation of highly exotic amino acids, such as D- and β-amino acids, is yet challenging, and particularly the synthesis of peptides bearing non-carbonyl backbone structures remains a demanding task. To upgrade the RaPID system to the next generation, we have engaged in intensive manipulation of the FIT system to expand the structural diversity of peptides accessible by our in vitro biosynthesis strategy. Semilogical engineering of tRNA body sequences led to a new suppressor tRNA (tRNAPro1E2) capable of effectively recruiting translation factors, particularly EF-Tu and EF-P. The use of tRNAPro1E2 in the FIT system allows for not only single but also consecutive and multiple elongation of exotic amino acids, such as D-, β-, and γ-amino acids as well as aminobenzoic acids. Moreover, the integration of the FIT system with various chem. or enzymic posttranslational modifications enables us to expand the range of accessible backbone structures to non-carbonyl moieties prominent in natural products and peptidomimetics. In such systems, FIT-expressed peptides undergo multistep backbone conversions in a one-pot manner to yield designer peptides composed of modified backbones such as azolines, azoles, and ring-closing pyridines. Our current research endeavors focus on applying such in vitro biosynthesis systems for the discovery of bioactive de novo pseudo-natural products.
- 115Schultz, P. Expanding the Genetic Code. Protein Sci. 2023, 32 (1), e4488 DOI: 10.1002/pro.4488There is no corresponding record for this reference.
- 116Neumann, H.; Wang, K.; Davis, L.; Garcia-Alai, M.; Chin, J. W. Encoding Multiple Unnatural Amino Acids via Evolution of a Quadruplet-Decoding Ribosome. Nature 2010, 464 (7287), 441– 444, DOI: 10.1038/nature08817116Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosomeNeumann, Heinz; Wang, Kaihang; Davis, Lloyd; Garcia-Alai, Maria; Chin, Jason W.Nature (London, United Kingdom) (2010), 464 (7287), 441-444CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The in vivo, genetically programmed incorporation of designer amino acids allows the properties of proteins to be tailored with mol. precision. The Methanococcus jannaschii tyrosyl-transfer-RNA synthetase-tRNACUA (MjTyrRS-tRNACUA) and the Methanosarcina barkeri pyrrolysyl-tRNA synthetase-tRNACUA (MbPylRS-tRNACUA) orthogonal pairs have been evolved to incorporate a range of unnatural amino acids in response to the amber codon in Escherichia coli. However, the potential of synthetic genetic code expansion is generally limited to the low efficiency incorporation of a single type of unnatural amino acid at a time, because every triplet codon in the universal genetic code is used in encoding the synthesis of the proteome. To encode efficiently many distinct unnatural amino acids into proteins we require blank codons and mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs that recognize unnatural amino acids and decode the new codons. Here we synthetically evolve an orthogonal ribosome (ribo-Q1) that efficiently decodes a series of quadruplet codons and the amber codon, providing several blank codons on an orthogonal mRNA, which it specifically translates. By creating mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs and combining them with ribo-Q1 we direct the incorporation of distinct unnatural amino acids in response to two of the new blank codons on the orthogonal mRNA. Using this code, we genetically direct the formation of a specific, redox-insensitive, nanoscale protein cross-link by the bio-orthogonal cycloaddn. of encoded azide- and alkyne-contg. amino acids. Because the synthetase-tRNA pairs used have been evolved to incorporate numerous unnatural amino acids, it will be possible to encode more than 200 unnatural amino acid combinations using this approach. As ribo-Q1 independently decodes a series of quadruplet codons, this work provides foundational technologies for the encoded synthesis and synthetic evolution of unnatural polymers in cells.
- 117Robertson, W. E.; Funke, L. F. H.; de la Torre, D.; Fredens, J.; Elliott, T. S.; Spinck, M.; Christova, Y.; Cervettini, D.; Böge, F. L.; Liu, K. C. Sense Codon Reassignment Enables Viral Resistance and Encoded Polymer Synthesis. Science 2021, 372 (6546), 1057– 1062, DOI: 10.1126/science.abg3029117Sense codon reassignment enables viral resistance and encoded polymer synthesisRobertson, Wesley E.; Funke, Louise F. H.; de la Torre, Daniel; Fredens, Julius; Elliott, Thomas S.; Spinck, Martin; Christova, Yonka; Cervettini, Daniele; Boge, Franz L.; Liu, Kim C.; Buse, Salvador; Maslen, Sarah; Salmond, George P. C.; Chin, Jason W.Science (Washington, DC, United States) (2021), 372 (6546), 1057-1062CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)It is widely hypothesized that removing cellular tRNAs (tRNAs)-making their cognate codons unreadable-might create a genetic firewall to viral infection and enable sense codon reassignment. However, it has been impossible to test these hypotheses. In this work, following synonymous codon compression and lab. evolution in Escherichia coli, we deleted the tRNAs and release factor 1, which normally decode two sense codons and a stop codon; the resulting cells could not read the canonical genetic code and were completely resistant to a cocktail of viruses. We reassigned these codons to enable the efficient synthesis of proteins contg. three distinct noncanonical amino acids. Notably, we demonstrate the facile reprogramming of our cells for the encoded translation of diverse noncanonical heteropolymers and macrocycles.
- 118Wang, L.; Brock, A.; Herberich, B.; Schultz, P. G. Expanding the Genetic Code of Escherichia coli. Science 2001, 292 (5516), 498– 500, DOI: 10.1126/science.1060077118Expanding the genetic code of Escherichia coliWang, Lei; Brock, Ansgar; Herberich, Brad; Schultz, Peter G.Science (Washington, DC, United States) (2001), 292 (5516), 498-500CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A unique tRNA/aminoacyl-tRNA synthetase pair has been generated that expands the no. of genetically encoded amino acids in Escherichia coli. When introduced into E. coli, this pair leads to the in vivo incorporation of the synthetic amino acid O-methyl-L-tyrosine into protein in response to an amber nonsense codon. The fidelity of translation is greater than 99%, as detd. by anal. of dihydrofolate reductase contg. the unnatural amino acid. This approach should provide a general method for increasing the genetic repertoire of living cells to include a variety of amino acids with novel structural, chem., and phys. properties not found in the common 20 amino acids.
- 119Dunkelmann, D. L.; Piedrafita, C.; Dickson, A.; Liu, K. C.; Elliott, T. S.; Fiedler, M.; Bellini, D.; Zhou, A.; Cervettini, D.; Chin, J. W. Adding α,α-Disubstituted and β-Linked Monomers to the Genetic Code of an Organism. Nature 2024, 625 (7995), 603– 610, DOI: 10.1038/s41586-023-06897-6There is no corresponding record for this reference.
- 120Bryson, D. I. Continuous Directed Evolution of Aminoacyl-tRNA Synthetases. Nat. Chem. Biol. 2017, 13, 1253, DOI: 10.1038/nchembio.2474120Continuous directed evolution of aminoacyl-tRNA synthetasesBryson, David I.; Fan, Chenguang; Guo, Li-Tao; Miller, Corwin; Soll, Dieter; Liu, David R.Nature Chemical Biology (2017), 13 (12), 1253-1260CODEN: NCBABT; ISSN:1552-4450. (Nature Research)Directed evolution of orthogonal aminoacyl-tRNA synthetases (AARSs) enables site-specific installation of noncanonical amino acids (ncAAs) into proteins. Traditional evolution techniques typically produce AARSs with greatly reduced activity and selectivity compared to their wild-type counterparts. We designed phage-assisted continuous evolution (PACE) selections to rapidly produce highly active and selective orthogonal AARSs through hundreds of generations of evolution. PACE of a chimeric Methanosarcina spp. pyrrolysyl-tRNA synthetase (PylRS) improved its enzymic efficiency (kcat/KMtRNA) 45-fold compared to the parent enzyme. Transplantation of the evolved mutations into other PylRS-derived synthetases improved yields of proteins contg. noncanonical residues up to 9.7-fold. Simultaneous pos. and neg. selection PACE over 48 h greatly improved the selectivity of a promiscuous Methanocaldococcus jannaschii tyrosyl-tRNA synthetase variant for site-specific incorporation of p-iodo-L-phenylalanine. These findings offer new AARSs that increase the utility of orthogonal translation systems and establish the capability of PACE to efficiently evolve orthogonal AARSs with high activity and amino acid specificity.
- 121Krahn, N.; Tharp, J. M.; Crnković, A.; Söll, D. Chapter Twelve - Engineering Aminoacyl-tRNA Synthetases for Use in Synthetic Biology. In The Enzymes; Ribas de Pouplana, L., Kaguni, L. S., Eds.; Academic Press, 2020; Vol. 48, pp 351– 395. DOI: 10.1016/bs.enz.2020.06.004There is no corresponding record for this reference.
- 122Amiram, M.; Haimovich, A. D.; Fan, C.; Wang, Y.-S.; Aerni, H.-R.; Ntai, I.; Moonan, D. W.; Ma, N. J.; Rovner, A. J.; Hong, S. H. Evolution of Translation Machinery in Recoded Bacteria Enables Multi-Site Incorporation of Nonstandard Amino Acids. Nat. Biotechnol. 2015, 33 (12), 1272– 1279, DOI: 10.1038/nbt.3372122Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acidsAmiram, Miriam; Haimovich, Adrian D.; Fan, Chenguang; Wang, Yane-Shih; Aerni, Hans-Rudolf; Ntai, Ioanna; Moonan, Daniel W.; Ma, Natalie J.; Rovner, Alexis J.; Hong, Seok Hoon; Kelleher, Neil L.; Goodman, Andrew L.; Jewett, Michael C.; Soll, Dieter; Rinehart, Jesse; Isaacs, Farren J.Nature Biotechnology (2015), 33 (12), 1272-1279CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Expansion of the genetic code with nonstandard amino acids (nsAAs) has enabled biosynthesis of proteins with diverse new chemistries. However, this technol. has been largely restricted to proteins contg. a single or few nsAA instances. Here we describe an in vivo evolution approach in a genomically recoded Escherichia coli strain for the selection of orthogonal translation systems capable of multi-site nsAA incorporation. We evolved chromosomal aminoacyl-tRNA synthetases (aaRSs) with up to 25-fold increased protein prodn. for p-acetyl-L-phenylalanine and p-azido-L-phenylalanine (pAzF). We also evolved aaRSs with tunable specificities for 14 nsAAs, including an enzyme that efficiently charges pAzF while excluding 237 other nsAAs. These variants enabled prodn. of elastin-like-polypeptides with 30 nsAA residues at high yields (∼50 mg/L) and high accuracy of incorporation (>95%). This approach to aaRS evolution should accelerate and expand our ability to produce functionalized proteins and sequence-defined polymers with diverse chemistries.
- 123Hohl, A.; Karan, R.; Akal, A.; Renn, D.; Liu, X.; Ghorpade, S.; Groll, M.; Rueping, M.; Eppinger, J. Engineering a Polyspecific Pyrrolysyl-tRNA Synthetase by a High Throughput FACS Screen. Sci. Rep. 2019, 9 (1), 11971, DOI: 10.1038/s41598-019-48357-0123Engineering a Polyspecific Pyrrolysyl-tRNA Synthetase by a High Throughput FACS ScreenHohl Adrian; Karan Ram; Akal Anastassja; Renn Dominik; Liu Xuechao; Ghorpade Seema; Rueping Magnus; Eppinger Jorg; Hohl Adrian; Akal Anastassja; Renn Dominik; Groll MichaelScientific reports (2019), 9 (1), 11971 ISSN:.The Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA(Pyl) are extensively used to add non-canonical amino acids (ncAAs) to the genetic code of bacterial and eukaryotic cells. However, new ncAAs often require a cumbersome de novo engineering process to generate an appropriate PylRS/tRNA(Pyl) pair. We here report a strategy to predict a PylRS variant with novel properties. The designed polyspecific PylRS variant HpRS catalyzes the aminoacylation of 31 structurally diverse ncAAs bearing clickable, fluorinated, fluorescent, and for the first time biotinylated entities. Moreover, we demonstrated a site-specific and copper-free conjugation strategy of a nanobody by the incorporation of biotin. The design of polyspecific PylRS variants offers an attractive alternative to existing screening approaches and provides insights into the complex PylRS-substrate interactions.
- 124Wang, L.; Xie, J.; Schultz, P. G. Expanding the Genetic Code. Annu. Rev. Biophys. Biomol. Struct. 2006, 35, 225– 249, DOI: 10.1146/annurev.biophys.35.101105.121507124Expanding the genetic codeWang, Lei; Xie, Jianming; Schultz, Peter G.Annual Review of Biophysics and Biomolecular Structure (2006), 35 (), 225-249CODEN: ABBSE4; ISSN:1056-8700. (Annual Reviews Inc.)A review. Recently, a general method was developed that makes it possible to genetically encode unnatural amino acids with diverse phys., chem., or biol. properties in Escherichia coli, yeast, and mammalian cells. More than 30 unnatural amino acids have been incorporated into proteins with high fidelity and efficiency by means of a unique codon and corresponding tRNA/aminoacyl-tRNA synthetase pair. These include fluorescent, glycosylated, metal-ion-binding, and redox-active amino acids, as well as amino acids with unique chem. and photochem. reactivity. This methodol. provides a powerful tool both for exploring protein structure and function in vitro and in vivo and for generating proteins with new or enhanced properties.
- 125Santoro, S.; Wang, L.; Herberich, B.; King, D. S.; Schultz, P. G. An Efficient System for the Evolution of Aminoacyl-tRNA Synthetase Specificity. Nat. Biotechnol. 2002, 20, 1044– 1048, DOI: 10.1038/nbt742125An efficient system for the evolution of aminoacyl-tRNA synthetase specificitySantoro, Stephen W.; Wang, Lei; Herberich, Brad; King, David S.; Schultz, Peter G.Nature Biotechnology (2002), 20 (10), 1044-1048CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)A variety of strategies to incorporate unnatural amino acids into proteins have been pursued, but all have limitations with respect to tech. accessibility, scalability, applicability to in vivo studies, or site specificity of amino acid incorporation. The ability to selectively introduce unnatural functional groups into specific sites within proteins, in vivo, provides a potentially powerful approach to the study of protein function and to large-scale prodn. of novel proteins. Here the authors describe a combined genetic selection and screen that allows the rapid evolution of aminoacyl-tRNA synthetase substrate specificity. The authors' strategy involves the use of an "orthogonal" aminoacyl-tRNA synthetase and tRNA pair that cannot interact with any of the endogenous synthetase-tRNA pairs in Escherichia coli. A chloramphenicol-resistance (Cmr) reporter is used to select highly active synthetase variants, and an amplifiable fluorescence reporter is used together with fluorescence-activated cell sorting (FACS) to screen for variants with the desired change in amino acid specificity. Both reporters are contained within a single genetic construct, eliminating the need for plasmid shuttling and allowing the evolution to be completed in a matter of days. Following evolution, the amplifiable fluorescence reporter allows visual and fluorimetric evaluation of synthetase activity and selectivity. Using this system to explore the evolvability of an amino acid binding pocket of a tyrosyl-tRNA synthetase, the authors identified three new variants that allow the selective incorporation of amino-, isopropyl-, and allyl-contg. tyrosine analogs into a desired protein. The new enzymes can be used to produce milligram-per-liter quantities of unnatural amino acid-contg. protein in E. coli.
- 126Dumas, A.; Lercher, L.; Spicer, C. D.; Davis, B. G. Designing Logical Codon Reassignment - Expanding the Chemistry in Biology. Chem. Sci. 2015, 6 (1), 50– 69, DOI: 10.1039/C4SC01534G126Designing logical codon reassignment - Expanding the chemistry in biologyDumas, Anaelle; Lercher, Lukas; Spicer, Christopher D.; Davis, Benjamin G.Chemical Science (2015), 6 (1), 50-69CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)A review. Over the last decade, the ability to genetically encode unnatural amino acids (UAAs) has evolved rapidly. The programmed incorporation of UAAs into recombinant proteins relies on the reassignment or suppression of canonical codons with an amino-acyl tRNA synthetase/tRNA (aaRS/tRNA) pair, selective for the UAA of choice. In order to achieve selective incorporation, the aaRS should be selective for the designed tRNA and UAA over the endogenous amino acids and tRNAs. Enhanced selectivity has been achieved by transferring an aaRS/tRNA pair from another kingdom to the organism of interest, and subsequent aaRS evolution to acquire enhanced selectivity for the desired UAA. Today, over 150 non-canonical amino acids have been incorporated using such methods. This enables the introduction of a large variety of structures into proteins, in organisms ranging from prokaryote, yeast and mammalian cells lines to whole animals, enabling the study of protein function at a level that could not previously be achieved. While most research to date has focused on the suppression of 'non-sense' codons, recent developments are beginning to open up the possibility of quadruplet codon decoding and the more selective reassignment of sense codons, offering a potentially powerful tool for incorporating multiple amino acids. Here, we aim to provide a focused review of methods for UAA incorporation with an emphasis in particular on the different tRNA synthetase/tRNA pairs exploited or developed, focusing upon the different UAA structures that have been incorporated and the logic behind the design and future creation of such systems. Our hope is that this will help rationalize the design of systems for incorporation of unexplored unnatural amino acids, as well as novel applications for those already known.
- 127Wan, W.; Tharp, J. M.; Liu, W. R. Pyrrolysyl-tRNA Synthetase: An Ordinary Enzyme but an Outstanding Genetic Code Expansion Tool. Biochim. Biophys. Acta Proteins Proteomics 2014, 1844 (6), 1059– 1070, DOI: 10.1016/j.bbapap.2014.03.002127Pyrrolysyl-tRNA synthetase: An ordinary enzyme but an outstanding genetic code expansion toolWan, Wei; Tharp, Jeffery M.; Liu, Wenshe R.Biochimica et Biophysica Acta, Proteins and Proteomics (2014), 1844 (6), 1059-1070CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B. V.)A review. The genetic incorporation of the 22nd proteinogenic amino acid, pyrrolysine (Pyl) at the amber codon is achieved by the action of pyrrolysyl-tRNA synthetase (PylRS) together with its cognate tRNAPyl. Unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side-chain promiscuity, low selectivity toward its substrate α-amine, and low selectivity toward the anticodon of tRNAPyl. These unique but ordinary features of PylRS as an aminoacyl-tRNA synthetase allow the Pyl incorporation machinery to be easily engineered for the genetic incorporation of >100 noncanonical amino acids (NCAAs) or α-hydroxy acids into proteins at the amber codon and the reassignment of other codons such as ochre UAA, opal UGA, and 4-base AGGA codons to code NCAAs.
- 128Ryu, Y.; Schultz, P. G. Efficient Incorporation of Unnatural Amino Acids into Proteins in Escherichia coli. Nat. Methods 2006, 3 (4), 263– 265, DOI: 10.1038/nmeth864128Efficient incorporation of unnatural amino acids into proteins in Escherichia coliRyu, Youngha; Schultz, Peter G.Nature Methods (2006), 3 (4), 263-265CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)We have developed a single-plasmid system for the efficient bacterial expression of mutant proteins contg. unnatural amino acids at specific sites designated by amber nonsense codons. In this system, multiple copies of a gene encoding an amber suppressor tRNA derived from a Methanocaldococcus jannaschii tyrosyl-tRNA (MjtRNATyrCUA) are expressed under control of the proK promoter and terminator, and a gene encoding the desired mutant M. jannaschii tyrosyl-tRNA synthetase (MjTyrRS) is expressed under control of a mutant glnS (glnS') promoter.
- 129Srinivasan, G.; James, C. M.; Krzycki, J. A. Pyrrolysine Encoded by UAG in Archaea: Charging of a UAG-Decoding Specialized tRNA. Science 2002, 296 (5572), 1459– 1462, DOI: 10.1126/science.1069588129Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNASrinivasan, Gayathri; James, Carey M.; Krzycki, Joseph A.Science (Washington, DC, United States) (2002), 296 (5572), 1459-1462CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Pyrrolysine is a lysine deriv. encoded by the UAG codon in methylamine methyltransferase genes of Methanosarcina barkeri. Near a methyltransferase gene cluster is the pylT gene, which encodes an unusual tRNA with a CUA anticodon. The adjacent pylS gene encodes a class II aminoacyl-tRNA synthetase that charges the pylT-derived tRNA with lysine but is not closely related to known lysyl-tRNA synthetases. Homologs of pylS and pylT are found in a Gram-pos. bacterium. Charging a tRNACUA with lysine is a likely first step in translating UAG amber codons as pyrrolysine in certain methanogens. Our results indicate that pyrrolysine is the 22nd genetically encoded natural amino acid.
- 130Hao, B.; Gong, W.; Ferguson, T. K.; James, C. M.; Krzycki, J. A.; Chan, M. K. A New UAG-Encoded Residue in the Structure of a Methanogen Methyltransferase. Science 2002, 296 (5572), 1462– 1466, DOI: 10.1126/science.1069556130A new UAG-encoded residue in the structure of a methanogen methyltransferaseHao, Bing; Gong, Weimin; Ferguson, Tsuneo K.; James, Carey M.; Krzycki, Joseph A.; Chan, Michael K.Science (Washington, DC, United States) (2002), 296 (5572), 1462-1466CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Genes encoding methanogenic methylamine methyltransferases all contain an in-frame amber (UAG) codon that is read through during translation. We have identified the UAG-encoded residue in a 1.55 angstrom resoln. structure of the Methanosarcina barkeri monomethylamine methyltransferase (MtmB). This structure reveals a homohexamer comprised of individual subunits with a TIM barrel fold. The electron d. for the UAG-encoded residue is distinct from any of the 21 natural amino acids. Instead it appears consistent with a lysine in amide-linkage to (4R,5R)-4-substituted-pyrroline-5-carboxylate. We suggest that this amino acid be named L-pyrrolysine.
- 131Chin, J. W. Expanding and Reprogramming the Genetic Code of Cells and Animals. Annu. Rev. Biochem. 2014, 83 (1), 379– 408, DOI: 10.1146/annurev-biochem-060713-035737131Expanding and reprogramming the genetic code of cells and animalsChin, Jason W.Annual Review of Biochemistry (2014), 83 (), 379-408CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)Genetic code expansion and reprogramming enable the site-specific incorporation of diverse designer amino acids into proteins produced in cells and animals. Recent advances are enhancing the efficiency of unnatural amino acid incorporation by creating and evolving orthogonal ribosomes and manipulating the genome. Increasing the no. of distinct amino acids that can be site-specifically encoded has been facilitated by the evolution of orthogonal quadruplet decoding ribosomes and the discovery of mutually orthogonal synthetase/tRNA pairs. Rapid progress in moving genetic code expansion from bacteria to eukaryotic cells and animals (C. elegans and D. melanogaster) and the incorporation of useful unnatural amino acids has been aided by the development and application of the pyrrolysyl-tRNA (tRNA) synthetase/tRNA pair for unnatural amino acid incorporation. Combining chemoselective reactions with encoded amino acids has facilitated the installation of posttranslational modifications, as well as rapid derivatization with diverse fluorophores for imaging.
- 132Ranaghan, K. E.; Hung, J. E.; Bartlett, G. J.; Mooibroek, T. J.; Harvey, J. N.; Woolfson, D. N.; van der Donk, W. A.; Mulholland, A. J. A Catalytic Role for Methionine Revealed by a Combination of Computation and Experiments on Phosphite Dehydrogenase. Chem. Sci. 2014, 5 (6), 2191– 2199, DOI: 10.1039/C3SC53009D132A catalytic role for methionine revealed by a combination of computation and experiments on phosphite dehydrogenaseRanaghan, Kara E.; Hung, John E.; Bartlett, Gail J.; Mooibroek, Tiddo J.; Harvey, Jeremy N.; Woolfson, Derek N.; van der Donk, Wilfred A.; Mulholland, Adrian J.Chemical Science (2014), 5 (6), 2191-2199CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Combined quantum mechanics/mol. mechanics (QM/MM) simulations of the reaction catalyzed by phosphite dehydrogenase (PTDH) identify Met53 as important for catalysis. This catalytic role is verified by expts. (including replacement by norleucine and selenomethionine), which show that mutation of this residue significantly affects kcat, without changing KM for phosphite. QM/MM and ab initio QM calcns. show that the catalytic effect is electrostatic in nature. The side chain of Met53 specifically stabilizes the transition state for the hydride transfer step of the reaction catalyzed by PTDH, forming a 'face-on' interaction with His292. To our knowledge, a defined catalytic role for methionine in an enzyme (as opposed to a steric or binding effect, or interaction with a metal ion) has not previously been identified. Analyses of the Protein Data Bank and Cambridge Structural Database indicate that this type of interaction may be relatively widespread, with implications for enzyme-catalyzed reaction mechanisms and protein structure.
- 133Ekanayake, K. B.; Mahawaththa, M. C.; Qianzhu, H.; Abdelkader, E. H.; George, J.; Ullrich, S.; Murphy, R. B.; Fry, S. E.; Johansen-Leete, J.; Payne, R. J. Probing Ligand Binding Sites on Large Proteins by Nuclear Magnetic Resonance Spectroscopy of Genetically Encoded Non-Canonical Amino Acids. J. Med. Chem. 2023, 66 (7), 5289– 5304, DOI: 10.1021/acs.jmedchem.3c00222There is no corresponding record for this reference.
- 134Ellman, J. A.; Volkman, B. F.; Mendel, D.; Schulz, P. G.; Wemmer, D. E. Site-Specific Isotopic Labeling of Proteins for NMR Studies. J. Am. Chem. Soc. 1992, 114 (20), 7959– 7961, DOI: 10.1021/ja00046a080134Site-specific isotopic labeling of proteins for NMR studiesEllman, Jonathan A.; Volkman, Brian F.; Mendel, David; Schulz, Peter G.; Wemmer, David E.Journal of the American Chemical Society (1992), 114 (20), 7959-61CODEN: JACSAT; ISSN:0002-7863.A single 13C-labeled alanine was site-specifically incorporated at position 82 of T4 lysozyme by in vitro suppression of an Ala 82 → TAG nonsense mutation with a chem. aminoacylated suppressor tRNA. The 13C-filtered proton NMR spectra obtained for this protein in both the native and denatured states clearly shows the Cα proton and Me group. The general methodol. described here should make possible a variety of detailed NMR studies of larger proteins, including the detn. of chem. shifts, pKA values, and relaxation parameters for individual amino acids in both the native and denatured states.
- 135Schmidt, M. J.; Borbas, J.; Drescher, M.; Summerer, D. A Genetically Encoded Spin Label for Electron Paramagnetic Resonance Distance Measurements. J. Am. Chem. Soc. 2014, 136 (4), 1238– 1241, DOI: 10.1021/ja411535q135A Genetically Encoded Spin Label for Electron Paramagnetic Resonance Distance MeasurementsSchmidt, Moritz J.; Borbas, Julia; Drescher, Malte; Summerer, DanielJournal of the American Chemical Society (2014), 136 (4), 1238-1241CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The authors report the genetic encoding of a noncanonical, spin-labeled amino acid in Escherichia coli. This enables the intracellular biosynthesis of spin-labeled proteins and obviates the need for any chem. labeling step usually required for protein EPR studies. The amino acid can be introduced at multiple, user-defined sites of a protein and is stable in E. coli even for prolonged expression times. It can report intramol. distance distributions in proteins by double-electron electron resonance measurements. Moreover, the signal of spin-labeled protein can be selectively detected in cells. This provides elegant new perspectives for in-cell EPR studies of endogenous proteins.
- 136Fafarman, A. T.; Boxer, S. G. Nitrile Bonds as Infrared Probes of Electrostatics in Ribonuclease S. J. Phys. Chem. B 2010, 114 (42), 13536– 13544, DOI: 10.1021/jp106406p136Nitrile Bonds as Infrared Probes of Electrostatics in Ribonuclease SFafarman, Aaron T.; Boxer, Steven G.Journal of Physical Chemistry B (2010), 114 (42), 13536-13544CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)Three different nitrile-contg. amino acids, p-cyanophenylalanine, m-cyanophenylalanine, and S-cyanohomocysteine, have been introduced near the active site of the semisynthetic enzyme RNase S to serve as probes of electrostatic fields. Vibrational Stark spectra, measured directly on the probe-modified proteins, confirm the predominance of the linear Stark tuning rate in describing the sensitivity of the nitrile stretch to external elec. fields, a necessary property for interpreting obsd. frequency shifts as a quant. measure of local elec. fields that can be compared with simulations. The x-ray structures of these nitrile-modified RNase variants and enzymic assays demonstrate minimal perturbation to the structure and function, resp., by the probes and provide a context for understanding the influence of the environment on the nitrile stretching frequency. The authors examine the ability of simulation techniques to recapitulate the spectroscopic properties of these nitriles as a means to directly test a computational electrostatic model for proteins, specifically that in the ubiquitous Amber-99 force field. Although qual. agreement between theory and expt. is obsd. for the largest shifts, substantial discrepancies are obsd. in some cases, highlighting the ongoing need for exptl. metrics to inform the development of theor. models of electrostatic fields in proteins.
- 137Xie, J.; Wang, L.; Wu, N.; Brock, A.; Spraggon, G.; Schultz, P. G. The Site-Specific Incorporation of p-Iodo-L-Phenylalanine into Proteins for Structure Determination. Nat. Biotechnol. 2004, 22 (10), 1297– 1301, DOI: 10.1038/nbt1013There is no corresponding record for this reference.
- 138Summerer, D.; Chen, S.; Wu, N.; Deiters, A.; Chin, J. W.; Schultz, P. G. A Genetically Encoded Fluorescent Amino Acid. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (26), 9785– 9789, DOI: 10.1073/pnas.0603965103138A genetically encoded fluorescent amino acidSummerer, Daniel; Chen, Shuo; Wu, Ning; Deiters, Alexander; Chin, Jason W.; Schultz, Peter G.Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (26), 9785-9789CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The ability to introduce fluorophores selectively into proteins provides a powerful tool to study protein structure, dynamics, localization, and biomol. interactions both in vitro and in vivo. Here, we report a strategy for the selective and efficient biosynthetic incorporation of a low-mol.-wt. fluorophore into proteins at defined sites. The fluorescent amino acid 2-amino-3-(5-(dimethylamino)naphthalene-1-sulfonamide)propanoic acid (dansylalanine) was genetically encoded in Saccharomyces cerevisiae by using an amber nonsense codon and corresponding orthogonal tRNA/aminoacyl-tRNA synthetase pair. This environmentally sensitive fluorophore was selectively introduced into human superoxide dismutase and used to monitor unfolding of the protein in the presence of guanidinium chloride. The strategy described here should be applicable to a no. of different fluorophores in both prokaryotic and eukaryotic organisms, and it should facilitate both biochem. and cellular studies of protein structure and function.
- 139Lang, K.; Davis, L.; Wallace, S.; Mahesh, M.; Cox, D. J.; Blackman, M. L.; Fox, J. M.; Chin, J. W. Genetic Encoding of Bicyclononynes and trans-Cyclooctenes for Site-Specific Protein Labeling in Vitro and in Live Mammalian Cells via Rapid Fluorogenic Diels-Alder Reactions. J. Am. Chem. Soc. 2012, 134 (25), 10317– 10320, DOI: 10.1021/ja302832g139Genetic Encoding of Bicyclononynes and trans-Cyclooctenes for Site-Specific Protein Labeling in Vitro and in Live Mammalian Cells via Rapid Fluorogenic Diels-Alder ReactionsLang, Kathrin; Davis, Lloyd; Wallace, Stephen; Mahesh, Mohan; Cox, Daniel J.; Blackman, Melissa L.; Fox, Joseph M.; Chin, Jason W.Journal of the American Chemical Society (2012), 134 (25), 10317-10320CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Rapid, site-specific labeling of proteins with diverse probes remains an outstanding challenge for chem. biologists. Enzyme-mediated labeling approaches may be rapid but use protein or peptide fusions that introduce perturbations into the protein under study and may limit the sites that can be labeled, while many "bioorthogonal" reactions for which a component can be genetically encoded are too slow to effect quant. site-specific labeling of proteins on a time scale that is useful for studying many biol. processes. We report a fluorogenic reaction between bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) and tetrazines that is 3-7 orders of magnitude faster than many bioorthogonal reactions. Unlike the reactions of strained alkenes, including trans-cyclooctenes and norbornenes, with tetrazines, the BCN-tetrazine reaction gives a single product of defined stereochem. We have discovered aminoacyl-tRNA synthetase/tRNA pairs for the efficient site-specific incorporation of a BCN-contg. amino acid, 1, and a trans-cyclooctene-contg. amino acid 2 (which also reacts extremely rapidly with tetrazines) into proteins expressed in Escherichia coli and mammalian cells. We demonstrate the rapid fluorogenic labeling of proteins contg. 1 and 2 in vitro, in E. coli, and in live mammalian cells. These approaches may be extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on labeling and imaging studies.
- 140Plass, T.; Milles, S.; Koehler, C.; Schultz, C.; Lemke, E. A. Genetically Encoded Copper-Free Click Chemistry. Angew. Chem. Int. Ed. 2011, 50 (17), 3878– 3881, DOI: 10.1002/anie.201008178140Genetically Encoded Copper-Free Click ChemistryPlass, Tilman; Milles, Sigrid; Koehler, Christine; Schultz, Carsten; Lemke, Edward A.Angewandte Chemie, International Edition (2011), 50 (17), 3878-3881, S3878/1-S3878/13CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)One of the most potent functional groups for in vivo chem. has been genetically encoded into E. coli, and its basic utility for in vivo labeling as well as high-resoln. single-mol. measurements has been demonstrated. SPAAC (strain-promoted azide-alkyne cycloaddn.) chem. is now available to site-specifically and noninvasively modify proteins in living cells. As the tRNA'/pylRSA showed no obvious dependence on linker length (1 vs. 2), it is conceivable that slightly altered derivs., such as mono- and difluorinated cyclooctynes, and possibly bicyclonones, could be directly used in this system. Other enhanced cyclooctynes, such as dibenzocycloctynes, could pose substantial challenges to the synthetase and/or the host translational machinery owing to their larger size. As pylRS from M. mazei is orthogonal in a variety of eukaryotic organisms, we are now evaluating the transfer of this system to mammalian cells, where the technique would not only greatly expand our abilities to track proteins in living specimen but also to introduce other type of functional groups, such as cross-linkers or spin-labels for NMR spectroscopy and magnetic resonance imaging (MRI) in living specimens.
- 141Brustad, E. M.; Lemke, E. A.; Schultz, P. G.; Deniz, A. A. A General and Efficient Method for the Site-Specific Dual-Labeling of Proteins for Single Molecule Fluorescence Resonance Energy Transfer. J. Am. Chem. Soc. 2008, 130 (52), 17664– 17665, DOI: 10.1021/ja807430h141A General and Efficient Method for the Site-Specific Dual-Labeling of Proteins for Single Molecule Fluorescence Resonance Energy TransferBrustad, Eric M.; Lemke, Edward A.; Schultz, Peter G.; Deniz, Ashok A.Journal of the American Chemical Society (2008), 130 (52), 17664-17665CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A general strategy for the site-specific dual-labeling of proteins for single-mol. fluorescence resonance energy transfer is presented. A genetically encoded unnatural ketone amino acid was labeled with a hydroxylamine-contg. fluorophore with high yield (>95%) and specificity. This methodol. was used to construct dual-labeled T4 lysozyme variants, allowing the study of T4 lysozyme folding at single-mol. resoln. The presented strategy is anticipated to expand the scope of single-mol. protein structure and function studies.
- 142Loving, G.; Imperiali, B. A Versatile Amino Acid Analogue of the Solvatochromic Fluorophore 4-N,N-Dimethylamino-1,8-naphthalimide: A Powerful Tool for the Study of Dynamic Protein Interactions. J. Am. Chem. Soc. 2008, 130 (41), 13630– 13638, DOI: 10.1021/ja804754y142A Versatile Amino Acid Analogue of the Solvatochromic Fluorophore 4-N,N-Dimethylamino-1,8-naphthalimide: A Powerful Tool for the Study of Dynamic Protein InteractionsLoving, Galen; Imperiali, BarbaraJournal of the American Chemical Society (2008), 130 (41), 13630-13638CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The authors have developed a new unnatural amino acid based on the solvatochromic fluorophore 4-N,N-dimethylamino-1,8-naphthalimide (4-DMN) for application in the study of protein-protein interactions. The fluorescence quantum yield of this chromophore is highly sensitive to changes in the local solvent environment, demonstrating "switch-like" emission properties characteristic of the dimethylaminophthalimide family of fluorophores. In particular, this new species possesses a no. of significant advantages over related fluorophores, including greater chem. stability under a wide range of conditions, a longer wavelength of excitation (408 nm), and improved synthetic accessibility. This amino acid has been prepd. as an Fmoc-protected building block and may readily be incorporated into peptides via std. solid-phase peptide synthesis. A series of comparative studies are presented to demonstrate the advantageous properties of the 4-DMN amino acid relative to those of the previously reported 4-N,N-dimethylaminophthalimidoalanine and 6-N,N-dimethylamino-2,3-naphthalimidoalanine amino acids. Other com. available solvatochromic fluorophores are also include in these studies. The potential of this new probe as a tool for the study of protein-protein interactions is demonstrated by introducing it into a peptide that is recognized by calcium-activated calmodulin. The binding interaction between these two components yields an increase in fluorescence emission greater than 900-fold.
- 143Mendes, K. R.; Martinez, J. A.; Kantrowitz, E. R. Asymmetric Allosteric Signaling in Aspartate Transcarbamoylase. ACS Chem. Biol. 2010, 5 (5), 499– 506, DOI: 10.1021/cb9003207143Asymmetric Allosteric Signaling in Aspartate TranscarbamoylaseMendes, Kimberly R.; Martinez, Jessica A.; Kantrowitz, Evan R.ACS Chemical Biology (2010), 5 (5), 499-506CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Here we use the fluorescence from a genetically encoded unnatural amino acid, L-(7-hydroxycoumarin-4-yl)ethylglycine (HCE-Gly), replacing an amino acid in the regulatory site of Escherichia coli aspartate transcarbamoylase (ATCase) to decipher the mol. details of regulation of this allosteric enzyme. The fluorescence of HCE-Gly is exquisitely sensitive to the binding of all four nucleotide effectors. Although ATP and CTP are primarily responsible for influencing enzyme activity, the results of our fluorescent binding studies indicate that UTP and GTP bind with similar affinities, suggesting a dissocn. between nucleotide binding and control of enzyme activity. Furthermore, while CTP is the strongest regulator of enzyme activity, it binds selectively to only a fraction of regulatory sites, allowing UTP to effectively fill the residual ones. Our results suggest that CTP and UTP are not competing for the same binding sites, but instead reveal an asymmetry between the two allosteric sites on the regulatory subunit of the enzyme. Correlation of binding and activity measurements explain how ATCase uses asym. allosteric sites to achieve regulatory sensitivity over a broad range of heterotropic effector concns.
- 144Dean, S. F.; Whalen, K. L.; Spies, M. A. Biosynthesis of a Novel Glutamate Racemase Containing a Site-Specific 7-Hydroxycoumarin Amino Acid: Enzyme-Ligand Promiscuity Revealed at the Atomistic Level. ACS Cent. Sci. 2015, 1 (7), 364– 373, DOI: 10.1021/acscentsci.5b00211144Biosynthesis of a Novel Glutamate Racemase Containing a Site-Specific 7-Hydroxycoumarin Amino Acid: Enzyme-Ligand Promiscuity Revealed at the Atomistic LevelDean, Sondra F.; Whalen, Katie L.; Spies, M. AshleyACS Central Science (2015), 1 (7), 364-373CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)Glutamate racemase (GR) catalyzes the cofactor independent stereoinversion of L- to D-glutamate for biosynthesis of bacterial cell walls. Because of its essential nature, this enzyme is under intense scrutiny as a drug target for the design of novel antimicrobial agents. However, the flexibility of the enzyme has made inhibitor design challenging. Previous steered mol. dynamics (MD), docking, and exptl. studies have suggested that the enzyme forms highly varied complexes with different competitive inhibitor scaffolds. The current study employs a mutant orthogonal tRNA/aminoacyl-tRNA synthetase pair to genetically encode a non-natural fluorescent amino acid, L-(7-hydroxycoumarin-4-yl) ethylglycine (7HC), into a region (Tyr53) remote from the active site (previously identified by MD studies as undergoing ligand-assocd. changes) to generate an active mutant enzyme (GRY53/7HC). The GRY53/7HC enzyme is an active racemase, which permitted us to examine the nature of these idiosyncratic ligand-assocd. phenomena. One type of competitive inhibitor resulted in a dose-dependent quenching of the fluorescence of GRY53/7HC, while another type of competitive inhibitor resulted in a dose-dependent increase in fluorescence of GRY53/7HC. In order to investigate the environmental changes of the 7HC ring system that are distinctly assocd. with each of the GRY53/7HC-ligand complexes, and thus the source of the disparate quenching phenomena, a parallel computational study is described, which includes essential dynamics, ensemble docking and MD simulations of the relevant GRY53/7HC-ligand complexes. The changes in the solvent exposure of the 7HC ring system due to ligand-assocd. GR changes are consistent with the exptl. obsd. quenching phenomena. This study describes an approach for rationally predicting global protein allostery resulting from enzyme ligation to distinctive inhibitor scaffolds. The implications for fragment-based drug discovery and high throughput screening are discussed.
- 145Wang, J.; Xie, J.; Schultz, P. G. A Genetically Encoded Fluorescent Amino Acid. J. Am. Chem. Soc. 2006, 128 (27), 8738– 8739, DOI: 10.1021/ja062666k145A Genetically Encoded Fluorescent Amino AcidWang, Jiangyun; Xie, Jianming; Schultz, Peter G.Journal of the American Chemical Society (2006), 128 (27), 8738-8739CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The fluorescent amino acid L-(7-hydroxycoumarin-4-yl) ethylglycine 1 has been genetically encoded in E. coli in response to the amber TAG codon. Because of its high fluorescence quantum yield, relatively large Stoke's shift, and sensitivity to both pH and polarity, this amino acid should provide a useful probe of protein localization and trafficking, protein conformation changes, and protein-protein interactions.
- 146Li, M.; Peng, T. Genetic Encoding of a Fluorescent Noncanonical Amino Acid as a FRET Donor for the Analysis of Deubiquitinase Activities. In Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols; Tsai, Y.-H., Elsässer, S. J., Eds.; Springer: US, 2023; pp 55- 67. DOI: 10.1007/978-1-0716-3251-2_4There is no corresponding record for this reference.
- 147Miyake-Stoner, S. J.; Miller, A. M.; Hammill, J. T.; Peeler, J. C.; Hess, K. R.; Mehl, R. A.; Brewer, S. H. Probing Protein Folding Using Site-Specifically Encoded Unnatural Amino Acids as FRET Donors with Tryptophan. Biochemistry 2009, 48 (25), 5953– 5962, DOI: 10.1021/bi900426d147Probing Protein Folding Using Site-Specifically Encoded Unnatural Amino Acids as FRET Donors with TryptophanMiyake-Stoner, Shigeki J.; Miller, Andrew M.; Hammill, Jared T.; Peeler, Jennifer C.; Hess, Kenneth R.; Mehl, Ryan A.; Brewer, Scott H.Biochemistry (2009), 48 (25), 5953-5962CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The exptl. study of protein folding is enhanced by the use of nonintrusive probes that are sensitive to local conformational changes in the protein structure. Here, we report the selection of an aminoacyl-tRNA synthetase/tRNA pair for the cotranslational, site-specific incorporation of two unnatural amino acids that can function as fluorescence resonance energy transfer (FRET) donors with Trp to probe the disruption of the hydrophobic core upon protein unfolding. L-4-Cyanophenylalanine (pCNPhe) and 4-ethynylphenylalanine (pENPhe) were incorporated into the hydrophobic core of the 171-residue protein, T4 lysozyme. The FRET donor ability of pCNPhe and pENPhe is evident by the overlap of the emission spectra of pCNPhe and pENPhe with the absorbance spectrum of Trp. The incorporation of both unnatural amino acids in place of a phenylalanine in the hydrophobic core of T4 lysozyme was well tolerated by the protein, due in part to the small size of the cyano and ethynyl groups. The hydrophobic nature of the ethynyl group of pENPhe suggests that this unnatural amino acid is a more conservative substitution into the hydrophobic core of the protein compared to pCNPhe. The urea-induced disruption of the hydrophobic core of the protein was probed by the change in FRET efficiency between either pCNPhe or pENPhe and the Trp residues in T4 lysozyme. The methodol. for the study of protein conformational changes using FRET presented here is of general applicability to the study of protein structural changes, since the incorporation of the unnatural amino acids is not inherently limited by the size of the protein.
- 148Bergfors, T. M. Protein crystallization. Internat’l University Line 2009. ISBN: 978–0-9720774–4-6.There is no corresponding record for this reference.
- 149Sakamoto, K.; Murayama, K.; Oki, K.; Iraha, F.; Kato-Murayama, M.; Takahashi, M.; Ohtake, K.; Kobayashi, T.; Kuramitsu, S.; Shirouzu, M. Genetic Encoding of 3-Iodo-l-Tyrosine in Escherichia coli for Single-Wavelength Anomalous Dispersion Phasing in Protein Crystallography. Structure 2009, 17 (3), 335– 344, DOI: 10.1016/j.str.2009.01.008There is no corresponding record for this reference.
- 150Lee, H. S.; Spraggon, G.; Schultz, P. G.; Wang, F. Genetic Incorporation of a Metal-Ion Chelating Amino Acid into Proteins as a Biophysical Probe. J. Am. Chem. Soc. 2009, 131 (7), 2481– 2483, DOI: 10.1021/ja808340b150Genetic Incorporation of a Metal-Ion Chelating Amino Acid into Proteins as a Biophysical ProbeLee, Hyun Soo; Spraggon, Glen; Schultz, Peter G.; Wang, FengJournal of the American Chemical Society (2009), 131 (7), 2481-2483CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A metal-ion chelating amino acid, (8-hydroxyquinolin-3-yl)alanine, was genetically encoded in Escherichia coli by an amber nonsense codon and corresponding orthogonal tRNA/aminoacyl-tRNA synthetase pair. The amino acid was incorporated into TM0665 protein, and the mutant protein was cocrystd. with Zn2+ to det. the structure by SAD phasing. The structure showed a high occupancy of the heavy metal bound to the HQ-Ala residue, and the heavy metal provided excellent phasing power to det. the structure. This method also facilitates the de novo design of metalloproteins with novel structures and functions, including fluorescent sensors.
- 151Nogly, P.; Weinert, T.; James, D.; Carbajo, S.; Ozerov, D.; Furrer, A.; Gashi, D.; Borin, V.; Skopintsev, P.; Jaeger, K. Retinal Isomerization in Bacteriorhodopsin Captured by a Femtosecond X-Ray Laser. Science 2018, 361 (6398), eaat0094 DOI: 10.1126/science.aat0094There is no corresponding record for this reference.
- 152Tenboer, J.; Basu, S.; Zatsepin, N.; Pande, K.; Milathianaki, D.; Frank, M.; Hunter, M.; Boutet, S.; Williams, G. J.; Koglin, J. E. Time-Resolved Serial Crystallography Captures High-Resolution Intermediates of Photoactive Yellow Protein. Science 2014, 346 (6214), 1242– 1246, DOI: 10.1126/science.1259357152Time-resolved serial crystallography captures high-resolution intermediates of photoactive yellow proteinTenboer, Jason; Basu, Shibom; Zatsepin, Nadia; Pande, Kanupriya; Milathianaki, Despina; Frank, Matthias; Hunter, Mark; Boutet, Sebastien; Williams, Garth J.; Koglin, Jason E.; Oberthuer, Dominik; Heymann, Michael; Kupitz, Christopher; Conrad, Chelsie; Coe, Jesse; Roy-Chowdhury, Shatabdi; Weierstall, Uwe; James, Daniel; Wang, Dingjie; Grant, Thomas; Barty, Anton; Yefanov, Oleksandr; Scales, Jennifer; Gati, Cornelius; Seuring, Carolin; Srajer, Vukica; Henning, Robert; Schwander, Peter; Fromme, Raimund; Ourmazd, Abbas; Moffat, Keith; Van Thor, Jasper J.; Spence, John C. H.; Fromme, Petra; Chapman, Henry N.; Schmidt, MariusScience (Washington, DC, United States) (2014), 346 (6214), 1242-1246CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Serial femtosecond crystallog. using ultrashort pulses from x-ray free electron lasers (XFELs) enables studies of the light-triggered dynamics of biomols. The authors used microcrystals of photoactive yellow protein (a bacterial blue light photoreceptor) as a model system and obtained high-resoln., time-resolved difference electron d. maps of excellent quality with strong features; these allowed the detn. of structures of reaction intermediates to a resoln. of 1.6 Å. The authors' results open the way to the study of reversible and nonreversible biol. reactions on time scales as short as femtoseconds under conditions that maximize the extent of reaction initiation throughout the crystal.
- 153Suga, M.; Akita, F.; Sugahara, M.; Kubo, M.; Nakajima, Y.; Nakane, T.; Yamashita, K.; Umena, Y.; Nakabayashi, M.; Yamane, T. Light-Induced Structural Changes and the Site of O = O Bond Formation in PSII Caught by XFEL. Nature 2017, 543 (7643), 131– 135, DOI: 10.1038/nature21400153Light-induced structural changes and the site of O=O bond formation in PSII caught by XFELSuga, Michihiro; Akita, Fusamichi; Sugahara, Michihiro; Kubo, Minoru; Nakajima, Yoshiki; Nakane, Takanori; Yamashita, Keitaro; Umena, Yasufumi; Nakabayashi, Makoto; Yamane, Takahiro; Nakano, Takamitsu; Suzuki, Mamoru; Masuda, Tetsuya; Inoue, Shigeyuki; Kimura, Tetsunari; Nomura, Takashi; Yonekura, Shinichiro; Yu, Long-Jiang; Sakamoto, Tomohiro; Motomura, Taiki; Chen, Jing-Hua; Kato, Yuki; Noguchi, Takumi; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Hatsui, Takaki; Nango, Eriko; Tanaka, Rie; Naitow, Hisashi; Matsuura, Yoshinori; Yamashita, Ayumi; Yamamoto, Masaki; Nureki, Osamu; Yabashi, Makina; Ishikawa, Tetsuya; Iwata, So; Shen, Jian-RenNature (London, United Kingdom) (2017), 543 (7643), 131-135CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total mol. mass of 350 kDa for a monomer. It catalyzes light-driven water oxidn. at its catalytic center, the oxygen-evolving complex (OEC). The structure of PSII has been analyzed at 1.9 Å resoln. by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asym., 'distorted-chair' form. This structure was further analyzed with femtosecond X-ray free electron lasers (XFEL), providing the 'radiation damage-free' structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temp. at a resoln. of 2.35 Å using time-resolved serial femtosecond crystallog. with an XFEL provided by the SPring-8 angstrom compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water mol. located 3.5 Å from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water mol. and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent pos. peak around O5, a unique μ4-oxo-bridge located in the quasi-center of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously.
- 154Dods, R.; Båth, P.; Morozov, D.; Gagnér, V. A.; Arnlund, D.; Luk, H. L.; Kübel, J.; Maj, M.; Vallejos, A.; Wickstrand, C. Ultrafast Structural Changes within a Photosynthetic Reaction Centre. Nature 2021, 589 (7841), 310– 314, DOI: 10.1038/s41586-020-3000-7There is no corresponding record for this reference.
- 155Chapman, H. N. X-Ray Free-Electron Lasers for the Structure and Dynamics of Macromolecules. Annu. Rev. Biochem. 2019, 88 (1), 35– 58, DOI: 10.1146/annurev-biochem-013118-110744There is no corresponding record for this reference.
- 156Kern, J.; Chatterjee, R.; Young, I. D.; Fuller, F. D.; Lassalle, L.; Ibrahim, M.; Gul, S.; Fransson, T.; Brewster, A. S.; Alonso-Mori, R. Structures of the Intermediates of Kok’s Photosynthetic Water Oxidation Clock. Nature 2018, 563 (7731), 421– 425, DOI: 10.1038/s41586-018-0681-2156Structures of the intermediates of Kok's photosynthetic water oxidation clockKern, Jan; Chatterjee, Ruchira; Young, Iris D.; Fuller, Franklin D.; Lassalle, Louise; Ibrahim, Mohamed; Gul, Sheraz; Fransson, Thomas; Brewster, Aaron S.; Alonso-Mori, Roberto; Hussein, Rana; Zhang, Miao; Douthit, Lacey; de Lichtenberg, Casper; Cheah, Mun Hon; Shevela, Dmitry; Wersig, Julia; Seuffert, Ina; Sokaras, Dimosthenis; Pastor, Ernest; Weninger, Clemens; Kroll, Thomas; Sierra, Raymond G.; Aller, Pierre; Butryn, Agata; Orville, Allen M.; Liang, Mengning; Batyuk, Alexander; Koglin, Jason E.; Carbajo, Sergio; Boutet, Sebastien; Moriarty, Nigel W.; Holton, James M.; Dobbek, Holger; Adams, Paul D.; Bergmann, Uwe; Sauter, Nicholas K.; Zouni, Athina; Messinger, Johannes; Yano, Junko; Yachandra, Vittal K.Nature (London, United Kingdom) (2018), 563 (7731), 421-425CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed addnl. expts. and proposed a five-state kinetic model for photosynthetic oxygen evolution, known as Kok's S-state clock or cycle1,2. The model comprises four (meta)stable intermediates (S0, S1, S2 and S3) and one transient S4 state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn4CaO5) cluster in the oxygen-evolving complex. This reaction is coupled to the two-step redn. and protonation of the mobile plastoquinone QB at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallog. and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temp., we visualize all (meta)stable states of Kok's cycle as high-resoln. structures (2.04-2.08 Å). In addn., we report structures of two transient states at 150 and 400 μs, revealing notable structural changes including the binding of one addnl. 'water', Ox, during the S2→S3 state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the addnl. oxygen Ox in the S3 state between Ca and Mn1 supports O-O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O2 release. Thus, our results exclude peroxo-bond formation in the S3 state, and the nucleophilic attack of W3 onto W2 is unlikely.
- 157Nango, E.; Royant, A.; Kubo, M.; Nakane, T.; Wickstrand, C.; Kimura, T.; Tanaka, T.; Tono, K.; Song, C.; Tanaka, R. A Three-Dimensional Movie of Structural Changes in Bacteriorhodopsin. Science 2016, 354 (6319), 1552– 1557, DOI: 10.1126/science.aah3497157A three-dimensional movie of structural changes in bacteriorhodopsinNango, Eriko; Royant, Antoine; Kubo, Minoru; Nakane, Takanori; Wickstrand, Cecilia; Kimura, Tetsunari; Tanaka, Tomoyuki; Tono, Kensuke; Song, Changyong; Tanaka, Rie; Arima, Toshi; Yamashita, Ayumi; Kobayashi, Jun; Hosaka, Toshiaki; Mizohata, Eiichi; Nogly, Przemyslaw; Sugahara, Michihiro; Nam, Daewoong; Nomura, Takashi; Shimamura, Tatsuro; Im, Dohyun; Fujiwara, Takaaki; Yamanaka, Yasuaki; Jeon, Byeonghyun; Nishizawa, Tomohiro; Oda, Kazumasa; Fukuda, Masahiro; Andersson, Rebecka; Bath, Petra; Dods, Robert; Davidsson, Jan; Matsuoka, Shigeru; Kawatake, Satoshi; Murata, Michio; Nureki, Osamu; Owada, Shigeki; Kameshima, Takashi; Hatsui, Takaki; Joti, Yasumasa; Schertler, Gebhard; Yabashi, Makina; Bondar, Ana-Nicoleta; Standfuss, Joerg; Neutze, Richard; Iwata, SoScience (Washington, DC, United States) (2016), 354 (6319), 1552-1557CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Bacteriorhodopsin is a membrane protein that harvests the energy content from light to transport protons out of the cell against a transmembrane potential. Here, the authors used time-resolved serial femtosecond crystallog. at an x-ray free electron laser to provide 13 structural snapshots of the conformational changes that occur in the nanoseconds to milliseconds after photoactivation. These changes began at the active site, propagated toward the extracellular side of the protein, and mediated internal protonation exchanges that achieved proton transport.
- 158Liu, X.; Liu, P.; Li, H.; Xu, Z.; Jia, L.; Xia, Y.; Yu, M.; Tang, W.; Zhu, X.; Chen, C. Excited-State Intermediates in a Designer Protein Encoding a Phototrigger Caught by an X-Ray Free-Electron Laser. Nat. Chem. 2022, 14 (9), 1054– 1060, DOI: 10.1038/s41557-022-00992-3There is no corresponding record for this reference.
- 159Hosaka, T.; Katsura, K.; Ishizuka-Katsura, Y.; Hanada, K.; Ito, K.; Tomabechi, Y.; Inoue, M.; Akasaka, R.; Takemoto, C.; Shirouzu, M. Crystal Structure of an Archaeal Tyrosyl-tRNA Synthetase Bound to Photocaged L-Tyrosine and Its Potential Application to Time-Resolved X-ray Crystallography. Int. J. Mol. Sci. 2022, 23 (18), 10399, DOI: 10.3390/ijms231810399There is no corresponding record for this reference.
- 160Markley, J. L.; Putter, I.; Jardetzky, O. High-Resolution Nuclear Magnetic Resonance Spectra of Selectively Deuterated Staphylococcal Nuclease. Science 1968, 161 (3847), 1249– 1251, DOI: 10.1126/science.161.3847.1249There is no corresponding record for this reference.
- 161Deiters, A.; Geierstanger, B. H.; Schultz, P. G. Site-Specific in vivo Labeling of Proteins for NMR Studies. ChemBioChem 2005, 6 (1), 55– 58, DOI: 10.1002/cbic.200400319There is no corresponding record for this reference.
- 162Jones, D. H.; Cellitti, S. E.; Hao, X.; Zhang, Q.; Jahnz, M.; Summerer, D.; Schultz, P. G.; Uno, T.; Geierstanger, B. H. Site-Specific Labeling of Proteins with NMR-Active Unnatural Amino Acids. J. Biomol. NMR 2010, 46 (1), 89– 100, DOI: 10.1007/s10858-009-9365-4There is no corresponding record for this reference.
- 163Abdelkader, E. H.; Qianzhu, H.; Huber, T.; Otting, G. Genetic Encoding of 7-Aza-l-tryptophan: Isoelectronic Substitution of a Single CH-Group in a Protein for a Nitrogen Atom for Site-Selective Isotope Labeling. ACS Sensors 2023, 8 (11), 4402– 4406, DOI: 10.1021/acssensors.3c01904There is no corresponding record for this reference.
- 164Cellitti, S. E.; Jones, D. H.; Lagpacan, L.; Hao, X.; Zhang, Q.; Hu, H.; Brittain, S. M.; Brinker, A.; Caldwell, J.; Bursulaya, B. In vivo Incorporation of Unnatural Amino Acids to Probe Structure, Dynamics, and Ligand Binding in a Large Protein by Nuclear Magnetic Resonance Spectroscopy. J. Am. Chem. Soc. 2008, 130 (29), 9268– 9281, DOI: 10.1021/ja801602q164In vivo Incorporation of Unnatural Amino Acids to Probe Structure, Dynamics, and Ligand Binding in a Large Protein by Nuclear Magnetic Resonance SpectroscopyCellitti, Susan E.; Jones, David H.; Lagpacan, Leanna; Hao, Xueshi; Zhang, Qiong; Hu, Huiyong; Brittain, Scott M.; Brinker, Achim; Caldwell, Jeremy; Bursulaya, Badry; Spraggon, Glen; Brock, Ansgar; Ryu, Youngha; Uno, Tetsuo; Schultz, Peter G.; Geierstanger, Bernhard H.Journal of the American Chemical Society (2008), 130 (29), 9268-9281CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of NMR spectra. Incorporation is accomplished by coexpressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (OCF3Phe), 13C/15N-labeled p-methoxyphenylalanine (OMePhe), and 15N-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF3Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the resp. aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of Escherichia coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compd. Chem. shift changes in 1H-15N HSQC, 1H-13C HSQC, and 19F NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF3Phe mutants of an active site tyrosine inhibited binding; incorporating 15N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr reestablished binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with any other method.
- 165Lampe, J. N.; Floor, S. N.; Gross, J. D.; Nishida, C. R.; Jiang, Y.; Trnka, M. J.; Ortiz de Montellano, P. R. Ligand-Induced Conformational Heterogeneity of Cytochrome P450 CYP119 Identified by 2D NMR Spectroscopy with the Unnatural Amino Acid (13)C-p-Methoxyphenylalanine. J. Am. Chem. Soc. 2008, 130 (48), 16168– 16169, DOI: 10.1021/ja8071463165Ligand-Induced Conformational Heterogeneity of Cytochrome P450 CYP119 Identified by 2D NMR Spectroscopy with the Unnatural Amino Acid 13C-p-MethoxyphenylalanineLampe, Jed N.; Floor, Stephen N.; Gross, John D.; Nishida, Clinton R.; Jiang, Yongying; Trnka, Michael J.; Ortiz de Montellano, Paul R.Journal of the American Chemical Society (2008), 130 (48), 16168-16169CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Conformational dynamics are thought to play an important role in ligand binding and catalysis by cytochrome P 450 enzymes, but few techniques exist to examine them in mol. detail. Using a unique isotopic labeling strategy, we have site specifically inserted a 13C-labeled unnatural amino acid residue, 13C-p-methoxyphenylalanine (MeOF), into two different locations in the substrate binding region of the thermophilic cytochrome P 450 enzyme CYP119. Surprisingly, in both cases the resonance signal from the ligand-free protein is represented by a doublet in the 1H,13C-HSQC spectrum. Upon binding of 4-phenylimidazole, the signals from the initial resonances are reduced in favor of a single new resonance, in the case of the F162MeOF mutant, or two new resonances, in the case of the F153MeOF mutant. This represents the first direct phys. evidence for the ligand-dependent existence of multiple P 450 conformers simultaneously in soln. This general approach may be used to further illuminate the role that conformational dynamics plays in the complex enzymic phenomena exhibited by P 450 enzymes.
- 166Hull, W. E.; Sykes, B. D. Fluorotyrosine Alkaline Phosphatase. Fluorine-19 Nuclear Magnetic Resonance Relaxation Times and Molecular Motion of the Individual Fluorotyrosines. Biochemistry 1974, 13 (17), 3431– 3437, DOI: 10.1021/bi00714a002166Fluorotyrosine alkaline phosphatase. Fluorine-19 nuclear magnetic resonance relaxation times and molecular motion of the individual fluorotyrosinesHull, William E.; Sykes, Brian D.Biochemistry (1974), 13 (17), 3431-7CODEN: BICHAW; ISSN:0006-2960.Alk. phosphatase from Escherichia coli was labeled in vivo withm-fluorotyrosine and the 19F NMR spectrum of the fully activelabeled protein showed 11 resolvable resonances corresponding to the 11 known tyrosines/subunit. Nuclear spin relaxation times T1 and T2 were detd. for each 19F resonance. Consideration of the theory of dipole-dipole relaxation between unlike spins (1H and 19F) results in the following conclusions. First, the relaxation times are insensitive to internal rotation about the Cβ-arom. ring bond. Secondly, the data require that motion about the Cα-Cβ bond have a correlation time of ≥10-6 sec; hence, such motion does not contribute significantly to relaxation. All of the relaxation data are well represented by a model which assumes (1) isotropic motion of the protein as a whole with a rotational correlation time τc ≃ 70 nsec and (2) a varying degree of intermol. contribution to the 19F relaxation in tyrosine residues by protons on nearby residues. Finally, the intermol. relaxation exhibited a strong correlation with the 19F chem. shift; the contribution of intermol. relaxation was roughly proportional to the shift of a tyrosine from the position of the denatured protein resonance. Thus, 19F NMR is a very useful tool for studying the general tertiary or quaternary structure of a protein, its motional properties, and differences in the local environments of particular residues.
- 167Gamcsik, M. P.; Gerig, J. T. NMR Studies of Fluorophenylalanine-Containing Carbonic Anhydrase. FEBS Lett. 1986, 196 (1), 71– 74, DOI: 10.1016/0014-5793(86)80216-2There is no corresponding record for this reference.
- 168Jackson, J. C.; Hammill, J. T.; Mehl, R. A. Site-Specific Incorporation of a 19F-Amino Acid into Proteins as an NMR Probe for Characterizing Protein Structure and Reactivity. J. Am. Chem. Soc. 2007, 129 (5), 1160– 1166, DOI: 10.1021/ja064661t168Site-Specific Incorporation of a 19F-Amino Acid into Proteins as an NMR Probe for Characterizing Protein Structure and ReactivityJackson, Jennifer C.; Hammill, Jared T.; Mehl, Ryan A.Journal of the American Chemical Society (2007), 129 (5), 1160-1166CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)19F NMR is a powerful tool for monitoring protein conformational changes and interactions; however, the inability to site-specifically introduce fluorine labels into proteins of biol. interest severely limits its applicability. Using methods for genetically directing incorporation of unnatural amino acids, the authors have inserted trifluoromethyl-L-phenylalanine (tfm-Phe) into proteins in vivo at TAG nonsense codons with high translational efficiency and fidelity. The binding of substrates, inhibitors, and cofactors, as well as reactions in enzymes, were studied by selective introduction of tfm-Phe and subsequent monitoring of the 19F NMR chem. shifts. Subtle protein conformational changes were detected near the active site and at long distances (25 Å). 19F signal sensitivity and resoln. was also sufficient to differentiate protein environments in vivo. Since there has been interest in using 19F-labeled proteins in solid-state membrane protein studies, folding studies, and in vivo studies, this general method for genetically incorporating a 19F-label into proteins of any size in Escherichia coli should have broad application beyond that of monitoring protein conformational changes.
- 169Hammill, J. T.; Miyake-Stoner, S.; Hazen, J. L.; Jackson, J. C.; Mehl, R. A. Preparation of Site-Specifically Labeled Fluorinated Proteins for 19F-NMR Structural Characterization. Nat. Protoc. 2007, 2 (10), 2601– 2607, DOI: 10.1038/nprot.2007.379There is no corresponding record for this reference.
- 170Wacks, D. B.; Schachman, H. K. 19F Nuclear Magnetic Resonance Studies of Fluorotyrosine-Labeled Aspartate Transcarbamoylase. Properties of the Enzyme and Its Catalytic and Regulatory Subunits. J. Biol. Chem. 1985, 260 (21), 11651– 11658, DOI: 10.1016/S0021-9258(17)39080-4There is no corresponding record for this reference.
- 171Gerig, J. T. Fluorine NMR of Proteins. Prog. Nucl. Magn. Reson. Spectrosc. 1994, 26, 293– 370, DOI: 10.1016/0079-6565(94)80009-X171Fluorine NMR of proteinsGerig, J. T.Progress in Nuclear Magnetic Resonance Spectroscopy (1994), 26 (4), 293-370CODEN: PNMRAT; ISSN:0079-6565.A review with 409 refs. demonstrating the scope of current applications of fluorine NMR to studies of protein structure and function. The authors focus on work that has been done over the past 10 yr. Topics covered include: receptor proteins, enzymes, reactions of proteins with fluorinated reagents, protein-fluorinated small mol. complexes, etc.
- 172Furter, R. Expansion of the Genetic Code: Site-Directed p-Fluoro-Phenylalanine Incorporation in Escherichia coli. Protein Sci. 1998, 7 (2), 419– 426, DOI: 10.1002/pro.5560070223172Expansion of the genetic code: site-directed p-fluoro-phenylalanine incorporation in Escherichia coliFurter, RolfProtein Science (1998), 7 (2), 419-426CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)Site-directed incorporation of the amino acid analog p-fluoro-phenylalanine (p-F-Phe) was achieved in Escherichia coli. A yeast suppressor tRNAamberPhe/phenylalanyl-tRNA synthetase pair was expressed in an analog-resistant E. coli strain to direct analog incorporation at a programmed amber stop codon in the DHFR marker protein. The programmed position was translated to 64-75% as p-F-Phe and the remainder as phenylalanine and lysine. Depending on the expression conditions, the p-F-Phe incorporation was 11-21-fold higher at the programmed position than the background incorporation at phenylalanine codons, showing high specificity of analog incorporation. Protein expression yields of 8-12 mg/L of culture, corresponding to about two thirds of the expression level of the wild-type DHFR protein, are sufficient to provide fluorinated proteins suitable for 19F-NMR spectroscopy and other sample-intensive methods. The use of a nonessential "21st" tRNA/synthetase pair will permit incorporation of a wide range of analogs, once the synthetase specificity has been modified accordingly.
- 173Kim, H.-W.; Perez, J. A.; Ferguson, S. J.; Campbell, I. D. The Specific Incorporation of Labelled Aromatic Amino Acids into Proteins through Growth of Bacteria in the Presence of Glyphosate. FEBS Lett. 1990, 272 (1–2), 34– 36, DOI: 10.1016/0014-5793(90)80442-LThere is no corresponding record for this reference.
- 174Niu, W.; Shu, Q.; Chen, Z.; Mathews, S.; Di Cera, E.; Frieden, C. The Role of Zn2+ on the Structure and Stability of Murine Adenosine Deaminase. J. Phys. Chem. B 2010, 114 (49), 16156– 16165, DOI: 10.1021/jp106041v174The role of Zn2+ on the structure and stability of murine adenosine deaminaseNiu, Weiling; Shu, Qin; Chen, Zhiwei; Mathews, Scott; Di Cera, Enrico; Frieden, CarlJournal of Physical Chemistry B (2010), 114 (49), 16156-16165CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)Adenosine deaminase (ADA) is a key enzyme in purine metab. and crucial for normal immune competence. It is a 40-kDa monomeric TIM-barrel protein contg. a tightly bound Zn2+, which is required for activity. Here, the authors investigated the role of Zn2+ with respect to ADA structure and stability. After removing Zn2+, the crystallog. structure of the protein remained highly ordered and similar to that of the holoprotein with structural changes limited to regions capping the active site pocket. The stability of the protein, however, was decreased significantly in the absence of Zn2+. Denaturation with urea showed the midpoint to be about 3.5M for the apoenzyme, compared with 6.4M for the holoenzyme. ADA contained 4 Trp residues distant from the Zn2+site; 19F NMR studies in the presence and absence of Zn2+ were carried out after incorporation of 6-19F-tryptophan. Chem. shift differences were obsd. for 3 of the 4 Trp residues, suggesting that, in contrast to the x-ray data, Zn2+-induced structural changes are propagated throughout the protein. Changes throughout the structure as suggested by the NMR data may explain the lower stability of the Zn2+-free protein. Real-time 19F NMR spectroscopy measuring the loss of Zn2+ showed that structural changes correlated with the loss of enzymic activity.
- 175Ruben, E. A.; Gandhi, P. S.; Chen, Z.; Koester, S. K.; DeKoster, G. T.; Frieden, C.; Di Cera, E. 19F NMR Reveals the Conformational Properties of Free Thrombin and Its Zymogen Precursor Prethrombin-2. J. Biol. Chem. 2020, 295 (24), 8227– 8235, DOI: 10.1074/jbc.RA120.013419There is no corresponding record for this reference.
- 176Duewel, H.; Daub, E.; Robinson, V.; Honek, J. F. Incorporation of Trifluoromethionine into a Phage Lysozyme: Implications and a New Marker for Use in Protein 19F NMR. Biochemistry 1997, 36 (11), 3404– 3416, DOI: 10.1021/bi9617973176Incorporation of Trifluoromethionine into a Phage Lysozyme: Implications and a New Marker for Use in Protein 19F NMRDuewel, Henry; Daub, Elisabeth; Robinson, Valerie; Honek, John F.Biochemistry (1997), 36 (11), 3404-3416CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Much interest is currently focused on understanding the detailed contribution that particular amino acid residues make in protein structure and function. Although the use of site-directed mutagenesis has greatly contributed to this goal, the approach is limited to the std. repertoire of twenty amino acids. Fluorinated amino acids have been utilized successfully to probe protein structure and dynamics as well as point to the importance of specific residues to biol. function. In our continuing investigations on the importance of the amino acid methionine in biol. systems, the successful incorporation of L-S-trifluoromethylhomocysteine (L-trifluoromethionine; L-TFM) into bacteriophage λ lysozyme (LaL), an enzyme contg. three methionine residues, is reported. The L isomer of TFM was synthesized in an overall yield of 33% from N-acetyl-D,L-homocysteine thiolactone and trifluoromethyl iodide. An expression plasmid giving strong overprodn. of LaL was prepd. and transformed into an Escherichia coli strain auxotrophic for methionine permitting the expression of LaL in the presence of L-TFM. The analog would not support growth of the auxotroph and was found to be inhibitory to cell growth. However, cells that were initially grown in a Met-rich media followed by protein induction under careful control of the resp. concns. of L-Met and L-TFM in the media, were able to overexpress TFM-labeled LaL (TFM-LaL) at both high (70%) and low (31%) levels of TFM incorporation. TFM-LaL at both levels of incorporation exhibited analogous activity to the wild type enzyme and were inhibited by chitooligosaccharides indicating that incorporation of the analog did not hinder enzyme function. Interestingly, the 19F soln. NMR spectra of the TFM-labeled enzymes consisted of four sharp resonances spanning a chem. shift range of 0.9 ppm, with three of the resonances showing very modest shielding changes on binding of chitopentaose. The 19F NMR anal. of TFM-LaL at both high and low levels of incorporation suggested that one of the methionine positions gives rise to two sep. resonances. The intensities of these two resonances were influenced by the extent of incorporation which was interpreted as an indication that subtle conformational changes in protein structure are induced by incorporated TFM. The similarities and differences between Met and TFM were analyzed using ab initio MO calcns. The methodol. presented offers promise as a new approach to the study of protein-ligand interactions as well as for future investigations into the functional importance of methionine in proteins.
- 177Cleve, P.; Robinson, V.; Duewel, H. S.; Honek, J. F. Difluoromethionine as a Novel 19F NMR Structural Probe for Internal Amino Acid Packing in Proteins. J. Am. Chem. Soc. 1999, 121 (37), 8475– 8478, DOI: 10.1021/ja9911418177Difluoromethionine as a Novel 19F NMR Structural Probe for Internal Amino Acid Packing in ProteinsVaughan, Mark D.; Cleve, Paul; Robinson, Valerie; Duewel, Henry S.; Honek, John F.Journal of the American Chemical Society (1999), 121 (37), 8475-8478CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The successful incorporation of difluoromethionine (DFM), a novel 19F NMR probe of internal amino acid packing, into the three methionine positions (1, 14, and 107) of a recombinant protein, the lysozyme from bacteriophage λ (LaL), is reported. The anisochronous 19F NMR signals of the diastereotopic fluorines showed a variation in the degree of chem. shift difference when present at relatively free surface positions (Met1 and Met107) vs. the tightly packed protein core (Met14), with the anisochronicity greatly enhanced for DFM incorporated at this latter position. The increased magnetic nonequivalence of the two fluorines at position 14 is thought to be a consequence of the restricted environment of DFM at this position. The anisochronicity of these two fluorines is further manifested in a differential chem. shift change for these two fluorines upon binding of an oligosaccharide inhibitor to LaL, with one of the two fluorines experiencing a significant upfield shift compared to the other. This differential variation is thought to be assocd. with a very subtle change in the protein conformation surrounding one fluorine at position 14, which is not significantly translated to the environment of the other fluorine.
- 178Holzberger, B.; Rubini, M.; Möller, H. M.; Marx, A. A Highly Active DNA Polymerase with a Fluorous Core. Angew. Chem. Int. Ed. 2010, 49 (7), 1324– 1327, DOI: 10.1002/anie.200905978178A highly active DNA polymerase with a fluorous coreHolzberger, Bastian; Rubini, Marina; Moeller, Heiko M.; Marx, AndreasAngewandte Chemie, International Edition (2010), 49 (7), 1324-1327, S1324/1-S1324/8CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)DNA polymerases catalyze all DNA synthesis in the cell and are key tools in important mol. biol. core technologies. Apart from naturally available DNA polymerases, several modified DNA polymerases with new characteristics have been developed. To date, directed evolution using the 20 natural amino acids is a promising method for the creation of nucleic acid polymerases with modified properties. Yet, the incorporation of non-natural amino acids may lead to enhanced chem. and biol. diversity of protein structures and properties by introduction of functional groups that are not represented by the natural amino acids. Herein, the authors present the generation of a multifluorinated DNA polymerase. The N-terminally truncated version of DNA polymerase I from Thermus aquaticus (KlenTaq) is a thermophilic DNA polymerase composed of 540 amino acids (63 kDa), including 13 methionine (Met) residues that were globally replaced by trifluoromethionine (TFM) with a substitution level of approx. 82%. The multifluorinated KlenTaq was highly active and exhibited a similar selectivity as the wild-type (wt.) enzyme. Moreover, the introduction of the NMR-active nucleus 19F offers the possibility to study DNA polymerase dynamics by 19F NMR spectroscopy. Despite its large size of 63 kDa, at least nine individual 19F resonances are obsd., which allow us to distinguish different states of the DNA polymerase on the way to incorporating a canonical or a noncanonical nucleotide. To our knowledge, this is by far the largest enzymically active protein with Met globally replaced by TFM.
- 179Orton, H. W.; Qianzhu, H.; Abdelkader, E. H.; Habel, E. I.; Tan, Y. J.; Frkic, R. L.; Jackson, C. J.; Huber, T.; Otting, G. Through-Space Scalar 19F-19F Couplings between Fluorinated Noncanonical Amino Acids for the Detection of Specific Contacts in Proteins. J. Am. Chem. Soc. 2021, 143 (46), 19587– 19598, DOI: 10.1021/jacs.1c10104There is no corresponding record for this reference.
- 180Miao, Q.; Nitsche, C.; Orton, H.; Overhand, M.; Otting, G.; Ubbink, M. Paramagnetic Chemical Probes for Studying Biological Macromolecules. Chem. Rev. 2022, 122 (10), 9571– 9642, DOI: 10.1021/acs.chemrev.1c00708180Paramagnetic Chemical Probes for Studying Biological MacromoleculesMiao, Qing; Nitsche, Christoph; Orton, Henry; Overhand, Mark; Otting, Gottfried; Ubbink, MarcellusChemical Reviews (Washington, DC, United States) (2022), 122 (10), 9571-9642CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Paramagnetic chem. probes have been used in ESR (EPR) and NMR (NMR) spectroscopy for more than four decades. Recent years witnessed a great increase in the variety of probes for the study of biol. macromols. (proteins, nucleic acids, and oligosaccharides). This Review aims to provide a comprehensive overview of the existing paramagnetic chem. probes, including chem. synthetic approaches, functional properties, and selected applications. Recent developments have seen, in particular, a rapid expansion of the range of lanthanoid probes with anisotropic magnetic susceptibilities for the generation of structural restraints based on residual dipolar couplings and pseudocontact shifts in soln. and solid state NMR spectroscopy, mostly for protein studies. Also many new isotropic paramagnetic probes, suitable for NMR measurements of paramagnetic relaxation enhancements, as well as EPR spectroscopic studies (in particular double resonance techniques) have been developed and employed to investigate biol. macromols. Notwithstanding the large no. of reported probes, only few have found broad application and further development of probes for dedicated applications is foreseen.
- 181Fanucci, G. E.; Cafiso, D. S. Recent advances and applications of site-directed spin labeling. Curr. Opin. Struct. Biol. 2006, 16 (5), 644– 653, DOI: 10.1016/j.sbi.2006.08.008181Recent advances and applications of site-directed spin labelingFanucci, Gail E.; Cafiso, David S.Current Opinion in Structural Biology (2006), 16 (5), 644-653CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. Site-directed spin labeling has become a popular biophys. tool for the characterization of protein structure, dynamics and conformational change. This method is well suited and widely used to study small sol. proteins, membrane proteins and large protein complexes. Recent advances in site-directed spin labeling methodol. have occurred in two areas. The first involves an understanding of the conformations and local dynamics of the spin-labeled side chain, including the features of proteins that influence ESR lineshape. The second advance is the application of pulse techniques to det. long-range distances and distance distributions in proteins. During the past two years, these tech. developments have been used to address several important problems concerning the mol. function of proteins.
- 182Braun, T.; Drescher, M.; Summerer, D. Expanding the Genetic Code for Site-Directed Spin-Labeling. Int. J. Mol. Sci. 2019, 20 (2), 373, DOI: 10.3390/ijms20020373There is no corresponding record for this reference.
- 183Kálai, T.; Fleissner, M. R.; Jeko, J.; Hubbell, W. L.; Hideg, K. Synthesis of New Spin Labels for Cu-Free Click Conjugation. Tetrahedron Lett. 2011, 52 (21), 2747– 2749, DOI: 10.1016/j.tetlet.2011.03.077There is no corresponding record for this reference.
- 184Kugele, A.; Braun, T. S.; Widder, P.; Williams, L.; Schmidt, M. J.; Summerer, D.; Drescher, M. Site-Directed Spin Labelling of Proteins by Suzuki-Miyaura Coupling via a Genetically Encoded Aryliodide Amino Acid. Chem. Commun. 2019, 55 (13), 1923– 1926, DOI: 10.1039/C8CC09325C184Site-directed spin labelling of proteins by Suzuki-Miyaura coupling via a genetically encoded aryliodide amino acidKugele Anandi; Braun Theresa Sophie; Widder Pia; Williams Lara; Schmidt Moritz Johannes; Summerer Daniel; Drescher MalteChemical communications (Cambridge, England) (2019), 55 (13), 1923-1926 ISSN:.We report site-directed protein spin labelling via Suzuki-Miyaura coupling of a nitroxide boronic acid label with the genetically encoded amino acid 4-iodo-l-phenylalanine. The resulting spin label bears a rigid biphenyl linkage with lower flexibility than spin label R1. It is suitable to obtain defined electron paramagnetic resonance distance distributions and to report protein-membrane interactions and conformational transitions of α-synuclein.
- 185Jana, S.; Evans, E. G. B.; Jang, H. S.; Zhang, S.; Zhang, H.; Rajca, A.; Gordon, S. E.; Zagotta, W. N.; Stoll, S.; Mehl, R. A. Ultrafast Bioorthogonal Spin-Labeling and Distance Measurements in Mammalian Cells Using Small, Genetically Encoded Tetrazine Amino Acids. J. Am. Chem. Soc. 2023, 145 (27), 14608– 14620, DOI: 10.1021/jacs.3c00967185Ultrafast Bioorthogonal Spin-Labeling and Distance Measurements in Mammalian Cells Using Small, Genetically Encoded Tetrazine Amino AcidsJana, Subhashis; Evans, Eric G. B.; Jang, Hyo Sang; Zhang, Shuyang; Zhang, Hui; Rajca, Andrzej; Gordon, Sharona E.; Zagotta, William N.; Stoll, Stefan; Mehl, Ryan A.Journal of the American Chemical Society (2023), 145 (27), 14608-14620CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Site-directed spin-labeling (SDSL)-in combination with double electron-electron resonance (DEER) spectroscopy-has emerged as a powerful technique for detg. both the structural states and the conformational equil. of biomacromols. DEER combined with in situ SDSL in live cells is challenging since current bioorthogonal labeling approaches are too slow to allow for complete labeling with low concns. of spin label prior to loss of signal from cellular redn. Here, we overcome this limitation by genetically encoding a novel family of small, tetrazine-bearing noncanonical amino acids (Tet-v4.0) at multiple sites in proteins expressed in Escherichia coli and in human HEK293T cells. We achieved specific and quant. spin-labeling of Tet-v4.0-contg. proteins by developing a series of strained trans-cyclooctene (sTCO)-functionalized nitroxides-including a gem-diethyl-substituted nitroxide with enhanced stability in cells-with rate consts. that can exceed 106 M-1 s-1. The remarkable speed of the Tet-v4.0/sTCO reaction allowed efficient spin-labeling of proteins in live cells within minutes, requiring only sub-micromolar concns. of sTCO-nitroxide. DEER recorded from intact cells revealed distance distributions in good agreement with those measured from proteins purified and labeled in vitro. Furthermore, DEER was able to resolve the maltose-dependent conformational change of Tet-v4.0-incorporated and spin-labeled MBP in vitro and support assignment of the conformational state of an MBP mutant within HEK293T cells. We anticipate the exceptional reaction rates of this system, combined with the relatively short and rigid side chains of the resulting spin labels, will enable structure/function studies of proteins directly in cells, without any requirements for protein purifn.
- 186Fleissner, M. R.; Brustad, E. M.; Kálai, T.; Altenbach, C.; Cascio, D.; Peters, F. B.; Hideg, K.; Peuker, S.; Schultz, P. G.; Hubbell, W. L. Site-Directed Spin Labeling of a Genetically Encoded Unnatural Amino Acid. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (51), 21637– 21642, DOI: 10.1073/pnas.0912009106186Site-directed spin labeling of a genetically encoded unnatural amino acidFleissner, Mark R.; Brustad, Eric M.; Kalai, Tamas; Altenbach, Christian; Cascio, Duilio; Peters, Francis B.; Hideg, Kalman; Schultz, Peter G.; Hubbell, Wayne L.Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (51), 21637-21642, S21637/1-S21637/10CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The traditional site-directed spin labeling (SDSL) method, which utilizes cysteine residues and sulfhydryl-reactive nitroxide reagents, can be challenging for proteins that contain functionally important native cysteine residues or disulfide bonds. To make SDSL amenable to any protein, we introduce an orthogonal labeling strategy, i.e., one that does not rely on any of the functional groups found in the common 20 amino acids. In this method, the genetically encoded unnatural amino acid p-acetyl-L-phenylalanine (p-AcPhe) is reacted with a hydroxylamine reagent to generate a nitroxide side chain (K1). The utility of this scheme was demonstrated with seven mutants of T4 lysozyme, each contg. a single p-AcPhe at a solvent-exposed helix site; the mutants were expressed in amts. qual. similar to the wild-type protein. In general, the EPR spectra of the resulting K1 mutants reflect higher nitroxide mobilities than the spectra of analogous mutants contg. the more constrained disulfide-linked side chain (R1) commonly used in SDSL. Despite this increased flexibility, site dependence of the EPR spectra suggests that K1 will be a useful sensor of local structure and of conformational changes in soln. Distance measurements between pairs of K1 residues using double electron electron resonance (DEER) spectroscopy indicate that K1 will also be useful for distance mapping.
- 187Nguyen, D. P.; Lusic, H.; Neumann, H.; Kapadnis, P. B.; Deiters, A.; Chin, J. W. Genetic Encoding and Labeling of Aliphatic Azides and Alkynes in Recombinant Proteins via a Pyrrolysyl-tRNA Synthetase/tRNACUA Pair and Click Chemistry. J. Am. Chem. Soc. 2009, 131 (25), 8720– 8721, DOI: 10.1021/ja900553w187Genetic Encoding and Labeling of Aliphatic Azides and Alkynes in Recombinant Proteins via a Pyrrolysyl-tRNA Synthetase/tRNACUA Pair and Click ChemistryNguyen, Duy P.; Lusic, Hrvoje; Neumann, Heinz; Kapadnis, Prashant B.; Deiters, Alexander; Chin, Jason W.Journal of the American Chemical Society (2009), 131 (25), 8720-8721CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We demonstrate that an orthogonal Methanosarcina barkeri MS pyrrolysyl-tRNA synthetase/tRNACUA pair directs the efficient, site-specific incorporation of N6-[(2-propynyloxy)carbonyl]-L-lysine, contg. a carbon-carbon triple bond, and N6-[(2-azidoethoxy)carbonyl]-L-lysine, contg. an azido group, into recombinant proteins in Escherichia coli. Proteins contg. the alkyne functional group are labeled with an azido biotin and an azido fluorophore, via copper catalyzed [3+2] cycloaddn. reactions, to produce the corresponding triazoles in good yield. The methods reported are useful for the site-specific labeling of recombinant proteins and may be combined with mutually orthogonal methods of introducing unnatural amino acids into proteins as well as with chem. orthogonal methods of protein labeling. This should allow the site specific incorporation of multiple distinct probes into proteins and the control of protein topol. and structure by intramol. orthogonal conjugation reactions.
- 188Chin, J. W.; Santoro, S. W.; Martin, A. B.; King, D. S.; Wang, L.; Schultz, P. G. Addition of p-Azido-l-phenylalanine to the Genetic Code of Escherichia coli. J. Am. Chem. Soc. 2002, 124 (31), 9026– 9027, DOI: 10.1021/ja027007w188Addition of p-Azido-L-phenylalanine to the Genetic Code of Escherichia coliChin, Jason W.; Santoro, Stephen W.; Martin, Andrew B.; King, David S.; Wang, Lei; Schultz, Peter G.Journal of the American Chemical Society (2002), 124 (31), 9026-9027CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We report the selection of a new orthogonal aminoacyl tRNA synthetase/tRNA pair for the in vivo incorporation of a photocrosslinker, p-azido-L-phenylalanine, into proteins in response to the amber codon, TAG. The amino acid is incorporated in good yield with high fidelity and can be used to crosslink interacting proteins.
- 189Kucher, S.; Korneev, S.; Tyagi, S.; Apfelbaum, R.; Grohmann, D.; Lemke, E. A.; Klare, J. P.; Steinhoff, H.-J.; Klose, D. Orthogonal Spin Labeling Using Click Chemistry for in vitro and in vivo Applications. J. Magn. Reson. 2017, 275, 38– 45, DOI: 10.1016/j.jmr.2016.12.001189Orthogonal spin labeling using click chemistry for in vitro and in vivo applicationsKucher, Svetlana; Korneev, Sergei; Tyagi, Swati; Apfelbaum, Ronja; Grohmann, Dina; Lemke, Edward A.; Klare, Johann P.; Steinhoff, Heinz-Juergen; Klose, DanielJournal of Magnetic Resonance (2017), 275 (), 38-45CODEN: JMARF3; ISSN:1090-7807. (Elsevier B.V.)Site-directed spin labeling for EPR- and NMR spectroscopy has mainly been achieved exploiting the specific reactivity of cysteines. For proteins with native cysteines or for in vivo applications, an alternative coupling strategy is required. In these cases click chem. offers major benefits by providing a fast and highly selective, biocompatible reaction between azide and alkyne groups. Here, we establish click chem. as a tool to target unnatural amino acids in vitro and in vivo using azide- and alkyne-functionalized spin labels. The approach is compatible with a variety of labels including redn.-sensitive nitroxides. Comparing spin labeling efficiencies from the copper-free with the strongly reducing copper(I)-catalyzed azide-alkyne click reaction, we find that the faster kinetics for the catalyzed reaction outrun redn. of the labile nitroxide spin labels and allow quant. labeling yields within short reaction times. Inter-spin distance measurements demonstrate that the novel side chain is suitable for paramagnetic NMR- or EPR-based conformational studies of macromol. complexes.
- 190Abdelkader, E. H.; Feintuch, A.; Yao, X.; Adams, L. A.; Aurelio, L.; Graham, B.; Goldfarb, D.; Otting, G. Protein Conformation by EPR Spectroscopy Using Gadolinium Tags Clicked to Genetically Encoded p-Azido-L-Phenylalanine. Chem. Commun. 2015, 51 (88), 15898– 15901, DOI: 10.1039/C5CC07121FThere is no corresponding record for this reference.
- 191Loh, C. T.; Ozawa, K.; Tuck, K. L.; Barlow, N.; Huber, T.; Otting, G.; Graham, B. Lanthanide Tags for Site-Specific Ligation to an Unnatural Amino Acid and Generation of Pseudocontact Shifts in Proteins. Bioconjug. Chem. 2013, 24 (2), 260– 268, DOI: 10.1021/bc300631zThere is no corresponding record for this reference.
- 192Mahawaththa, M. C.; Lee, M. D.; Giannoulis, A.; Adams, L. A.; Feintuch, A.; Swarbrick, J. D.; Graham, B.; Nitsche, C.; Goldfarb, D.; Otting, G. Small Neutral Gd(iii) Tags for Distance Measurements in Proteins by Double Electron-Electron Resonance Experiments. Phys. Chem. Chem. Phys. 2018, 20 (36), 23535– 23545, DOI: 10.1039/C8CP03532FThere is no corresponding record for this reference.
- 193Yang, H.; Yang, S.; Kong, J.; Dong, A.; Yu, S. Obtaining Information About Protein Secondary Structures in Aqueous Solution Using Fourier Transform IR Spectroscopy. Nat. Protoc. 2015, 10 (3), 382– 396, DOI: 10.1038/nprot.2015.024193Obtaining information about protein secondary structures in aqueous solution using Fourier transform IR spectroscopyYang, Huayan; Yang, Shouning; Kong, Jilie; Dong, Aichun; Yu, ShaoningNature Protocols (2015), 10 (3), 382-396CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Fourier transform IR (FTIR) spectroscopy is a nondestructive technique for structural characterization of proteins and polypeptides. The IR spectral data of polymers are usually interpreted in terms of the vibrations of a structural repeat. The repeat units in proteins give rise to nine characteristic IR absorption bands (amides A, B and I-VII). Amide I bands (1,700-1,600 cm-1) are the most prominent and sensitive vibrational bands of the protein backbone, and they relate to protein secondary structural components. In this protocol, we have detailed the principles that underlie the detn. of protein secondary structure by FTIR spectroscopy, as well as the basic steps involved in protein sample prepn., instrument operation, FTIR spectra collection and spectra anal. in order to est. protein secondary-structural components in aq. (both H2O and deuterium oxide (D2O)) soln. using algorithms, such as second-deriv., deconvolution and curve fitting. Small amts. of high-purity (>95%) proteins at high concns. (>3 mg ml-1) are needed in this protocol; typically, the procedure can be completed in 1-2 d.
- 194Lorenz-Fonfria, V. A. Infrared Difference Spectroscopy of Proteins: From Bands to Bonds. Chem. Rev. 2020, 120 (7), 3466– 3576, DOI: 10.1021/acs.chemrev.9b00449194Infrared Difference Spectroscopy of Proteins: From Bands to BondsLorenz-Fonfria, Victor A.Chemical Reviews (Washington, DC, United States) (2020), 120 (7), 3466-3576CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. IR difference spectroscopy probes vibrational changes of proteins upon their perturbation. Compared with other spectroscopic methods it stands out by its sensitivity to the protonation state, H-bonding and the conformation of different groups in proteins, including the peptide backbone, amino acid side chains, internal water mols. or cofactors. In particular, the detection of protonation and H-bonding changes in a time-resolved manner, not easily obtained by other techniques, is one of the most successful applications of IR difference spectroscopy. The present review deals with the use of perturbations designed to specifically change the protein between two (or more) functionally relevant states, a strategy often referred to as reaction-induced IR difference spectroscopy. In the first half of this contribution I review the technique of reaction-induced IR difference spectroscopy of proteins, with special emphasis given to the prepn. of suitable samples and their characterization; strategies for protein perturbations; and methodologies for time-resolved measurements (from nanoseconds to minutes). The second half of this contribution focus on the spectral interpretation. It starts by reviewing how changes in H-bonding, medium polarity, and vibrational coupling affect vibrational frequencies, intensities and bandwidths. It is followed by band assignments, a crucial aspect mostly performed with the help of isotopic labeling and site-directed mutagenesis, and completed by integrating and interpreting the results in the context of the studied protein, an aspect increasingly supported by spectral calcns. Selected examples from the literature, predominately but not exclusively from retinal proteins, were used to illustrate the topics covered in this review.
- 195Chung, J. K.; Thielges, M. C.; Fayer, M. D. Dynamics of the Folded and Unfolded Villin Headpiece (HP35) Measured with Ultrafast 2D IR Vibrational Echo Spectroscopy. Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (9), 3578– 3583, DOI: 10.1073/pnas.1100587108195Dynamics of the folded and unfolded villin headpiece (HP35) measured with ultrafast 2D IR vibrational echo spectroscopyChung, Jean K.; Thielges, Megan C.; Fayer, Michael D.Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (9), 3578-3583, S3578/1-S3578/5CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A series of two-dimensional IR vibrational echo expts. performed on nitrile-labeled villin headpiece [HP35-(CN)2] is described. HP35 is a small peptide composed of three alpha helixes in the folded configuration. The dynamics of the folded HP35-(CN)2 are compared to that of the guanidine-induced unfolded peptide, as well as the nitrile-functionalized phenylalanine (PheCN), which is used to differentiate the peptide dynamic contributions to the observables from those of the water solvent. Because the viscosity of solvent has a significant effect on fast dynamics, the viscosity of the solvent is held const. by adding glycerol. For the folded peptide, the addn. of glycerol to the water solvent causes observable slowing of the peptide's dynamics. Holding the viscosity const. as GuHCl is added, the dynamics of unfolded peptide are much faster than those of the folded peptide, and they are very similar to that of PheCN. These observations indicate that the local environment of the nitrile in the unfolded peptide resembles that of PheCN, and the dynamics probed by the CN are dominated by the fluctuations of the solvent mols., in contrast to the observations on the folded peptide.
- 196Chung, J. K.; Thielges, M. C.; Fayer, M. D. Conformational Dynamics and Stability of HP35 Studied with 2D IR Vibrational Echoes. J. Am. Chem. Soc. 2012, 134 (29), 12118– 12124, DOI: 10.1021/ja303017d196Conformational Dynamics and Stability of HP35 Studied with 2D IR Vibrational EchoesChung, Jean K.; Thielges, Megan C.; Fayer, Michael D.Journal of the American Chemical Society (2012), 134 (29), 12118-12124CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Two-dimensional IR (2D IR) vibrational echo spectroscopy was used to measure the fast dynamics of two variants of chicken villin headpiece 35 (HP35). The CN of cyanophenylalanine residues inserted in the hydrophobic core was used as a vibrational probe. Expts. were performed on both singly (HP35-P) and doubly CN-labeled peptide (HP35-P2) within the wild-type sequence, as well as on HP-35 contg. a singly labeled cyanophenylalanine and two norleucine mutations (HP35-P NleNle). There is a remarkable similarity between the dynamics measured in singly and doubly CN-labeled HP35, demonstrating that the presence of an addnl. CN vibrational probe does not significantly alter the dynamics of the small peptide. The substitution of two lysine residues by norleucines markedly improves the stability of HP35 by replacing charged with nonpolar residues, stabilizing the hydrophobic core. The results of the 2D IR expts. reveal that the dynamics of HP35-P are significantly faster than those of HP35-P NleNle. These observations suggest that the slower structural fluctuations in the hydrophobic core, indicating a more tightly structured core, may be an important contributing factor to HP35-P NleNle's increased stability.
- 197Urbanek, D. C.; Vorobyev, D. Y.; Serrano, A. L.; Gai, F.; Hochstrasser, R. M. The Two-Dimensional Vibrational Echo of a Nitrile Probe of the Villin HP35 Protein. J. Phys. Chem. Lett. 2010, 1 (23), 3311– 3315, DOI: 10.1021/jz101367d197The Two-Dimensional Vibrational Echo of a Nitrile Probe of the Villin HP35 ProteinUrbanek, Diana C.; Vorobyev, Dmitriy Yu.; Serrano, Arnaldo L.; Gai, Feng; Hochstrasser, Robin M.Journal of Physical Chemistry Letters (2010), 1 (23), 3311-3315CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)Two-dimensional (2D) IR spectroscopy was used to probe the hydrophobic core structure of the 35-residue villin headpiece subdomain, HP35, by monitoring the C≡N vibrational stretching band of a cyano-substituted phenylalanine (Phe). The presence of two humps in the vibrational frequency distribution in the folded equil. state is revealed. They represent two states that exchange more slowly than ca. 10 ps. The two CN stretch mode peak frequencies (and their equil. populations) are 2228.7 (44%) and 2234.5 cm-1 (56%). The two CN modes have different frequency-frequency correlation times of 7.4 and 1.6 ps, resp. These results suggest that the population with the higher frequency CN group is partly exposed, whereas the other CN mode experiences a hydrophobic-like environment.
- 198Bagchi, S.; Boxer, S. G.; Fayer, M. D. Ribonuclease S Dynamics Measured Using a Nitrile Label with 2D IR Vibrational Echo Spectroscopy. J. Phys. Chem. B 2012, 116 (13), 4034– 4042, DOI: 10.1021/jp2122856198Ribonuclease S dynamics measured using a nitrile label with 2D IR vibrational echo spectroscopyBagchi, Sayan; Boxer, Steven G.; Fayer, Michael D.Journal of Physical Chemistry B (2012), 116 (13), 4034-4042CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)A nitrile-labeled amino acid, p-cyanophenylalanine, is introduced near the active site of the semisynthetic enzyme, RNase S, to serve as a probe of protein dynamics and fluctuations. RNase S is the limited proteolysis product of subtilisin acting on RNase A, and consists of a small fragment including amino acids 1-20 (the S-peptide) and a larger fragment including residues 21-124 (the S-protein). A series of 2-dimensional vibrational echo expts. performed on the nitrile-labeled S-peptide and RNase S are described. The time-dependent changes in the 2-dimensional IR vibrational echo line shapes were analyzed using the center line slope method to obtain the frequency-frequency correlation function (FFCF). The observations showed that the nitrile probe in the S-peptide had dynamics that were similar to, but faster than, those of the single amino acid p-cyanophenylalanine in water. In contrast, the dynamics of the nitrile label when the peptide was bound to form RNase S were dominated by homogeneous dephasing (motionally narrowed) contributions with only a small contribution from very fast inhomogeneous structural dynamics. These results provided insights into the nature of the structural dynamics of the RNase S complex. The equil. dynamics of the nitrile-labeled S-peptide and the RNase S complex were also investigated by mol. dynamics (MD) simulations. The exptl. detd. FFCFs are compared to the FFCFs obtained from the MD simulations, thereby testing the capacity of simulations to det. the amplitudes and time scales of protein structural fluctuations on fast time scales under thermal equil. conditions.
- 199Tharp, J. M.; Wang, Y.-S.; Lee, Y.-J.; Yang, Y.; Liu, W. R. Genetic Incorporation of Seven ortho-Substituted Phenylalanine Derivatives. ACS Chem. Biol. 2014, 9 (4), 884– 890, DOI: 10.1021/cb400917a199Genetic incorporation of seven ortho-substituted phenylalanine derivativesTharp, Jeffery M.; Wang, Yane-Shih; Lee, Yan-Jiun; Yang, Yanyan; Liu, Wenshe R.ACS Chemical Biology (2014), 9 (4), 884-890CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Seven phenylalanine derivs. with small ortho substitutions were genetically encoded in Escherichia coli and mammalian cells at an amber codon using a previously reported, rationally designed pyrrolysyl-tRNA synthetase mutant (PylRS(N346A/C348A)) coupled with tRNACUAPyl. Ortho substitutions of the phenylalanine derivs. reported here included 3 halides, Me, methoxy, nitro, and nitrile. These compds. have the potential for use in multiple biochem. and biophys. applications. Specifically, the authors demonstrated that o-cyanophenylalanine could be used as a selective sensor to probe the local environment of proteins and applied this to study protein folding/unfolding. For 6 of these compds. this constitutes the 1st report of their genetic incorporation in living cells. With these compds. the total no. of substrates available for PylRS(N346A/C348A) is increased to nearly 40, which demonstrates that PylRS(N346A/C348A) is able to recognize phenylalanine with a substitution at any side-chain arom. position as a substrate. To the authors' knowledge, PylRS(N346A/C348A) is the only aminoacyl-tRNA synthetase with such a high substrate promiscuity.
- 200van Wilderen, L. J. G. W.; Kern-Michler, D.; Müller-Werkmeister, H. M.; Bredenbeck, J. Vibrational Dynamics and Solvatochromism of the Label SCN in Various Solvents and Hemoglobin by Time Dependent IR and 2D-IR Spectroscopy. Phys. Chem. Chem. Phys. 2014, 16 (36), 19643– 19653, DOI: 10.1039/C4CP01498G200Vibrational dynamics and solvatochromism of the label SCN in various solvents and hemoglobin by time dependent IR and 2D-IR spectroscopyvan Wilderen, Luuk J. G. W.; Kern-Michler, Daniela; Mueller-Werkmeister, Henrike M.; Bredenbeck, JensPhysical Chemistry Chemical Physics (2014), 16 (36), 19643-19653CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)We investigated the characteristics of the thiocyanate (SCN) functional group as a probe of local structural dynamics for 2D-IR spectroscopy of proteins, exploiting the dependence of vibrational frequency on the environment of the label. Steady-state and time-resolved IR spectroscopy are performed on the model compd. methylthiocyanate (MeSCN) in solvents of different polarity, and compared to data obtained on SCN as a local probe introduced as cyanylated cysteine in the protein bovine Hb. The vibrational lifetime of the protein label is detd. to be 37 ps, and its anharmonicity is obsd. to be lower than that of the model compd. (which itself exhibits solvent-independent anharmonicity). The vibrational lifetime of MeSCN generally correlates with the solvent polarity, i.e. longer lifetimes in less polar solvents, with the longest lifetime being 158 ps. However, the capacity of the solvent to form hydrogen bonds complicates this simplified picture. The long lifetime of the SCN vibration is in contrast to commonly used azide labels or isotopically-labeled amide I and better suited to monitor structural rearrangements by 2D-IR spectroscopy. We present time-dependent 2D-IR data on the labeled protein which reveal an initially inhomogeneous structure around the CN oscillator. The distribution becomes homogeneous after 5 ps so that spectral diffusion has effectively erased the 'memory' of the CN stretching frequency. Therefore, the 2D-IR data of the label incorporated in Hb demonstrate how SCN can be utilized to sense rearrangements in the local structure on a picosecond timescale.
- 201Bloem, R.; Koziol, K.; Waldauer, S. A.; Buchli, B.; Walser, R.; Samatanga, B.; Jelesarov, I.; Hamm, P. Ligand Binding Studied by 2D IR Spectroscopy Using the Azidohomoalanine Label. J. Phys. Chem. B 2012, 116 (46), 13705– 13712, DOI: 10.1021/jp3095209201Ligand Binding Studied by 2D IR Spectroscopy Using the Azidohomoalanine LabelBloem, Robbert; Koziol, Klemens; Waldauer, Steven A.; Buchli, Brigitte; Walser, Reto; Samatanga, Brighton; Jelesarov, Ilian; Hamm, PeterJournal of Physical Chemistry B (2012), 116 (46), 13705-13712CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)We explore the capability of the azidohomoalanine (Aha) as a vibrational label for 2D IR spectroscopy to study the binding of the target peptide to the PDZ2 domain. The Aha label responds sensitively to its local environment and its peak extinction coeff. of 350-400 M-1 cm-1 is high enough to routinely measure it in the low millimolar concn. regime. The central frequency, inhomogeneous width and spectral diffusion times deduced from the 2D IR line shapes of the Aha label at various positions in the peptide sequence is discussed in relationship to the known X-ray structure of the peptide bound to the PDZ2 domain. The results suggest that the Aha label introduces only a small perturbation to the overall structure of the peptide in the binding pocket. Finally, Aha is a methionine analog that can be incorporated also into larger proteins at essentially any position using protein expression. Altogether, Aha thus fulfills the requirements a versatile label should have for studies of protein structure and dynamics by 2D IR spectroscopy.
- 202Thielges, M. C.; Axup, J. Y.; Wong, D.; Lee, H. S.; Chung, J. K.; Schultz, P. G.; Fayer, M. D. Two-Dimensional IR Spectroscopy of Protein Dynamics Using Two Vibrational Labels: A Site-Specific Genetically Encoded Unnatural Amino Acid and an Active Site Ligand. J. Phys. Chem. B 2011, 115 (38), 11294– 11304, DOI: 10.1021/jp206986v202Two-Dimensional IR Spectroscopy of Protein Dynamics Using Two Vibrational Labels: A Site-Specific Genetically Encoded Unnatural Amino Acid and an Active Site LigandThielges, Megan C.; Axup, Jun Y.; Wong, Daryl; Lee, Hyun Soo; Chung, Jean K.; Schultz, Peter G.; Fayer, Michael D.Journal of Physical Chemistry B (2011), 115 (38), 11294-11304CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)Protein dynamics and interactions in myoglobin (Mb) were characterized via two vibrational dynamics labels (VDLs): a genetically incorporated site-specific azide (Az) bearing unnatural amino acid (AzPhe43) and an active site CO ligand. The Az-labeled protein was studied using ultrafast two-dimensional IR (2D IR) vibrational echo spectroscopy. CO bound at the active site of the heme serves as a second VDL located nearby. Therefore, it was possible to use Fourier transform IR (FT-IR) and 2D IR spectroscopic expts. on the Az in unligated Mb and in Mb bound to CO (MbAzCO) and on the CO in MbCO and MbAzCO to investigate the environment and motions of different states of one protein from the perspective of two spectrally resolved VDLs. A very broad bandwidth 2D IR spectrum, encompassing both the Az and CO spectral regions, found no evidence of direct coupling between the two VDLs. In MbAzCO, both VDLs reported similar time scale motions: very fast homogeneous dynamics, fast, ∼1 ps dynamics, and dynamics on a much slower time scale. Therefore, each VDL reports independently on the protein dynamics and interactions, and the measured dynamics are reflective of the protein motions rather than intrinsic to the chem. nature of the VDL. The AzPhe VDL also permitted study of oxidized Mb dynamics, which could not be accessed previously with 2D IR spectroscopy. The expts. demonstrate that the combined application of 2D IR spectroscopy and site-specific incorporation of VDLs can provide information on dynamics, structure, and interactions at virtually any site throughout any protein.
- 203Ye, S.; Zaitseva, E.; Caltabiano, G.; Schertler, G. F. X.; Sakmar, T. P.; Deupi, X.; Vogel, R. Tracking G-Protein-Coupled Receptor Activation Using Genetically Encoded Infrared Probes. Nature 2010, 464 (7293), 1386– 1389, DOI: 10.1038/nature08948203Tracking G-protein-coupled receptor activation using genetically encoded infrared probesYe, Shixin; Zaitseva, Ekaterina; Caltabiano, Gianluigi; Schertler, Gebhard F. X.; Sakmar, Thomas P.; Deupi, Xavier; Vogel, ReinerNature (London, United Kingdom) (2010), 464 (7293), 1386-1389CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Rhodopsin is a prototypical heptahelical family A G-protein-coupled receptor (GPCR) responsible for dim-light vision. Light isomerizes rhodopsin's retinal chromophore and triggers concerted movements of transmembrane helixes, including an outward tilting of helix 6 (H6) and a smaller movement of H5, to create a site for G-protein binding and activation. However, the precise temporal sequence and mechanism underlying these helix rearrangements is unclear. We used site-directed non-natural amino acid mutagenesis to engineer rhodopsin with p-azido-l-phenylalanine residues incorporated at selected sites, and monitored the azido vibrational signatures using IR spectroscopy as rhodopsin proceeded along its activation pathway. Here we report significant changes in electrostatic environments of the azido probes even in the inactive photoproduct Meta I, well before the active receptor state was formed. These early changes suggest a significant rotation of H6 and movement of the cytoplasmic part of H5 away from H3. Subsequently, a large outward tilt of H6 leads to opening of the cytoplasmic surface to form the active receptor photoproduct Meta II. Thus, our results reveal early conformational changes that precede larger rigid-body helix movements, and provide a basis to interpret recent GPCR crystal structures and to understand conformational sub-states obsd. during the activation of other GPCRs.
- 204Le Sueur, A. L.; Horness, R. E.; Thielges, M. C. Applications of Two-Dimensional Infrared Spectroscopy. Analyst 2015, 140 (13), 4336– 4349, DOI: 10.1039/C5AN00558B204Applications of two-dimensional infrared spectroscopyLe Sueur, Amanda L.; Horness, Rachel E.; Thielges, Megan C.Analyst (Cambridge, United Kingdom) (2015), 140 (13), 4336-4349CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)A review. Two-dimensional IR (2D IR) spectroscopy has recently emerged as a powerful tool with applications in many areas of scientific research. The inherent high time resoln. coupled with bond-specific spatial resoln. of IR spectroscopy enable direct characterization of rapidly interconverting species and fast processes, even in complex systems found in chem. and biol. In this minireview, we briefly outline the fundamental principles and exptl. procedures of 2D IR spectroscopy. Using illustrative example studies, we explain the important features of 2D IR spectra and their capability to elucidate mol. structure and dynamics. Primarily, this minireview aims to convey the scope and potential of 2D IR spectroscopy by highlighting select examples of recent applications including the use of innate or introduced vibrational probes for the study of nucleic acids, peptides/proteins, and materials.
- 205Smith, E. E.; Linderman, B. Y.; Luskin, A. C.; Brewer, S. H. Probing Local Environments with the Infrared Probe: l-4-Nitrophenylalanine. J. Phys. Chem. B 2011, 115 (10), 2380– 2385, DOI: 10.1021/jp109288j205Probing Local Environments with the Infrared Probe: L-4-NitrophenylalanineSmith, Emily E.; Linderman, Barton Y.; Luskin, Austin C.; Brewer, Scott H.Journal of Physical Chemistry B (2011), 115 (10), 2380-2385CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)The genetic incorporation of unnatural amino acids (UAAs) with high efficiency and fidelity is a powerful tool for the study of protein structure and dynamics with site-specificity in a relatively nonintrusive manner. Here, we illustrate the ability of L-4-nitrophenylalanine to serve as a sensitive IR probe of local protein environments in the 247 residue superfolder green fluorescent protein (sfGFP). Specifically, the nitro sym. stretching frequency of L-4-nitrophenylalanine was shown to be sensitive to both solvents that mimic different protein environments and 15N isotopic labeling of the three-atom nitro group of this UAA. 14NO2 and 15NO2 variants of this UAA were incorporated utilizing an engineered orthogonal aminoacyl-tRNA synthetase/tRNA pair into a solvent exposed and a partially buried position in sfGFP with high efficiency and fidelity. The combination of isotopic labeling and difference FTIR spectroscopy permitted the nitro sym. stretching frequency of L-4-nitrophenylalanine to be exptl. measured at either site in sfGFP. The 14NO2 sym. stretching frequency red-shifted 7.7 cm-1 between the solvent exposed and partially buried position, thus illustrating the ability of this UAA to serve as an effective IR probe of local protein environments.
- 206Kooter, I. M.; Moguilevsky, N.; Bollen, A.; van der Veen, L. A.; Otto, C.; Dekker, H. L.; Wever, R. The Sulfonium Ion Linkage in Myeloperoxidase: direct spectroscopic detection by isotopic labeling and effect of mutation. J. Biol. Chem. 1999, 274 (38), 26794– 26802, DOI: 10.1074/jbc.274.38.26794There is no corresponding record for this reference.
- 207Thielges, M. C.; Case, D. A.; Romesberg, F. E. Carbon-Deuterium Bonds as Probes of Dihydrofolate Reductase. J. Am. Chem. Soc. 2008, 130 (20), 6597– 6603, DOI: 10.1021/ja0779607207Carbon-Deuterium Bonds as Probes of Dihydrofolate ReductaseThielges, Megan C.; Case, David A.; Romesberg, Floyd E.Journal of the American Chemical Society (2008), 130 (20), 6597-6603CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Much effort has been directed toward understanding the contributions of electrostatics and dynamics to protein function and esp. to enzyme catalysis. Unfortunately, these studies have been limited by the absence of direct exptl. probes. We have been developing the use of carbon-deuterium bonds as probes of proteins and now report the application of the technique to the enzyme dihydrofolate reductase, which catalyzes a hydride transfer and has served as a paradigm for biol. catalysis. We observe that the stretching absorption frequency of (methyl-d3) methionine carbon-deuterium bonds shows an approx. linear dependence on solvent dielec. Solvent and computational studies support the empirical interpretation of the stretching frequency in terms of local polarity. To begin to explore the use of this technique to study enzyme function and mechanism, we report a preliminary anal. of (methyl-d3) methionine residues within dihydrofolate reductase. Specifically, we characterize the IR absorptions at Met16 and Met20, within the catalytically important Met20 loop, and Met42, which is located within the hydrophobic core of the enzyme. The results confirm the sensitivity of the carbon-deuterium bonds to their local protein environment, demonstrate that dihydrofolate reductase is electrostatically and dynamically heterogeneous, and lay the foundation for the direct characterization protein electrostatics and dynamics and, potentially, their contribution to catalysis.
- 208Le Sueur, A. L.; Schaugaard, R. N.; Baik, M.-H.; Thielges, M. C. Methionine Ligand Interaction in a Blue Copper Protein Characterized by Site-Selective Infrared Spectroscopy. J. Am. Chem. Soc. 2016, 138 (22), 7187– 7193, DOI: 10.1021/jacs.6b03916208Methionine Ligand Interaction in a Blue Copper Protein Characterized by Site-Selective Infrared SpectroscopyLe Sueur, Amanda L.; Schaugaard, Richard N.; Baik, Mu-Hyun; Thielges, Megan C.Journal of the American Chemical Society (2016), 138 (22), 7187-7193CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The reactivity of metal sites in proteins is tuned by protein-based ligands. For example, in blue copper proteins such as plastocyanin (Pc), the structure imparts a highly elongated bond between the Cu and a methionine (Met) axial ligand to modulate its redox properties. Despite extensive study, a complete understanding of the contribution of the protein to redox activity is challenged by exptl. accessing both redox states of metalloproteins. Using IR spectroscopy in combination with site-selective labeling with carbon-deuterium (C-D) vibrational probes, we characterized the localized changes at the Cu ligand Met97 in the oxidized and reduced states, as well as the Zn(II) or Co(II)-substituted, the pH-induced low-coordinate, the apoprotein, and the unfolded states. The IR absorptions of (d3-methyl)Met97 are highly sensitive to interaction of the sulfur-based orbitals with the metal center and are demonstrated to be useful reporters of its modulation in the different states. Unrestricted Kohn-Sham d. functional theory calcns. performed on a model of the Cu site of Pc confirm the obsd. dependence. IR spectroscopy was then applied to characterize the impact of binding to the physiol. redox partner cytochrome (cyt) f. The spectral changes suggest a slightly stronger Cu-S(Met97) interaction in the complex with cyt f that has potential to modulate the electron transfer properties. Besides providing direct, mol.-level comparison of the oxidized and reduced states of Pc from the perspective of the axial Met ligand and evidence for perturbation of the Cu site properties by redox partner binding, this study demonstrates the localized spatial information afforded by IR spectroscopy of selectively incorporated C-D probes.
- 209Horness, R. E.; Basom, E. J.; Thielges, M. C. Site-Selective Characterization of Src Homology 3 Domain Molecular Recognition with Cyanophenylalanine Infrared Probes. Anal. Methods 2015, 7 (17), 7234– 7241, DOI: 10.1039/C5AY00523J209Site-selective characterization of Src homology 3 domain molecular recognition with cyanophenylalanine infrared probesHorness, Rachel E.; Basom, Edward J.; Thielges, Megan C.Analytical Methods (2015), 7 (17), 7234-7241CODEN: AMNEGX; ISSN:1759-9679. (Royal Society of Chemistry)Local heterogeneity of microenvironments in proteins is important in biol. function, but difficult to characterize exptl. One approach is the combination of IR spectroscopy and site-selective incorporation of probe moieties with spectrally resolved IR absorptions that enable characterization within inherently congested protein IR spectra. We employed this method to study mol. recognition of a Src homol. 3 (SH3) domain from the yeast protein Sho1 for a peptide contg. the proline-rich recognition sequence of its physiol. binding partner Pbs2. Nitrile IR probes were introduced at four distinct sites in the protein by selective incorporation of p-cyanophenylalanine via the amber codon suppressor method and characterized by IR spectroscopy. Variation among the IR absorption bands reports on heterogeneity in local residue environments dictated by the protein structure, as well as on residue-dependent changes upon peptide binding. The study informs on the mol. recognition of SH3Sho1 and illustrates the speed and simplicity of this approach for characterization of select microenvironments within proteins.
- 210Basom, E. J.; Maj, M.; Cho, M.; Thielges, M. C. Site-Specific Characterization of Cytochrome P450cam Conformations by Infrared Spectroscopy. Anal. Chem. 2016, 88 (12), 6598– 6606, DOI: 10.1021/acs.analchem.6b01520There is no corresponding record for this reference.
- 211Bagchi, S.; Fried, S. D.; Boxer, S. G. A Solvatochromic Model Calibrates Nitriles’ Vibrational Frequencies to Electrostatic Fields. J. Am. Chem. Soc. 2012, 134 (25), 10373– 10376, DOI: 10.1021/ja303895k211A Solvatochromic Model Calibrates Nitriles' Vibrational Frequencies to Electrostatic FieldsBagchi, Sayan; Fried, Stephen D.; Boxer, Steven G.Journal of the American Chemical Society (2012), 134 (25), 10373-10376CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Electrostatic interactions provide a primary connection between a protein's three-dimensional structure and its function. IR probes are useful because vibrational frequencies of certain chem. groups, such as nitriles, are linearly sensitive to local electrostatic field and can serve as a mol. elec. field meter. IR spectroscopy has been used to study electrostatic changes or fluctuations in proteins, but measured peak frequencies have not been previously mapped to total elec. fields, because of the absence of a field-frequency calibration and the complication of local chem. effects such as H-bonds. The authors report a solvatochromic model that provides a means to assess the H-bonding status of arom. nitrile vibrational probes and calibrates their vibrational frequencies to electrostatic field. The anal. involves correlations between the nitrile's IR frequency and its 13C chem. shift, whose observation is facilitated by a robust method for introducing isotopes into arom. nitriles. The method is tested on the model protein RNase S contg. a labeled p-CN-Phe near the active site. Comparison of the measurements in RNase S against solvatochromic data gives an est. of the av. total electrostatic field at this location. The value detd. agrees quant. with mol. dynamics simulations, suggesting broader potential for the use of IR probes in the study of protein electrostatics.
- 212Wang, L.; Zhang, J.; Han, M.-J.; Zhang, L.; Chen, C.; Huang, A.; Xie, R.; Wang, G.; Zhu, J.; Wang, Y. A Genetically Encoded Two-Dimensional Infrared Probe for Enzyme Active-Site Dynamics. Angew. Chem. Int. Ed. 2021, 60 (20), 11143– 11147, DOI: 10.1002/anie.202016880There is no corresponding record for this reference.
- 213Huguenin-Dezot, N.; Alonzo, D. A.; Heberlig, G. W.; Mahesh, M.; Nguyen, D. P.; Dornan, M. H.; Boddy, C. N.; Schmeing, T. M.; Chin, J. W. Trapping Biosynthetic Acyl-Enzyme Intermediates with Encoded 2,3-Diaminopropionic Acid. Nature 2019, 565 (7737), 112– 117, DOI: 10.1038/s41586-018-0781-z213Trapping biosynthetic acyl-enzyme intermediates with encoded 2,3-diaminopropionic acidHuguenin-Dezot, Nicolas; Alonzo, Diego A.; Heberlig, Graham W.; Mahesh, Mohan; Nguyen, Duy P.; Dornan, Mark H.; Boddy, Christopher N.; Schmeing, T. Martin; Chin, Jason W.Nature (London, United Kingdom) (2019), 565 (7737), 112-117CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Many enzymes catalyze reactions that proceed through covalent acyl-enzyme (ester or thioester) intermediates. These enzymes include serine hydrolases (encoded by one per cent of human genes, and including serine proteases and thioesterases), cysteine proteases (including caspases), and many components of the ubiquitination machinery. Their important acyl-enzyme intermediates are unstable, commonly having half-lives of minutes to hours. In some cases, acyl-enzyme complexes can be stabilized using substrate analogs or active-site mutations but, although these approaches can provide valuable insight, they often result in complexes that are substantially non-native. Here we develop a strategy for incorporating 2,3-diaminopropionic acid (DAP) into recombinant proteins, via expansion of the genetic code. We show that replacing catalytic cysteine or serine residues of enzymes with DAP permits their first-step reaction with native substrates, allowing the efficient capture of acyl-enzyme complexes that are linked through a stable amide bond. For one of these enzymes, the thioesterase (TE) domain of valinomycin synthetase (Vlm), we elucidate the biosynthetic pathway by which it progressively oligomerizes tetradepsipeptidyl substrates to a dodecadepsipeptidyl intermediate, which it then cyclizes to produce valinomycin. By trapping the first and last acyl-thioesterase intermediates in the catalytic cycle as DAP conjugates, we provide structural insight into how conformational changes in thioesterase domains of such nonribosomal peptide synthetases (NRPSs) control the oligomerization and cyclization of linear substrates. The encoding of DAP will facilitate the characterization of diverse acyl-enzyme complexes, and may be extended to capturing the native substrates of transiently acylated proteins of unknown function.
- 214Tang, S.; Beattie, A. T.; Kafkova, L.; Petris, G.; Huguenin-Dezot, N.; Fiedler, M.; Freeman, M.; Chin, J. W. Mechanism-Based Traps Enable Protease and Hydrolase Substrate Discovery. Nature 2022, 602 (7898), 701– 707, DOI: 10.1038/s41586-022-04414-9214Mechanism-based traps enable protease and hydrolase substrate discoveryTang, Shan; Beattie, Adam T.; Kafkova, Lucie; Petris, Gianluca; Huguenin-Dezot, Nicolas; Fiedler, Marc; Freeman, Matthew; Chin, Jason W.Nature (London, United Kingdom) (2022), 602 (7898), 701-707CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)Hydrolase enzymes, including proteases, are encoded by 2-3% of the genes in the human genome and 14% of these enzymes are active drug targets1. However, the activities and substrate specificities of many proteases-esp. those embedded in membranes-and other hydrolases remain unknown. Here we report a strategy for creating mechanism-based, light-activated protease and hydrolase substrate traps in complex mixts. and live mammalian cells. The traps capture substrates of hydrolases, which normally use a serine or cysteine nucleophile. Replacing the catalytic nucleophile with genetically encoded 2,3-diaminopropionic acid allows the first step reaction to form an acyl-enzyme intermediate in which a substrate fragment is covalently linked to the enzyme through a stable amide bond2; this enables stringent purifn. and identification of substrates. We identify new substrates for proteases, including an intramembrane mammalian rhomboid protease RHBDL4 (refs. 3,4). We demonstrate that RHBDL4 can shed luminal fragments of endoplasmic reticulum-resident type I transmembrane proteins to the extracellular space, as well as promoting non-canonical secretion of endogenous sol. endoplasmic reticulum-resident chaperones. We also discover that the putative serine hydrolase retinoblastoma binding protein 9 (ref. 5) is an aminopeptidase with a preference for removing arom. amino acids in human cells. Our results exemplify a powerful paradigm for identifying the substrates and activities of hydrolase enzymes.
- 215Dorman, G.; Prestwich, G. D. Benzophenone Photophores in Biochemistry. Biochemistry 1994, 33 (19), 5661– 5673, DOI: 10.1021/bi00185a001215Benzophenone Photophores in BiochemistryDorman, Gyorgy; Prestwich, Glenn D.Biochemistry (1994), 33 (19), 5661-73CODEN: BICHAW; ISSN:0006-2960.A review, with ∼85 refs. The photoactivatable aryl ketone derivs. have been rediscovered as biochem. probes in the last 5 yr. The expanding use of benzophenone (BP) photoprobes can be attributed to three distinct chem. and biochem. advantages. First, BPs are chem. more stable than diazo esters, aryl azides, and diazirines. Second, BPs can be manipulated in ambient light and can be activated at 350-360 nm, avoiding protein-damaging wavelengths. Third, BPs react preferentially with unreactive C-H bonds, even in the presence of solvent water and bulk nucleophiles. These three properties combine to produce highly efficient covalent modifications of macromols., frequently with remarkable site specificity. This perspective includes a brief review of BP photochem. and a selection of specific applications of these photoprobes, which address questions in protein, nucleic acid, and lipid biochem.
- 216Chin, J. W.; Martin, A. B.; King, D. S.; Wang, L.; Schultz, P. G. Addition of a Photocrosslinking Amino Acid to the Genetic Code of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (17), 11020– 11024, DOI: 10.1073/pnas.172226299216Addition of a photocrosslinking amino acid to the genetic code of Escherichia coliChin, Jason W.; Martin, Andrew B.; King, David S.; Wang, Lei; Schultz, Peter G.Proceedings of the National Academy of Sciences of the United States of America (2002), 99 (17), 11020-11024CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Benzophenones are among the most useful photocrosslinking agents in biol. We have evolved an orthogonal aminoacyl-tRNA synthetase/tRNA pair that makes possible the in vivo incorporation of p-benzoyl-L-phenylalanine into proteins in Escherichia coli in response to the amber codon, TAG. This unnatural amino acid was incorporated with high translational efficiency and fidelity into the dimeric protein glutathione S-transferase. Irradn. resulted in efficient crosslinking (>50%) of the protein subunits. This methodol. may prove useful for discovering and defining protein interactions in vitro and in vivo.
- 217Farrell, I. S.; Toroney, R.; Hazen, J. L.; Mehl, R. A.; Chin, J. W. Photo-Cross-Linking Interacting Proteins with a Genetically Encoded Benzophenone. Nat. Methods 2005, 2 (5), 377– 384, DOI: 10.1038/nmeth0505-377217Photo-cross-linking interacting proteins with a genetically encoded benzophenoneFarrell, Ian S.; Toroney, Rebecca; Hazen, Jennifer L.; Mehl, Ryan A.; Chin, Jason W.Nature Methods (2005), 2 (5), 377-384CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A major challenge in understanding the networks of interactions that control cell and organism function is the definition of protein interactions. Solid-phase peptide synthesis has allowed the photo-crosslinkable amino acid p-benzoyl-L-phenylalanine to be site-specifically incorporated into peptide chains, to facilitate the definition of peptide-ligand complexes. The method, however, is limited to the in vitro study of peptides and small proteins. An innovative develpoment allows the incorporation of a site-specific photo-cross-linker into virtually any protein that can be expressed in Escherichia coli, thereby promoting in vivo or in vitro crosslinking of proteins. The method relies on an orthogonal aminoacyl tRNA synthetase-tRnACUA pair that incorporates pBpa at the position encoded by the amber codon (UAG) in any gene transformed into E. coli. The system described in this protocol uses two plasmids: a p15A-based plasmid to express the orthogonal tRNA and synthetase pair (pDULE) and a second plasmid contg. an amber mutant of the gene of interest. To produce the photo-cross-linker-contg. protein, cultures of E. coli carrying both plasmids are grown in the presence of the unnatural amino acid. To photo-cross-link the protein to its binding partner in vivo or in vitro, cells or purified proteins, resp., are exposed to UV light.
- 218Chin, J. W.; Schultz, P. G. In Vivo Photocrosslinking with Unnatural Amino Acid Mutagenesis. ChemBioChem 2002, 3 (11), 1135– 1137, DOI: 10.1002/1439-7633(20021104)3:11<1135::AID-CBIC1135>3.0.CO;2-M218In vivo photocrosslinking with unnatural amino acid mutagenesisChin, Jason W.; Schultz, Peter G.ChemBioChem (2002), 3 (11), 1135-1137CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)A method for the characterization of protein interactions in vivo by photocrosslinking with unnatural amino acid mutagenesis was developed. The method involves the replacement of a single amino acid in a protein with amino acid p-benzoyl-L-phenylalanine (pβpa) in vivo; irradn. of the cell with near UV-light to crosslink proteins proximal to the surface of the pβpa- contg. protein; cell lysis, purifn., and identification of the complex or complexes formed.
- 219Mori, H.; Ito, K. Different Modes of SecY-SecA Interactions Revealed by Site-Directed in vivo Photo-Cross-Linking. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (44), 16159– 16164, DOI: 10.1073/pnas.0606390103There is no corresponding record for this reference.
- 220Majmudar, C. Y.; Lee, L. W.; Lancia, J. K.; Nwokoye, A.; Wang, Q.; Wands, A. M.; Wang, L.; Mapp, A. K. Impact of Nonnatural Amino Acid Mutagenesis on the in Vivo Function and Binding Modes of a Transcriptional Activator. J. Am. Chem. Soc. 2009, 131 (40), 14240– 14242, DOI: 10.1021/ja904378z220Impact of Nonnatural Amino Acid Mutagenesis on the in Vivo Function and Binding Modes of a Transcriptional ActivatorMajmudar, Chinmay Y.; Lee, Lori W.; Lancia, Jody K.; Nwokoye, Adaora; Wang, Qian; Wands, Amberlyn M.; Wang, Lei; Mapp, Anna K.Journal of the American Chemical Society (2009), 131 (40), 14240-14242CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein-protein interactions play an essential role in cellular function, and methods to discover and characterize them in their native context are of paramount importance for gaining a deeper understanding of biol. networks. In this study, an enhanced nonsense suppression system was utilized to incorporate the non-natural amino acid p-benzoyl-L-phenylalanine (pBpa) throughout the transcriptional activation domain of the prototypical eukaryotic transcriptional activator Gal4 in vivo (Saccharomyces cerevisiae). Functional studies of the pBpa-contg. Gal4 mutants suggest that this essential binding interface of Gal4 is minimally impacted by these substitutions, with both transcriptional activity and sensitivity to growth conditions maintained. Further supporting this are in vivo crosslinking studies, including the detection of a key binding partner of Gal4, the inhibitor protein Gal80. Crosslinking with a range of pBpa-contg. mutants revealed a Gal4·Gal80 binding interface that extends beyond that previously predicted by conventional strategies. Thus, this approach can be broadened to the discovery of novel binding partners of transcription factors, information that will be crit. for the development of therapeutically useful small mol. modulators of these protein-protein interactions.
- 221Liu, C.; Young, A. L.; Starling-Windhof, A.; Bracher, A.; Saschenbrecker, S.; Rao, B. V.; Rao, K. V.; Berninghausen, O.; Mielke, T.; Hartl, F. U. Coupled Chaperone Action in Folding and Assembly of Hexadecameric Rubisco. Nature 2010, 463 (7278), 197– 202, DOI: 10.1038/nature08651There is no corresponding record for this reference.
- 222Ye, Z.; Bair, M.; Desai, H.; Williams, G. J. A Photocrosslinking Assay for Reporting Protein Interactions in Polyketide and Fatty Acid Synthases. Mol. BioSyst. 2011, 7 (11), 3152– 3156, DOI: 10.1039/c1mb05270eThere is no corresponding record for this reference.
- 223Ye, Z.; Williams, G. J. Mapping a Ketosynthase:Acyl Carrier Protein Binding Interface via Unnatural Amino Acid-Mediated Photo-Cross-Linking. Biochemistry 2014, 53 (48), 7494– 7502, DOI: 10.1021/bi500936u223Mapping a ketosynthase:acyl carrier protein binding interface via unnatural amino acid-mediated Photo-Cross-LinkingYe, Zhixia; Williams, Gavin J.Biochemistry (2014), 53 (48), 7494-7502CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Probing and interrogating protein interactions that involve acyl carrier proteins (ACP's) in fatty acid synthases and polyketide synthases are crit. to understanding the mol. basis for the programmed assembly of complex natural products. Here, we have used unnatural amino acid mutagenesis to site specifically install photo-crosslinking functionality into acyl carrier proteins from diverse systems and the ketosynthase FabF from the Escherichia coli type II fatty acid synthase. Subsequently, a photo-crosslinking assay was employed to systematically probe the ability of FabF to interact with a broad panel of ACP's, illustrating the expected orthogonality of ACP:FabF interactions and the role of charged residues in helix II of the ACP. In addn., FabF residues involved in the binding interaction with the cognate carrier protein were identified via surface scanning mutagenesis and photo-crosslinking. Furthermore, the ability to install the photo-crosslinking amino acid at virtually any position allowed interrogation of the role that carrier protein acylation plays in detg. the binding interface with FabF. A conserved carrier protein motif that includes the phosphopantetheinylation site was also shown to play an integral role in maintenance of the AcpP:FabF binding interaction. Our results provide unprecedented insight into the mol. details that describe the AcpP:FabF binding interface and demonstrate that unnatural amino acid based photo-crosslinking is a powerful tool for probing and interrogating protein interactions in complex biosynthetic systems.
- 224Pavic, K.; Rios, P.; Dzeyk, K.; Koehler, C.; Lemke, E. A.; Köhn, M. Unnatural Amino Acid Mutagenesis Reveals Dimerization As a Negative Regulatory Mechanism of VHR’s Phosphatase Activity. ACS Chem. Biol. 2014, 9 (7), 1451– 1459, DOI: 10.1021/cb500240n224Unnatural Amino Acid Mutagenesis Reveals Dimerization As a Negative Regulatory Mechanism of VHR's Phosphatase ActivityPavic, Karolina; Rios, Pablo; Dzeyk, Kristina; Koehler, Christine; Lemke, Edward A.; Koehn, MajaACS Chemical Biology (2014), 9 (7), 1451-1459CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Vaccinia H1-related (VHR) phosphatase (also known as DUSP3) is a dual specificity phosphatase that is required for cell-cycle progression and plays a role in cell growth of certain cancers. Therefore, it represents a potential drug target. VHR is structurally and biochem. well characterized, yet its regulatory principles are still poorly understood. Understanding its regulation is important, not only to comprehend VHR's biol. mechanisms and roles but also to det. its potential and druggability as a target in cancer. Here, we investigated the functional role of the unique "variable insert" region in VHR by selectively introducing the photo-cross-linkable amino acid para-benzoylphenylalanine (pBPA) using the amber suppression method. This approach led to the discovery of VHR dimerization, which was further confirmed using traditional chem. cross-linkers. Phe68 in VHR was discovered as a residue involved in the dimerization. We demonstrate that VHR can dimerize inside cells, and that VHR catalytic activity is reduced upon dimerization. Our results suggest that dimerization could occlude the active site of VHR, thereby blocking its accessibility to substrates. These findings indicate that the previously unknown transient self-assocn. of VHR acts as a means for the neg. regulation of its catalytic activity.
- 225Morozov, Y. I.; Agaronyan, K.; Cheung, A. C. M.; Anikin, M.; Cramer, P.; Temiakov, D. A Novel Intermediate in Transcription Initiation by Human Mitochondrial RNA Polymerase. Nucleic Acids Res. 2014, 42 (6), 3884– 3893, DOI: 10.1093/nar/gkt1356There is no corresponding record for this reference.
- 226Wong, H. E.; Kwon, I. Effects of Non-Natural Amino Acid Incorporation into the Enzyme Core Region on Enzyme Structure and Function. Int. J. Mol. Sci. 2015, 16 (9), 22735– 22753, DOI: 10.3390/ijms160922735226Effects of non-natural amino acid incorporation into the enzyme core region on enzyme structure and functionWong, H. Edward; Kwon, InchanInternational Journal of Molecular Sciences (2015), 16 (9), 22735-22753CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)Techniques to incorporate non-natural amino acids (NNAAs) have enabled biosynthesis of proteins contg. new building blocks with unique structures, chem., and reactivity that are not found in natural amino acids. It is crucial to understand how incorporation of NNAAs affects protein function because NNAA incorporation may perturb crit. function of a target protein. This study investigated how the site-specific incorporation of NNAAs affects the catalytic properties of an enzyme. L-3-(2-Naphthyl)alanine (2Nal), a NNAA with a hydrophobic and bulky side-chain, , was site-specifically incorporated at 6 different positions in the hydrophobic core of a model enzyme, murine dihydrofolate reductase (mDHFR). The mDHFR variants with a greater change in van der Waals vol. upon 2Nal incorporation exhibited a greater redn. in catalytic efficiency. Similarly, the steric incompatibility calcd. using RosettaDesign, a protein stability calcn. program, correlated with the changes in catalytic efficiency.
- 227Rappaport, F.; Boussac, A.; Force, D. A.; Peloquin, J.; Brynda, M.; Sugiura, M.; Un, S.; Britt, R. D.; Diner, B. A. Probing the Coupling between Proton and Electron Transfer in Photosystem II Core Complexes Containing a 3-Fluorotyrosine. J. Am. Chem. Soc. 2009, 131 (12), 4425– 4433, DOI: 10.1021/ja808604h227Probing the coupling between proton and electron transfer in photosystem II core complexes containing a 3-fluorotyrosineRappaport, Fabrice; Boussac, Alain; Force, Dee Ann; Peloquin, Jeffrey; Brynda, Marcin; Sugiura, Miwa; Un, Sun; Britt, R. David; Diner, Bruce A.Journal of the American Chemical Society (2009), 131 (12), 4425-4433CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The catalytic cycle of numerous enzymes involves the coupling between proton transfer and electron transfer. Yet, the understanding of this coordinated transfer in biol. systems remains limited, likely because its characterization relies on the controlled but exptl. challenging modifications of the free energy changes assocd. with either the electron or proton transfer. The authors have performed such a study here in photosystem II. The driving force for electron transfer from TyrZ to P680•+ has been decreased by ∼80 meV by mutating the axial ligand of P680, and that for proton transfer upon oxidn. of TyrZ by substituting a 3-fluorotyrosine (3F-TyrZ) for TyrZ. In Mn-depleted photosystem II, the dependence upon pH of the oxidn. rates of TyrZ and 3F-TyrZ were found to be similar. However, in the pH range where the phenolic hydroxyl of TyrZ is involved in a H-bond with a proton acceptor, the activation energy of the oxidn. of 3F-TyrZ is decreased by 110 meV, a value which correlates with the in vitro finding of a 90 meV stabilization energy to the phenolate form of 3F-Tyr when compared to Tyr. Thus, when the phenol of YZ acts as a H-bond donor, its oxidn. by P680•+ is controlled by its prior deprotonation. This contrasts with the situation prevailing at lower pH, where the proton acceptor is protonated and therefore unavailable, in which the oxidn.-induced proton transfer from the phenolic hydroxyl of TyrZ has been proposed to occur concertedly with the electron transfer to P680•+. This suggests a switch between a concerted proton/electron transfer at pHs < 7.5 to a sequential one at pHs > 7.5 and illustrates the roles of the H-bond and of the likely salt-bridge existing between the phenolate and the nearby proton acceptor in detg. the coupling between proton and electron transfer.
- 228Schlesinger, S.; Schlesinger, M. J. The Effect of Amino Acid Analogues on Alkaline Phosphatase Formation in Escherichia coli K-12: I. Substitution of Triazolealanine for Histidine. J. Biol. Chem. 1967, 242 (14), 3369– 3372, DOI: 10.1016/S0021-9258(18)95919-3There is no corresponding record for this reference.
- 229Fried, S. D.; Bagchi, S.; Boxer, S. G. Extreme Electric Fields Power Catalysis in the Active Site of Ketosteroid Isomerase. Science 2014, 346 (6216), 1510– 1514, DOI: 10.1126/science.1259802229Extreme electric fields power catalysis in the active site of ketosteroid isomeraseFried, Stephen D.; Bagchi, Sayan; Boxer, Steven G.Science (Washington, DC, United States) (2014), 346 (6216), 1510-1514CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Enzymes use protein architecture to impose specific electrostatic fields onto their bound substrates, but the magnitude and catalytic effect of these elec. fields have proven difficult to quantify with std. exptl. approaches. Here, using vibrational Stark effect spectroscopy, the authors found that the active site of ketosteroid isomerase (KSI) exerted an extremely large elec. field onto the C:O chem. bond that undergoes a charge rearrangement in the KSI rate-detg. step. Moreover, the authors found that the magnitude of the elec. field exerted by the active site strongly correlated with the enzyme's catalytic rate enhancement, enabling them to quantify the fraction of the catalytic effect that was electrostatic in origin. The measurements described here may help explain the role of electrostatics in many other enzymes and biomol. systems.
- 230Ortmayer, M.; Hardy, F. J.; Quesne, M. G.; Fisher, K.; Levy, C.; Heyes, D. J.; Catlow, C. R. A.; de Visser, S. P.; Rigby, S. E. J.; Hay, S.; Green, A. P. A Noncanonical Tryptophan Analogue Reveals an Active Site Hydrogen Bond Controlling Ferryl Reactivity in a Heme Peroxidase. JACS Au 2021, 1 (7), 913– 918, DOI: 10.1021/jacsau.1c00145There is no corresponding record for this reference.
- 231Kang, G.; Taguchi, A. T.; Stubbe, J.; Drennan, C. L. Structure of a Trapped Radical Transfer Pathway within a Ribonucleotide Reductase Holocomplex. Science 2020, 368 (6489), 424– 427, DOI: 10.1126/science.aba6794231Structure of a trapped radical transfer pathway within a ribonucleotide reductase holocomplexKang, Gyunghoon; Taguchi, Alexander T.; Stubbe, JoAnne; Drennan, Catherine L.Science (Washington, DC, United States) (2020), 368 (6489), 424-427CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Ribonucleotide reductases (RNRs) are a diverse family of enzymes that are alone capable of generating 2'-deoxynucleotides de novo and are thus crit. in DNA biosynthesis and repair. The nucleotide redn. reaction in all RNRs requires the generation of a transient active site thiyl radical, and in class I RNRs, this process involves a long-range radical transfer between two subunits, α and β. Because of the transient subunit assocn., an at. resoln. structure of an active α2β2 RNR complex has been elusive. We used a doubly substituted β2, E52Q/(2,3,5)-trifluorotyrosine122-β2, to trap wild-type α2 in a long-lived α2β2 complex. We report the structure of this complex by means of cryo-electron microscopy to 3.6-angstrom resoln., allowing for structural visualization of a 32-angstrom-long radical transfer pathway that affords RNR activity.
- 232Beiboer, S. H. W.; Berg, B. v. d.; Dekker, N.; Cox, R. C.; Verheij, H. M. Incorporation of an Unnatural Amino Acid in the Active Site of Porcine Pancreatic Phospholipase A2. Substitution of Histidine by l,2,4-Triazole-3-Alanine Yields an Enzyme with High Activity at Acidic pH. Protein Eng. Des. Sel. 1996, 9 (4), 345– 352, DOI: 10.1093/protein/9.4.345There is no corresponding record for this reference.
- 233Soumillion, P.; Fastrez, J. Incorporation of 1,2,4-Triazole-3-Alanine into a Mutant of Phage Lambda Lysozyme Containing a Single Histidine. Protein Eng. Des. Sel. 1998, 11 (3), 213– 217, DOI: 10.1093/protein/11.3.213There is no corresponding record for this reference.
- 234Blatter, N.; Prokup, A.; Deiters, A.; Marx, A. Modulating the pKa of a Tyrosine in KlenTaq DNA Polymerase that Is Crucial for Abasic Site Bypass by in Vivo Incorporation of a Non-canonical Amino Acid. Chem Bio Chem 2014, 15, 1735– 1737, DOI: 10.1002/cbic.201400051There is no corresponding record for this reference.
- 235Obeid, S.; Blatter, N.; Kranaster, R.; Schnur, A.; Diederichs, K.; Welte, W.; Marx, A. Replication Through an Abasic DNA Lesion: Structural Basis for Adenine Selectivity. EMBO J. 2010, 29 (10), 1738, DOI: 10.1038/emboj.2010.64There is no corresponding record for this reference.
- 236Greene, B. L.; Kang, G.; Cui, C.; Bennati, M.; Nocera, D. G.; Drennan, C. L.; Stubbe, J. Ribonucleotide Reductases: Structure, Chemistry, and Metabolism Suggest New Therapeutic Targets. Annu. Rev. Biochem. 2020, 89 (1), 45– 75, DOI: 10.1146/annurev-biochem-013118-111843236Ribonucleotide Reductases: Structure, Chemistry, and Metabolism Suggest New Therapeutic TargetsGreene, Brandon L.; Kang, Gyunghoon; Cui, Chang; Bennati, Marina; Nocera, Daniel G.; Drennan, Catherine L.; Stubbe, JoAnneAnnual Review of Biochemistry (2020), 89 (), 45-75CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)A review. Ribonucleotide reductases (RNRs) catalyze the de novo conversion of nucleotides to deoxynucleotides in all organisms, controlling their relative ratios and abundance. In doing so, they play an important role in fidelity of DNA replication and repair. RNRs' central role in nucleic acid metab. has resulted in five therapeutics that inhibit human RNRs. In this review, we discuss the structural, dynamic, and mechanistic aspects of RNR activity and regulation, primarily for the human and Escherichia coli class Ia enzymes. The unusual radical-based org. chem. of nucleotide redn., the inorg. chem. of the essential metallo-cofactor biosynthesis/maintenance, the transport of a radical over a long distance, and the dynamics of subunit interactions all present distinct entry points toward RNR inhibition that are relevant for drug discovery. We describe the current mechanistic understanding of small mols. that target different elements of RNR function, including downstream pathways that lead to cell cytotoxicity. We conclude by summarizing novel and emergent RNR targeting motifs for cancer and antibiotic therapeutics.
- 237Uhlin, U.; Eklund, H. Structure of Ribonucleotide Reductase Protein R1. Nature 1994, 370 (6490), 533– 539, DOI: 10.1038/370533a0237Structure of ribonucleotide reductase protein R1Uhlin, Ulla; Eklund, HansNature (London, United Kingdom) (1994), 370 (6490), 533-9CODEN: NATUAS; ISSN:0028-0836.The crystal structure ribonucleotide reductase (I) subunit R1 (in complex with subunit R2) at 2.5 Å is reported. The 3-dimensional structure of the R2 subunit was previously reported and refined at 2.2 Å. The R2 tyrosyl radical-based I reaction involves 5 cysteines. Two redox-active R1 cysteines (Cys-225 and Cys-462) are located at adjacent antiparallel strands in a new type of 10-stranded α/β-barrel, and 2 others (Cys-754 and Cys-759) at the C-terminal end in a flexible arm. The 5th cysteine (Cys-439), in a loop in the center of the barrel, is positioned to initiate the radical reaction.
- 238Reece, S. Y.; Seyedsayamdost, M. R.; Stubbe, J.; Nocera, D. G. Electron Transfer Reactions of Fluorotyrosyl Radicals. J. Am. Chem. Soc. 2006, 128 (42), 13654– 13655, DOI: 10.1021/ja0636688238Electron transfer reactions of fluorotyrosyl radicalsReece, Steven Y.; Seyedsayamdost, Mohammad R.; Stubbe, JoAnne; Nocera, Daniel G.Journal of the American Chemical Society (2006), 128 (42), 13654-13655CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The complex Re(bpy)(CO)3CN is an excited state oxidant of tyrosine upon deprotonation of the tyrosyl phenol. Re(bpy-FnY)(CO)3CN complexes ([Re]-FnY: [Re]-Y, [Re]-3-FY, [Re]-3,5-F2Y, [Re]-2,3-F2Y, [Re]-2,3,5-F3Y, [Re]-2,3,6-F3Y, and [Re]-F4Y) (FnY = Me tyrosinate and fluorotyrosinates) were prepd. so as to vary the FnY·/FnY- redn. potential and thus the driving force for electron transfer (ET) in this system. Time-resolved emission and nanosecond absorption spectroscopies were used to measure the rates for charge sepn. (CS), and charge recombination, CR, for each complex. A driving force anal. reveals that CS is well described by Marcus' theory for ET, is strongly driving force dependent (activated), and occurs in the normal region for ET. CR, however, is weakly driving force dependent (near activation-less) and occurs in the inverted region for ET. Fluorotyrosines will be powerful probes for unraveling charge transport mechanisms in enzymes that use tyrosyl radicals. An x-ray crystal structure detn. of Re(bpy)(CO)3CN·MeOH is also presented.
- 239Seyedsayamdost, M. R.; Yee, C. S.; Reece, S. Y.; Nocera, D. G.; Stubbe, J. pH Rate Profiles of FnY356-R2s (n = 2, 3, 4) in Escherichia coli Ribonucleotide Reductase: Evidence that Y356 Is a Redox-Active Amino Acid along the Radical Propagation Pathway. J. Am. Chem. Soc. 2006, 128 (5), 1562– 1568, DOI: 10.1021/ja055927j239pH Rate Profiles of FnY356-R2s (n = 2, 3, 4) in Escherichia coli Ribonucleotide Reductase: Evidence that Y356 Is a Redox-Active Amino Acid along the Radical Propagation PathwaySeyedsayamdost, Mohammad R.; Yee, Cyril S.; Reece, Steven Y.; Nocera, Daniel G.; Stubbe, JoAnneJournal of the American Chemical Society (2006), 128 (5), 1562-1568CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The Escherichia coli ribonucleotide reductase (RNR), composed of two subunits (R1 and R2), catalyzes the conversion of nucleotides to deoxynucleotides. Substrate redn. requires that a tyrosyl radical (Y122•) in R2 generate a transient cysteinyl radical (C439•) in R1 through a pathway thought to involve amino acid radical intermediates [Y122• → W48 → Y356 within R2 to Y731 → Y730 → C439 within R1]. To study this radical propagation process, the authors have synthesized R2 semisynthetically using intein technol. and replaced Y356 with a variety of fluorinated tyrosine analogs (2,3-F2Y, 3,5-F2Y, 2,3,5-F3Y, 2,3,6-F3Y, and F4Y) that have been described and characterized in the accompanying paper. These fluorinated tyrosine derivs. have potentials that vary from -50 to +270 mV relative to tyrosine over the accessible pH range for RNR and pKas that range from 5.6 to 7.8. The pH rate profiles of deoxynucleotide prodn. by these FnY356-R2s are reported. The results suggest that the rate-detg. step can be changed from a phys. step to the radical propagation step by altering the redn. potential of Y356• using these analogs. As the difference in potential of the FnY• relative to Y• becomes >80 mV, the activity of RNR becomes inhibited, and by 200 mV, RNR activity is no longer detectable. These studies support the model that Y356 is a redox-active amino acid on the radical-propagation pathway. On the basis of the authors' previous studies with 3-NO2Y356-R2, the authors assume that 2,3,5-F3Y356, 2,3,6-F3Y356, and F4Y356-R2s are all deprotonated at pH >7.5. The authors show that they all efficiently initiate nucleotide redn. If this assumption is correct, then a hydrogen-bonding pathway between W48 and Y356 of R2 and Y731 of R1 does not play a central role in triggering radical initiation nor is hydrogen-atom transfer between these residues obligatory for radical propagation.
- 240Seyedsayamdost, M. R.; Xie, J.; Chan, C. T. Y.; Schultz, P. G.; Stubbe, J. Site-Specific Insertion of 3-Aminotyrosine into Subunit α2 of E. coli Ribonucleotide Reductase: Direct Evidence for Involvement of Y730 and Y731 in Radical Propagation. J. Am. Chem. Soc. 2007, 129 (48), 15060– 15071, DOI: 10.1021/ja076043y240Site-Specific Insertion of 3-Aminotyrosine into Subunit α2 of E. coli Ribonucleotide Reductase: Direct Evidence for Involvement of Y730 and Y731 in Radical PropagationSeyedsayamdost, Mohammad R.; Xie, Jianming; Chan, Clement T. Y.; Schultz, Peter G.; Stubbe, JoAnneJournal of the American Chemical Society (2007), 129 (48), 15060-15071CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)E. coli ribonucleotide reductase (RNR) catalyzes the prodn. of deoxynucleotides using complex radical chem. Active RNR is composed of a 1:1 complex of two subunits: α2 and β2. α2 Binds nucleoside diphosphate substrates and deoxynucleotide/ATP allosteric effectors and is the site of nucleotide redn. β2 Contains the stable diiron tyrosyl radical (Y122·) cofactor that initiates deoxynucleotide formation. This process is proposed to involve reversible radical transfer over >35 Å between the Y122 in β2 and C439 in the active site of α2. A docking model of α2β2, based on structures of the individual subunits, suggests that radical initiation involves a pathway of transient, arom. amino acid radical intermediates, including Y730 and Y731 in α2. In this study the function of residues Y730 and Y731 is investigated by their site-specific replacement with 3-aminotyrosine (NH2Y). Using the in vivo suppressor tRNA/aminoacyl-tRNA synthetase method, Y730NH2Y-α2 and Y731NH2Y-α2 have been generated with high fidelity in yields of 4-6 mg/g of cell paste. These mutants have been examd. by stopped flow UV-vis and EPR spectroscopies in the presence of β2, CDP, and ATP. The results reveal formation of an NH2Y radical (NH2Y730· or NH2Y731·) in a kinetically competent fashion. Activity assays demonstrate that both NH2Y-α2s make deoxynucleotides. These results show that the NH2Y· can oxidize C439 suggesting a hydrogen atom transfer mechanism for the radical propagation pathway within α2. The obsd. NH2Y· may constitute the first detection of an amino acid radical intermediate in the proposed radical propagation pathway during turnover.
- 241Seyedsayamdost, M. R.; Chan, C. T. Y.; Mugnaini, V.; Stubbe, J.; Bennati, M. PELDOR Spectroscopy with DOPA-β2 and NH2Y-α2s: Distance Measurements between Residues Involved in the Radical Propagation Pathway of E. coli Ribonucleotide Reductase. J. Am. Chem. Soc. 2007, 129 (51), 15748– 15749, DOI: 10.1021/ja076459b241PELDOR Spectroscopy with DOPA-β2 and NH2Y-α2s: Distance Measurements between Residues Involved in the Radical Propagation Pathway of E. coli Ribonucleotide ReductaseSeyedsayamdost, Mohammad R.; Chan, Clement T. Y.; Mugnaini, Veronica; Stubbe, JoAnne; Bennati, MarinaJournal of the American Chemical Society (2007), 129 (51), 15748-15749CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Escherichia coli ribonucleotide reductase (RNR) catalyzes the redn. of nucleotides to 2'-deoxynucleotides. The active enzyme is a 1:1 complex of two homodimeric subunits, α2 and β2. The α2 is the site of nucleotide redn., and β2 harbors a diferric tyrosyl radical (Y122•) cofactor. Turnover requires formation of a cysteinyl radical (C439•) in the active site of α2 at the expense of the Y122• in β2. A docking model for the α2β2 interaction and a pathway for radical transfer from β2 to α2 have been proposed. This pathway contains three Ys: Y356 in β2 and Y731/Y730 in α2. We have previously incorporated 3-hydroxytyrosine and 3-aminotyrosine into these residues and showed that they act as radical traps. In this study, we use these α2/β2 variants and PELDOR spectroscopy to measure the distance between the Y122• in one αβ pair and the newly formed radical in the second αβ pair. The results yield distances that are similar to those predicted by the docking model for radical transfer. Further, they support a long-range radical initiation process for C439• generation and provide a structural constraint for residue Y356, which is thermally labile in all β2 structures solved to date.
- 242Seyedsayamdost, M. R.; Stubbe, J. Site-Specific Replacement of Y356 with 3,4-Dihydroxyphenylalanine in the β2 Subunit of E. coli Ribonucleotide Reductase. J. Am. Chem. Soc. 2006, 128 (8), 2522– 2523, DOI: 10.1021/ja057776q242Site-Specific Replacement of Y356 with 3,4-Dihydroxyphenylalanine in the β2 Subunit of E. coli Ribonucleotide ReductaseSeyedsayamdost, Mohammad R.; Stubbe, JoAnneJournal of the American Chemical Society (2006), 128 (8), 2522-2523CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)E. coli ribonucleotide reductase (RNR), composed of the homodimeric subunits α2 and β2, catalyzes the conversion of nucleotides to deoxynucleotides via complex radical chem. The radical initiation process involves a putative proton-coupled electron transfer (PCET) pathway over 35 Å between α2 and β2. Y356 in β2 has been proposed to lie on this pathway. To test this model, intein technol. has been used to make β2 semi-synthetically in which Y356 is replaced with a DOPA-amino acid. Anal. of this mutant with α2 and various combinations of substrate and effector by SF UV-vis spectroscopy and EPR methods demonstrates formation of a DOPA radical concomitant with disappearance of the tyrosyl radical, which initiates the reaction. The results reveal that Y356 lies on the PCET pathway and demonstrate the first kinetically competent conformational changes prior to ET. They further show that substrate binding brings about rapid conformational changes which place the complex into its active form(s) and suggest that the RNR complex is asym.
- 243Seyedsayamdost, M. R.; Stubbe, J. Forward and Reverse Electron Transfer with the Y356DOPA-β2 Heterodimer of E. coli Ribonucleotide Reductase. J. Am. Chem. Soc. 2007, 129 (8), 2226– 2227, DOI: 10.1021/ja0685607243Forward and Reverse Electron Transfer with the Y356DOPA-β2 Heterodimer of E. coli Ribonucleotide ReductaseSeyedsayamdost, Mohammad R.; Stubbe, JoAnneJournal of the American Chemical Society (2007), 129 (8), 2226-2227CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)E. coli ribonucleotide reductase catalyzes the conversion of nucleotides to deoxynucleotides, and consists of two subunits, α2 and β2. β2 Contains a stable diiron tyrosyl radical (Y122•) that is essential for catalysis. α2 harbors the active site, where nucleotide redn. occurs, as well as effector and activity sites which control substrate specificity and turnover rates. In this study, we have used intein methodol. to generate a heterodimer of β2 contg. the unnatural amino acid 3,4-dihydroxyphenylalanine (DOPA) at residue 356 (DOPA-ββ'). In this heterodimer, the β-monomer is full-length (residues 1-375), whereas the β'-monomer is truncated and only contains residues 1-353. DOPA-ββ', upon addn. of α2, CDP, and ATP effector, generates a DOPA• concomitant with loss of the Y122•. Anal. of DOPA• stability by EPR reveal that DOPA•-ββ' can reoxidize Y122 thereby regenerating the Y122•. These results, for the first time, directly demonstrate back electron transfer from residue 356 to Y122.
- 244Yokoyama, K.; Smith, A. A.; Corzilius, B.; Griffin, R. G.; Stubbe, J. Equilibration of Tyrosyl Radicals (Y356•, Y731•, Y730•) in the Radical Propagation Pathway of the Escherichia coli Class Ia Ribonucleotide Reductase. J. Am. Chem. Soc. 2011, 133 (45), 18420– 18432, DOI: 10.1021/ja207455k244Equilibration of Tyrosyl Radicals (Y356·, Y731·, Y730·) in the Radical Propagation Pathway of the Escherichia coli Class Ia Ribonucleotide ReductaseYokoyama, Kenichi; Smith, Albert A.; Corzilius, Bjorn; Griffin, Robert G.; Stubbe, JoAnneJournal of the American Chemical Society (2011), 133 (45), 18420-18432CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Escherichia coli ribonucleotide reductase (RNR) is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides using a diferric tyrosyl radical (Y122·) cofactor in β2 to initiate catalysis in α2. Each turnover requires reversible long-range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y122· ↹ [W48] ↹ Y356 within β to Y731 ↹ Y730 ↹ C439 within α). Previously, we reported that a β2 mutant with 3-nitrotyrosyl radical (NO2Y·; 1.2 radicals/β2) in place of Y122· in the presence of α2, CDP, and ATP catalyzes formation of 0.6 equiv of dCDP and accumulates 0.6 equiv of a new Y· proposed to be located on Y356 in β2. We now report three independent methods that establish that Y356 is the predominant location (85-90%) of the radical, with the remaining 10-15% delocalized onto Y731 and Y730 in α2. Pulsed electron-electron double-resonance spectroscopy on samples prepd. by rapid freeze quench (RFQ) methods identified three distances: 30 ± 0.4 Å (88% ± 3%) and 33 ± 0.4 and 38 ± 0.5 Å (12% ± 3%) indicative of NO2Y122·-Y356·, NO2Y122·-NO2Y122·, and NO2Y122·-Y731(730)·, resp. Radical distribution in α2 was supported by RFQ ESR (EPR) studies using Y731(3,5-F2Y) or Y730(3,5-F2Y)-α2, which revealed F2Y·, studies using globally incorporated [β-2H2]Y-α2, and anal. using parameters obtained from 140 GHz EPR spectroscopy. The amt. of Y· delocalized in α2 from these two studies varied from 6% to 15%. The studies together give the first insight into the relative redox potentials of the three transient Y· radicals in the PCET pathway and their conformations.
- 245Lin, C.-Y.; Muñoz Hernández, A. L.; Laremore, T. N.; Silakov, A.; Krebs, C.; Boal, A. K.; Bollinger, J. M., Jr Use of Noncanonical Tyrosine Analogues to Probe Control of Radical Intermediates during Endoperoxide Installation by Verruculogen Synthase (FtmOx1). ACS Catal. 2022, 12 (12), 6968– 6979, DOI: 10.1021/acscatal.2c01037There is no corresponding record for this reference.
- 246Taylor, A.; Heyes, D. J.; Scrutton, N. S. Catalysis by Nature’s photoenzymes. Curr. Opin. Struct. Biol. 2022, 77, 102491, DOI: 10.1016/j.sbi.2022.102491246Catalysis by Nature's photoenzymesTaylor, Aoife; Heyes, Derren J.; Scrutton, Nigel S.Current Opinion in Structural Biology (2022), 77 (), 102491CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. Photoenzymes use light to initiate biochem. reactions. Although rarely found in nature, their study has advanced understanding of how light energy can be harnessed to facilitate enzyme catalysis, which is also of importance to the design and engineering of man-made photocatalysts. Natural photoenzymes can be assigned to one of two families, based broadly on the nature of the light-sensing chromophores used, those being chlorophyll-like tetrapyrroles or flavins. In all cases, light absorption leads to excited state electron transfer, which in turn initiates photocatalysis. Reviewed here are recent findings relating to the structures and mechanisms of known photoenzymes. We highlight recent advances that have deepened understanding of mechanisms in biol. photocatalysis.
- 247Taylor, A.; Zhang, S.; Johannissen, L. O.; Sakuma, M.; Phillips, R. S.; Green, A. P.; Hay, S.; Heyes, D. J.; Scrutton, N. S. Mechanistic Implications of the Ternary Complex Structural Models for the Photoenzyme Protochlorophyllide Oxidoreductase. FEBS J. 2024, 291, 1404, DOI: 10.1111/febs.17025There is no corresponding record for this reference.
- 248Horowitz, S.; Adhikari, U.; Dirk, L. M. A.; Del Rizzo, P. A.; Mehl, R. A.; Houtz, R. L.; Al-Hashimi, H. M.; Scheiner, S.; Trievel, R. C. Manipulating Unconventional CH-Based Hydrogen Bonding in a Methyltransferase via Noncanonical Amino Acid Mutagenesis. ACS Chem. Biol. 2014, 9 (8), 1692– 1697, DOI: 10.1021/cb5001185248Manipulating Unconventional CH-Based Hydrogen Bonding in a Methyltransferase via Noncanonical Amino Acid MutagenesisHorowitz, Scott; Adhikari, Upendra; Dirk, Lynnette M. A.; Del Rizzo, Paul A.; Mehl, Ryan A.; Houtz, Robert L.; Al-Hashimi, Hashim M.; Scheiner, Steve; Trievel, Raymond C.ACS Chemical Biology (2014), 9 (8), 1692-1697CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Recent studies have demonstrated that the active sites of S-adenosylmethionine (AdoMet)-dependent methyltransferases form strong carbon-oxygen (CH···O) hydrogen bonds with the substrate's sulfonium group that are important in AdoMet binding and catalysis. To probe these interactions, we substituted the noncanonical amino acid p-aminophenylalanine (pAF) for the active site tyrosine in the lysine methyltransferase SET7/9, which forms multiple CH···O hydrogen bonds to AdoMet and is invariant in SET domain enzymes. Using quantum chem. calcns. to predict the mutation's effects, coupled with biochem. and structural studies, we obsd. that pAF forms a strong CH···N hydrogen bond to AdoMet that is offset by an energetically unfavorable amine group rotamer within the SET7/9 active site that hinders AdoMet binding and activity. Together, these results illustrate that the invariant tyrosine in SET domain methyltransferases functions as an essential hydrogen bonding hub and cannot be readily substituted by residues bearing other hydrogen bond acceptors.
- 249Kirsh, J. M.; Weaver, J. B.; Boxer, S. G.; Kozuch, J. Comprehensive Analysis of Nitrile Probe IR Shifts and Intensities in Proteins: Experiment and Critical Evaluation of Simulations. ChemRxiv 2024, DOI: 10.26434/chemrxiv-2023-j935v-v2There is no corresponding record for this reference.
- 250Weaver, J. B.; Kozuch, J.; Kirsh, J. M.; Boxer, S. G. Nitrile Infrared Intensities Characterize Electric Fields and Hydrogen Bonding in Protic, Aprotic, and Protein Environments. J. Am. Chem. Soc. 2022, 144 (17), 7562– 7567, DOI: 10.1021/jacs.2c00675250Nitrile Infrared Intensities Characterize Electric Fields and Hydrogen Bonding in Protic, Aprotic, and Protein EnvironmentsWeaver, Jared Bryce; Kozuch, Jacek; Kirsh, Jacob M.; Boxer, Steven G.Journal of the American Chemical Society (2022), 144 (17), 7562-7567CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Nitriles are widely used vibrational probes; however, the interpretation of their IR frequencies is complicated by hydrogen bonding (H-bonding) in protic environments. We report a new vibrational Stark effect (VSE) that correlates the elec. field projected on the -C≡N bond to the transition dipole moment and, by extension, the nitrile peak area or integrated intensity. This linear VSE applies to both H-bonding and non-H-bonding interactions. It can therefore be generally applied to det. elec. fields in all environments. Addnl., it allows for semiempirical extn. of the H-bonding contribution to the blueshift of the nitrile frequency. Nitriles were incorporated at H-bonding and non-H-bonding protein sites using amber suppression, and each nitrile variant was structurally characterized at high resoln. We exploited the combined information available from variations in frequency and integrated intensity and demonstrate that nitriles are a generally useful probe for elec. fields.
- 251Zheng, C.; Mao, Y.; Kozuch, J.; Atsango, A. O.; Ji, Z.; Markland, T. E.; Boxer, S. G. A Two-Directional Vibrational Probe Reveals Different Electric Field Orientations in Solution and an Enzyme Active Site. Nat. Chem. 2022, 14 (8), 891– 897, DOI: 10.1038/s41557-022-00937-w251A two-directional vibrational probe reveals different electric field orientations in solution and an enzyme active siteZheng, Chu; Mao, Yuezhi; Kozuch, Jacek; Atsango, Austin O.; Ji, Zhe; Markland, Thomas E.; Boxer, Steven G.Nature Chemistry (2022), 14 (8), 891-897CODEN: NCAHBB; ISSN:1755-4330. (Nature Portfolio)The catalytic power of an elec. field depends on its magnitude and orientation with respect to the reactive chem. species. Understanding and designing new catalysts for electrostatic catalysis thus requires methods to measure the elec. field orientation and magnitude at the mol. scale. Elec. field orientations can be extd. using a two-directional vibrational probe by exploiting the vibrational Stark effect of both the C:O and C-D stretches of a deuterated aldehyde. Combining spectroscopy with mol. dynamics and electronic structure partitioning methods, despite distinct polarities, solvents act similarly in their preference for electrostatically stabilizing large bond dipoles at the expense of destabilizing small ones. In contrast, for an active-site aldehyde inhibitor of liver alc. dehydrogenase, the elec. field orientation deviates markedly from that found in solvents, which provides direct evidence for the fundamental difference between the electrostatic environment of solvents and of a preorganized enzyme active site.
- 252Kedzierski, P.; Zaczkowska, M.; Sokalski, W. A. Extreme Catalytic Power of Ketosteroid Isomerase Related to the Reversal of Proton Dislocations in Hydrogen-Bond Network. J. Phys. Chem. B 2020, 124 (18), 3661– 3666, DOI: 10.1021/acs.jpcb.0c01489252Extreme catalytic power of ketosteroid isomerase related to the reversal of proton dislocations in hydrogen-bond networkKedzierski, Pawel; Zaczkowska, Maria; Sokalski, W. AndrzejJournal of Physical Chemistry B (2020), 124 (18), 3661-3666CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)Dynamic electrostatic catalytic field (DECF) vectors derived from transition state and reactant wavefunctions for the two-step reaction occurring within ketosteroid isomerase (KSI) have been calcd. using MP2/aug-cc-pVTZ and lower theory levels to det. the magnitude of the catalytic effect and the optimal directions of proton transfers in the KSI hydrogen-bond network. The most surprising and meaningful finding is that the KSI catalytic activity is enhanced by proton dislocations proceeding in opposite directions for each of the two consecutive reaction steps in the same hydrogen network. Such a mechanism allows an ultrafast switching of the catalytic proton wire environment, possibly related to the exceptionally high KSI catalytic power.
- 253Pollack, R. M. Enzymatic Mechanisms for Catalysis of Enolization: Ketosteroid Isomerase. Bioorg. Chem. 2004, 32 (5), 341– 353, DOI: 10.1016/j.bioorg.2004.06.005253Enzymatic mechanisms for catalysis of enolization: ketosteroid isomerasePollack, Ralph M.Bioorganic Chemistry (2004), 32 (5), 341-353CODEN: BOCMBM; ISSN:0045-2068. (Elsevier)A review. Breaking a C-H bond adjacent to a carbonyl group is a slow step in a large no. of chem. reactions. However, many enzymes are capable of catalyzing this reaction with great efficiency. One of the most proficient of these enzymes is 3-oxo-Δ5-steroid isomerase (KSI), which catalyzes the isomerization of a wide variety of 3-oxo-Δ5-steroids to their Δ4-conjugated isomers. Here, the reaction mechanism of KSI is discussed, with particular emphasis on energetic considerations. Both exptl. and theor. approaches are considered to explain the mechanistic details of the reaction.
- 254Wu, Y.; Boxer, S. G. A Critical Test of the Electrostatic Contribution to Catalysis with Noncanonical Amino Acids in Ketosteroid Isomerase. J. Am. Chem. Soc. 2016, 138 (36), 11890– 11895, DOI: 10.1021/jacs.6b06843254A Critical Test of the Electrostatic Contribution to Catalysis with Noncanonical Amino Acids in Ketosteroid IsomeraseWu, Yufan; Boxer, Steven G.Journal of the American Chemical Society (2016), 138 (36), 11890-11895CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The vibrational Stark effect (VSE) has been used to measure the elec. field in the active site of ketosteroid isomerase (KSI). These measured fields correlate with ΔG‡ in a series of conventional mutants yielding an est. for the electrostatic contribution to catalysis (Fried et al. Science, 2014, 346, 1510-1513). In this work we test this result with much more conservative variants in which individual Tyr residues in the active site are replaced by 3-chlorotyrosine via amber suppression. The elec. fields sensed at the position of the carbonyl bond involved in charge displacement during catalysis were characterized using the VSE, where the field sensitivity has been calibrated by vibrational Stark spectroscopy, solvatochromism, and MD simulations. A linear relationship is obsd. between the elec. field and ΔG‡ that interpolates between wild-type and more drastic conventional mutations, reinforcing the evaluation of the electrostatic contribution to catalysis in KSI. A simplified model and calcn. are developed to est. changes in the elec. field accompanying changes in the extended hydrogen-bond network in the active site. The results are consistent with a model in which the O-H group of a key active site tyrosine functions by imposing a static electrostatic potential onto the carbonyl bond. The model suggests that the contribution to catalysis from the active site hydrogen bonds is of similar wt. to the distal interactions from the rest of the protein. A similar linear correlation was also obsd. between the proton affinity of KSI's active site and the catalytic rate, suggesting a direct connection between the strength of the H-bond and the elec. field it exerts.
- 255Faraldos, J. A.; Antonczak, A. K.; González, V.; Fullerton, R.; Tippmann, E. M.; Allemann, R. K. Probing Eudesmane Cation-π Interactions in Catalysis by Aristolochene Synthase with Non-canonical Amino Acids. J. Am. Chem. Soc. 2011, 133 (35), 13906– 13909, DOI: 10.1021/ja205927u255Probing eudesmane cation-π interactions in catalysis by aristolochene synthase with non-canonical amino acidsFaraldos, Juan A.; Antonczak, Alicja K.; Gonzalez, Veronica; Fullerton, Rebecca; Tippmann, Eric M.; Allemann, Rudolf K.Journal of the American Chemical Society (2011), 133 (35), 13906-13909CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Stabilization of the reaction intermediate, eudesmane cation (I), through interaction with Trp-334 during catalysis by aristolochene synthase of Penicillium roqueforti was investigated by site-directed incorporation of proteinogenic and non-canonical arom. amino acids. The amt. of germacrene A (II) generated by the mutant enzymes served as a measure of the stabilization of I. II is a neutral intermediate, from which I is formed during PR-AS catalysis by protonation of the C6,C7 double bond. The replacement of Trp-334 with para-substituted Phe residues of increasing electron-withdrawing properties led to a progressive accumulation of II that showed a good correlation with the interaction energies of simple cations such as Na+ with substituted benzenes. These results provided compelling evidence for the stabilizing role played by Trp-334 in aristolochene synthase catalysis for the energetically demanding transformation of II to I.
- 256Morikubo, N.; Fukuda, Y.; Ohtake, K.; Shinya, N.; Kiga, D.; Sakamoto, K.; Asanuma, M.; Hirota, H.; Yokoyama, S.; Hoshino, T. Cation-π Interaction in the Polyolefin Cyclization Cascade Uncovered by Incorporating Unnatural Amino Acids into the Catalytic Sites of Squalene Cyclase. J. Am. Chem. Soc. 2006, 128 (40), 13184– 13194, DOI: 10.1021/ja063358p256Cation-π Interaction in the Polyolefin Cyclization Cascade Uncovered by Incorporating Unnatural Amino Acids into the Catalytic Sites of Squalene CyclaseMorikubo, Noriko; Fukuda, Yoriyuki; Ohtake, Kazumasa; Shinya, Naoko; Kiga, Daisuke; Sakamoto, Kensaku; Asanuma, Miwako; Hirota, Hiroshi; Yokoyama, Shigeyuki; Hoshino, TsutomuJournal of the American Chemical Society (2006), 128 (40), 13184-13194CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)It has been assumed that the π-electrons of arom. residues in the catalytic sites of triterpene cyclases stabilize the cationic intermediates formed during the polycyclization cascade of squalene or oxidosqualene, but no definitive exptl. evidence has been given. To validate this cation-π interaction, natural and unnatural arom. amino acids were site-specifically incorporated into squalene-hopene cyclase (SHC) from Alicyclobacillus acidocaldarius and the kinetic data of the mutants were compared with that of the wild-type SHC. The catalytic sites of Phe-365 and Phe-605 were substituted with O-methyltyrosine, tyrosine, and tryptophan, which have higher cation-π binding energies than phenylalanine. These replacements actually increased the SHC activity at low temp., but decreased the activity at high temp., as compared with the wild-type SHC. This decreased activity is due to the disorganization of the protein architecture caused by the introduction of the amino acids more bulky than phenylalanine. Then, mono-, di-, and trifluorophenylalanines were incorporated at positions 365 and 605; these amino acids reduce cation-π binding energies but have van der Waals radii similar to that of phenylalanine. The activities of the SHC variants with fluorophenylalanines were found to be inversely proportional to the no. of the fluorine atoms on the arom. ring and clearly correlated with the cation-π binding energies of the ring moiety. No serious structural alteration was obsd. for these variants even at high temp. These results unambiguously show that the π-electron d. of residues 365 and 605 has a crucial role for the efficient polycyclization reaction by SHC. This is the first report to demonstrate exptl. the involvement of cation-π interaction in triterpene biosynthesis.
- 257Herbst, R. W.; Guce, A.; Bryngelson, P. A.; Higgins, K. A.; Ryan, K. C.; Cabelli, D. E.; Garman, S. C.; Maroney, M. J. Role of Conserved Tyrosine Residues in NiSOD Catalysis: A Case of Convergent Evolution. Biochemistry 2009, 48 (15), 3354– 3369, DOI: 10.1021/bi802029t257Role of Conserved Tyrosine Residues in NiSOD Catalysis: A Case of Convergent EvolutionHerbst, Robert W.; Guce, Abigail; Bryngelson, Peter A.; Higgins, Khadine A.; Ryan, Kelly C.; Cabelli, Diane E.; Garman, Scott C.; Maroney, Michael J.Biochemistry (2009), 48 (15), 3354-3369CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Superoxide dismutases rely on protein structural elements to adjust the redox potential of the metallocenter to an optimum value near 300 mV (vs. NHE), to provide a source of protons for catalysis, and to control the access of anions to the active site. These aspects of the catalytic mechanism are examd. herein for recombinant prepns. of the nickel-dependent SOD (NiSOD) from Streptomyces coelicolor and for a series of mutants that affect a key tyrosine residue, Tyr9 (Y9F-, Y62F-, Y9F/Y62F-, and D3A-NiSOD). Structural aspects of the nickel sites are examd. by a combination of EPR and X-ray absorption spectroscopies, and by single-crystal X-ray diffraction at ∼1.9 Å resoln. in the case of Y9F- and D3A-NiSODs. The functional effects of the mutations are examd. by kinetic studies employing pulse radiolytic generation of O2- and by redox titrns. These studies reveal that although the structure of the nickel center in NiSOD is unique, the ligand environment is designed to optimize the redox potential at 290 mV and results in the oxidn. of 50% of the nickel centers in the oxidized hexamer. Kinetic investigations show that all of the mutant proteins have considerable activity. In the case of Y9F-NiSOD, the enzyme exhibits satn. behavior that is not obsd. in wild-type (WT) NiSOD and suggests that release of peroxide is inhibited. The crystal structure of Y9F-NiSOD reveals an anion binding site that is occupied by either Cl- or Br- and is located close to but not within bonding distance of the nickel center. The structure of D3A-NiSOD reveals that in addn. to affecting the interaction between subunits, this mutation repositions Tyr9 and leads to altered chem. with peroxide. Comparisons with Mn(SOD) and Fe(SOD) reveal that although different strategies for adjusting the redox potential and supply of protons are employed, NiSOD has evolved a similar strategy for controlling the access of anions to the active site.
- 258Campeciño, J. O.; Dudycz, L. W.; Tumelty, D.; Berg, V.; Cabelli, D. E.; Maroney, M. J. A Semisynthetic Strategy Leads to Alteration of the Backbone Amidate Ligand in the NiSOD Active Site. J. Am. Chem. Soc. 2015, 137 (28), 9044– 9052, DOI: 10.1021/jacs.5b03629258A Semisynthetic Strategy Leads to Alteration of the Backbone Amidate Ligand in the NiSOD Active SiteCampecino, Julius O.; Dudycz, Lech W.; Tumelty, David; Berg, Volker; Cabelli, Diane E.; Maroney, Michael J.Journal of the American Chemical Society (2015), 137 (28), 9044-9052CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Computational investigations have implicated the amidate ligand in nickel superoxide dismutase (NiSOD) in stabilizing Ni-centered redox catalysis and in preventing cysteine thiolate ligand oxidn. To test these predictions, we have used an exptl. approach utilizing a semisynthetic scheme that employs native chem. ligation of a pentapeptide (HCDLP) to recombinant S. coelicolor NiSOD lacking these N-terminal residues, NΔ5-NiSOD. Wild-type enzyme produced in this manner exhibits the characteristic spectral properties of recombinant WT-NiSOD and is as catalytically active. The semisynthetic scheme was also employed to construct a variant where the amidate ligand was converted to a secondary amine, H1*-NiSOD, a novel strategy that retains a backbone N-donor atom. The H1*-NiSOD variant was found to have only ∼1% of the catalytic activity of the recombinant wild-type enzyme, and had altered spectroscopic properties. X-ray absorption spectroscopy reveals a four-coordinate planar site with N2S2-donor ligands, consistent with electronic absorption spectroscopic results indicating that the Ni center in H1*-NiSOD is mostly reduced in the as-isolated sample, as opposed to 50:50 Ni(II)/Ni(III) mixt. that is typical for the recombinant wild-type enzyme. The EPR spectrum of as-isolated H1*-NiSOD accounts for ∼11% of the Ni in the sample and is similar to WT-NiSOD, but more axial, with gz < gx,y. 14N-hyperfine is obsd. on gz, confirming the addn. of the apical histidine ligand in the Ni(III) complex. The altered electronic properties and implications for redox catalysis are discussed in light of predictions based on synthetic and computational models.
- 259Evans, R. M.; Krahn, N.; Murphy, B. J.; Lee, H.; Armstrong, F. A.; Söll, D. Selective Cysteine-To-Selenocysteine Changes in a [NiFe]-Hydrogenase Confirm a Special Position for Catalysis and Oxygen Tolerance. Proc. Natl. Acad. Sci. U.S.A. 2021, 118 (13), e2100921118 DOI: 10.1073/pnas.2100921118There is no corresponding record for this reference.
- 260Mukai, T.; Sevostyanova, A.; Suzuki, T.; Fu, X.; Söll, D. A Facile Method for Producing Selenocysteine-Containing Proteins. Angew. Chem. Int. Ed. 2018, 57 (24), 7215– 7219, DOI: 10.1002/anie.201713215260A Facile Method for Producing Selenocysteine-Containing ProteinsMukai, Takahito; Sevostyanova, Anastasia; Suzuki, Tateki; Fu, Xian; Soell, DieterAngewandte Chemie, International Edition (2018), 57 (24), 7215-7219CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Selenocysteine (Sec, U) confers new chem. properties on proteins. Improved tools are thus required that enable Sec insertion into any desired position of a protein. We report a facile method for synthesizing selenoproteins with multiple Sec residues by expanding the genetic code of Escherichia coli. We recently discovered allo-tRNAs, tRNA species with unusual structure, that are as efficient serine acceptors as E. coli tRNASer. Ser-allo-tRNA was converted into Sec-allo-tRNA by Aeromonas salmonicida selenocysteine synthase (SelA). Sec-allo-tRNA variants were able to read through five UAG codons in the fdhF mRNA coding for E. coli formate dehydrogenase H, and produced active FDHH with five Sec residues in E. coli. Engineering of the E. coli selenium metab. along with mutational changes in allo-tRNA and SelA improved the yield and purity of recombinant human glutathione peroxidase 1 (to over 80 %). Thus, our allo-tRNAUTu system offers a new selenoprotein engineering platform.
- 261Snider, G. W.; Ruggles, E.; Khan, N.; Hondal, R. J. Selenocysteine Confers Resistance to Inactivation by Oxidation in Thioredoxin Reductase: Comparison of Selenium and Sulfur Enzymes. Biochemistry 2013, 52 (32), 5472– 5481, DOI: 10.1021/bi400462j261Selenocysteine Confers Resistance to Inactivation by Oxidation in Thioredoxin Reductase: Comparison of Selenium and Sulfur EnzymesSnider, Gregg W.; Ruggles, Erik; Khan, Nadeem; Hondal, Robert J.Biochemistry (2013), 52 (32), 5472-5481CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Mammalian thioredoxin reductase (TR) is a selenocysteine (Sec)-contg. homodimeric pyridine nucleotide oxidoreductase which catalyzes the redn. of oxidized thioredoxin. We have previously demonstrated the full-length mitochondrial mammalian TR (mTR3) enzyme to be resistant to inactivation from exposure to 50 mM H2O2. Because a Sec residue oxidizes more rapidly than a cysteine (Cys) residue, it has been previously thought that Sec-contg. enzymes are "sensitive to oxidn." compared to Cys-orthologues. Here we show for the first time a direct comparison of the abilities of Sec-contg. mTR3 and the Cys-orthologue from D. melanogaster (DmTR) to resist inactivation by oxidn. from a variety of oxidants including H2O2, hydroxyl radical, peroxynitrite, hypochlorous acid, hypobromous acid, and hypothiocyanous acid. The results show that the Sec-contg. TR is far superior to the Cys-orthologue TR in resisting inactivation by oxidn. To further test our hypothesis that the use of Sec confers strong resistance to inactivation by oxidn., we constructed a chimeric enzyme in which we replaced the active site Cys nucleophile of DmTR with a Sec residue using semisynthesis. The chimeric Sec-contg. enzyme has similar ability to resist inactivation by oxidn. as the wild type Sec-contg. TR from mouse mitochondria. The use of Sec in the chimeric enzyme "rescued" the enzyme from oxidant-induced inactivation for all of the oxidants tested in this study, in direct contrast to previous understanding. We discuss two possibilities for this rescue effect from inactivation under identical conditions of oxidative stress: (i) Sec resists overoxidn. and inactivation, whereas a Cys residue can be permanently overoxidized to the sulfinic acid form, and (ii) Sec protects the body of the enzyme from harmful oxidn. by allowing the enzyme to metabolize (turnover) various oxidants much better than a Cys-contg. TR.
- 262Wu, Z. P.; Hilvert, D. Selenosubtilisin as a Glutathione Peroxidase Mimic. J. Am. Chem. Soc. 1990, 112 (14), 5647– 5648, DOI: 10.1021/ja00170a043262Selenosubtilisin as a glutathione peroxidase mimicWu, Zhen Ping; Hilvert, DonaldJournal of the American Chemical Society (1990), 112 (14), 5647-8CODEN: JACSAT; ISSN:0002-7863.An artificial Se-contg. protein, selenolsubtilisin, mimics the redox properties of the naturally-occurring selenoenzyme glutathione peroxidase. It efficiently catalyzes the redn. of alkyl hydroperoxides by aryl thiols under mild aq. conditions. Kinetic studies suggest that the enzymic reaction proceeds via a ping pong mechanism with a covalent selenenyl sulfide deriv. as a key reaction intermediate. Comparison of the initial rates for the redn. of tert-Bu hydroperoxide by 3-carboxy-4-nitrobenzenethiol catalyzed by the selenoprotein and by di-Ph diselenide indicates that the protein binding site enhances the reaction rate ≥70,000-fold. Artificial peroxidases may be useful both as models for understanding the mechanism of action of the analogous natural enzymes and as antioxidant drugs in medicine or as practical catalysts in chem. synthesis.
- 263Hardy, F. J.; Ortmayer, M.; Green, A. P.; Noble, C. E. M.; Anderson, J. L. R. Recent Advances in Understanding, Enhancing and Creating Heme Peroxidases. In Reference Module in Chemistry, Molecular Sciences and Chemical Engineering; Elsevier, 2020. DOI: 10.1016/B978-0-08-102688-5.00021-0There is no corresponding record for this reference.
- 264Poulos, T. L. Heme Enzyme Structure and Function. Chem. Rev. 2014, 114, 3919– 3962, DOI: 10.1021/cr400415k264Heme Enzyme Structure and FunctionPoulos, Thomas L.Chemical Reviews (Washington, DC, United States) (2014), 114 (7), 3919-3962CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Metalloporphyrins are employed in various capacities throughout the biosphere, and of these, heme (iron protoporphyrin IX) is one of the most abundant and widely used. Heme is well-known for its roles in shuttling electrons between proteins as seen in mitochondrial respiration and in O2 storage as is the case with globins, but it also serves as a cofactor in multiple enzyme-mediated processes. Although heme enzymes can catalyze both reductive and oxidative reactions, the present review focuses primarily on those that catalyze oxidn. reactions, and esp. those for which crystal structures are available.
- 265Behan, R. K.; Green, M. T. On the Status of Ferryl Protonation. J. Inorg. Biochem. 2006, 100 (4), 448– 459, DOI: 10.1016/j.jinorgbio.2005.12.019265On the status of ferryl protonationBehan, Rachel K.; Green, Michael T.Journal of Inorganic Biochemistry (2006), 100 (4), 448-459CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier B.V.)A review. The authors examine the issue of ferryl protonation in heme proteins. An anal. of the results obtained from x-ray crystallog., resonance Raman spectroscopy, and extended x-ray absorption spectroscopy (EXAFS) is presented. Fe-O bond distances obtained from all three techniques are compared using Badger's rule. The long Fe-O bond lengths found in the ferryl crystal structures of myoglobin, cytochrome c peroxidase, horseradish peroxidase, and catalase deviate substantially from the values predict by Badger's rule, while the oxo-like distances obtained from EXAFS measurements are in good agreement with the empirical formula. D. functional calcns., which suggest that Moessbauer spectroscopy can be used to det. ferryl protonation states, are presented. The authors' calcns. indicate that the quadrupole splitting (ΔEq) changes significantly upon ferryl protonation. New resonance Raman data for horse-heart myoglobin compd. II (Mb-II, pH 4.5) are also presented. An Fe-O stretching frequency of 790 cm-1 (shifting to 754 cm-1 with 18O substitution) was obtained. This frequency provides a Badger distance of rFe-O = 1.66 Å. This distance is in agreement with the 1.69 Å Fe-O bond distance obtained from EXAFS measurements but is significantly shorter than the 1.93 Å bond found in the crystal structure of Mb-II (pH 5.2). In light of the available evidence, the authors conclude that the ferryl forms of myoglobin (pKa ≤ 4), horseradish peroxidase (pKa ≤ 4), cytochrome c peroxidase (pKa ≤ 4), and catalase (pKa ≤ 7) are not basic. They are authentic FeIV oxos with Fe-O bonds on the order of 1.65 Å.
- 266Sivaramakrishnan, S.; Ouellet, H.; Du, J.; McLean, K. J.; Medzihradszky, K. F.; Dawson, J. H.; Munro, A. W.; Ortiz de Montellano, P. R. A Novel Intermediate in the Reaction of Seleno CYP119 with m-Chloroperbenzoic Acid. Biochemistry 2011, 50 (14), 3014– 3024, DOI: 10.1021/bi101728y266A novel intermediate in the reaction of seleno CYP119 with m-chloroperbenzoic acidSivaramakrishnan, Santhosh; Ouellet, Hugues; Du, Jing; McLean, Kirsty J.; Medzihradszky, Katalin F.; Dawson, John H.; Munro, Andrew W.; Ortiz de Montellano, Paul R.Biochemistry (2011), 50 (14), 3014-3024CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Cytochrome P 450-mediated monooxygenation generally proceeds via a reactive ferryl intermediate coupled to a ligand radical [Fe(IV)≡O]+· termed Compd. I (Cpd I). The proximal cysteine thiolate ligand is a crit. determinant of the spectral and catalytic properties of P 450 enzymes. To explore the effect of an increased level of donation of electrons by the proximal ligand in the P 450 catalytic cycle, we recently reported successful incorporation of SeCys into the active site of CYP119, a thermophilic cytochrome P 450. Here we report relevant phys. properties of SeCYP119 and a detailed anal. of the reaction of SeCYP119 with m-chloroperbenzoic acid (mCPBA). Our results indicate that the selenolate anion reduces rather than stabilizes Cpd I and also protects the heme from oxidative destruction, leading to the generation of a new stable species with an absorbance max. at 406 nm. This stable intermediate can be returned to the normal ferric state by reducing agents and thiols, in agreement with oxidative modification of the selenolate ligand itself. Thus, in the seleno protein, the oxidative damage shifts from the heme to the proximal ligand, presumably because (a) an increased level of donation of electrons more efficiently quenches reactive species such as Cpd I and (b) the protection of the thiolate ligand provided by the protein active site structure is insufficient to shield the more oxidizable selenolate ligand.
- 267Aldag, C.; Gromov, I. A.; García-Rubio, I.; Von Koenig, K.; Schlichting, I.; Jaun, B.; Hilvert, D. Probing the Role of the Proximal Heme Ligand in Cytochrome P450cam by Recombinant Incorporation of Selenocysteine. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (14), 5481– 5486, DOI: 10.1073/pnas.0810503106There is no corresponding record for this reference.
- 268Jiang, Y.; Sivaramakrishnan, S.; Hayashi, T.; Cohen, S.; Moënne-Loccoz, P.; Shaik, S.; Ortiz de Montellano, P. R. Calculated and Experimental Spin State of Seleno Cytochrome P450. Angew. Chem. Int. Ed. 2009, 48 (39), 7193– 7195, DOI: 10.1002/anie.200901485268Calculated and Experimental Spin State of Seleno Cytochrome P450Jiang, Yongying; Sivaramakrishnan, Santhosh; Hayashi, Takahiro; Cohen, Shimrit; Moenne-Loccoz, Pierre; Shaik, Sason; Ortiz de Montellano, Paul R.Angewandte Chemie, International Edition (2009), 48 (39), 7193-7195, S7193/1-S7193/19CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The cysteine thiolate ligand coordinated to the heme iron atom in cytochrome P 450 is thought to be responsible for the unique spectroscopic and catalytic properties of these enzymes. To explore the role of the proximal ligand in these proteins, the cysteine has been replaced by a variety of other ligands by site-specific mutagenesis. Here, the expression and characterization of a seleno cytochrome P 450 in which the cysteine thiolate iron ligand is replaced by a selenocysteine is reported. CYP119 was used for this substitution because the proximal ligand is the only cysteine in its sequence. The seleno protein was expressed in a cysteine auxotroph BL21(DE3)CysE strain of Escherichia coli that cannot synthesize cysteine owing to a mutation in the CysE gene. A pCWori vector contg. the CYP119 gene encoding a 6-His tag at the C-terminus was transformed into the auxotrophic BL21(DE3)Cys cells, and the seleno protein was expressed in minimal media contg. L-selenocystine. The protein yield was 2.6 mg L-1 of culture after affinity purifn., which is approx. 8-10 times lower than that of the normal thiolate-ligated protein. This approach results in over 70 % replacement of the cysteine by a selenocysteine as judged by the relative peak intensities of the Cys and SeCys proteins by LC/ESI-MS. In conclusion, the first selenium-incorporated P 450 encoding was expressed and characterized. The preliminary results reveal that the spectral characteristics of this novel CYP119 are comparable to those of the corresponding WT protein, indicating the presence of a six-coordinate low-spin heme iron with water as a distal ligand. More importantly, the catalytic activity of the seleno-mutant is comparable to that of the WT enzyme. Furthermore, computational calcns. clearly support the exptl. assigned spin state. Future studies will focus on both examg. how this substitution affects the stability of the putative compd. I species and the development of novel catalysts.
- 269Onderko, E. L.; Silakov, A.; Yosca, T. H.; Green, M. T. Characterization of a Selenocysteine-Ligated P450 Compound I Reveals Direct Link Between Electron Donation and Reactivity. Nat. Chem. 2017, 9 (7), 623– 628, DOI: 10.1038/nchem.2781269Characterization of a selenocysteine-ligated P450 compound I reveals direct link between electron donation and reactivityOnderko, Elizabeth L.; Silakov, Alexey; Yosca, Timothy H.; Green, Michael T.Nature Chemistry (2017), 9 (7), 623-628CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)Strong electron-donation from the axial thiolate ligand of cytochrome P 450 has been proposed to increase the reactivity of compd. I with respect to C-H bond activation. However, it has proven difficult to test this hypothesis, and a direct link between reactivity and electron donation has yet to be established. To make this connection, we prepd. a selenolate-ligated cytochrome P 450 compd. I intermediate. This isoelectronic perturbation allowed for direct comparisons with the wild-type enzyme. Selenium incorporation was achieved using a cysteine auxotrophic Escherichia coli strain. The intermediate was prepd. with m-chloroperbenzoic acid and characterized by UV-visible, Moessbauer, and ESR spectroscopies. Measurements revealed increased asymmetry around the ferryl moiety, consistent with increased electron donation from the axial selenolate ligand. In line with this observation, we found that the selenolate-ligated compd. I cleaved C-H bonds more rapidly than the wild-type intermediate.
- 270Sivaramakrishnan, S.; Ouellet, H.; Matsumura, H.; Guan, S.; Moënne-Loccoz, P.; Burlingame, A. L.; Ortiz De Montellano, P. R. Proximal Ligand Electron Donation and Reactivity of the Cytochrome P450 Ferric-Peroxo Anion. J. Am. Chem. Soc. 2012, 134 (15), 6673– 6684, DOI: 10.1021/ja211499q270Proximal Ligand Electron Donation and Reactivity of the Cytochrome P450 Ferric-Peroxo AnionSivaramakrishnan, Santhosh; Ouellet, Hugues; Matsumura, Hirotoshi; Guan, Shenheng; Moenne-Loccoz, Pierre; Burlingame, Alma L.; Ortiz de Montellano, Paul R.Journal of the American Chemical Society (2012), 134 (15), 6673-6684CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)CYP125 from Mycobacterium tuberculosis catalyzes sequential oxidn. of the cholesterol side-chain terminal Me group to the alc., aldehyde, and finally acid. Here, we demonstrate that CYP125 simultaneously catalyzes the formation of five other products, all of which result from deformylation of the sterol side chain. The aldehyde intermediate is shown to be the precursor of both the conventional acid metabolite and the five deformylation products. The acid arises by protonation of the ferric-peroxo anion species and formation of the ferryl-oxene species, also known as Compd. I, followed by hydrogen abstraction and oxygen transfer. The deformylation products arise by addn. of the same ferric-peroxo anion to the aldehyde intermediate to give a peroxyhemiacetal that leads to C-C bond cleavage. This bifurcation of the catalytic sequence has allowed us to examine the effect of electron donation by the proximal ligand on the properties of the ferric-peroxo anion. Replacement of the cysteine thiolate iron ligand by a selenocysteine results in UV-vis, EPR, and resonance Raman spectral changes indicative of an increased electron donation from the proximal selenolate ligand to the iron. Anal. of the product distribution in the reaction of the selenocysteine substituted enzyme reveals a gain in the formation of the acid (Compd. I pathway) at the expense of deformylation products. These observations are consistent with an increase in the pKa of the ferric-peroxo anion, which favors its protonation and, therefore, Compd. I formation.
- 271Ortmayer, M.; Fisher, K.; Basran, J.; Wolde-Michael, E. M.; Heyes, D. J.; Levy, C.; Lovelock, S. L.; Anderson, J. L. R.; Raven, E. L.; Hay, S. Rewiring the “ Push-Pull ” Catalytic Machinery of a Heme Enzyme Using an Expanded Genetic Code. ACS Catal. 2020, 10, 2735– 2746, DOI: 10.1021/acscatal.9b05129271Rewiring the "Push-Pull" Catalytic Machinery of a Heme Enzyme Using an Expanded Genetic CodeOrtmayer, Mary; Fisher, Karl; Basran, Jaswir; Wolde-Michael, Emmanuel M.; Heyes, Derren J.; Levy, Colin; Lovelock, Sarah L.; Anderson, J. L. Ross; Raven, Emma L.; Hay, Sam; Rigby, Stephen E. J.; Green, Anthony P.ACS Catalysis (2020), 10 (4), 2735-2746CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Nature employs a limited no. of genetically encoded axial ligands to control diverse heme enzyme activities. Deciphering the functional significance of these ligands requires a quant. understanding of how their electron-donating capabilities modulate the structures and reactivities of the iconic ferryl intermediates compds. I and II. However, probing these relationships exptl. has proven to be challenging as ligand substitutions accessible via conventional mutagenesis do not allow fine tuning of electron donation and typically abolish catalytic function. Here, we exploit engineered translation components to replace the histidine ligand of cytochrome c peroxidase (CcP) by a less electron-donating Nδ-Me histidine (Me-His) with little effect on the enzyme structure. The rate of formation (k1) and the reactivity (k2) of compd. I are unaffected by ligand substitution. In contrast, proton-coupled electron transfer to compd. II (k3) is 10-fold slower in CcP Me-His, providing a direct link between electron donation and compd. II reactivity, which can be explained by weaker electron donation from the Me-His ligand ("the push") affording an electron-deficient ferryl oxygen with reduced proton affinity ("the pull"). The deleterious effects of the Me-His ligand can be fully compensated by introducing a W51F mutation designed to increase "the pull" by removing a hydrogen bond to the ferryl oxygen. Analogous substitutions in ascorbate peroxidase lead to similar activity trends to those obsd. in CcP, suggesting that a common mechanistic strategy is employed by enzymes using distinct electron transfer pathways. Our study highlights how noncanonical active site substitutions can be used to directly probe and deconstruct highly evolved bioinorg. mechanisms.
- 272Xiao, H.; Peters, F. B.; Yang, P.-Y.; Reed, S.; Chittuluru, J. R.; Schultz, P. G. Genetic Incorporation of Histidine Derivatives Using an Engineered Pyrrolysyl-tRNA Synthetase. ACS Chem. Biol. 2014, 9 (5), 1092– 1096, DOI: 10.1021/cb500032c272Genetic Incorporation of Histidine Derivatives Using an Engineered Pyrrolysyl-tRNA SynthetaseXiao, Han; Peters, Francis B.; Yang, Peng-Yu; Reed, Sean; Chittuluru, Johnathan R.; Schultz, Peter G.ACS Chemical Biology (2014), 9 (5), 1092-1096CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)A polyspecific amber suppressor aminoacyl-tRNA synthetase/tRNA pair was evolved that genetically encodes a series of histidine analogs in both Escherichia coli and mammalian cells. In combination with tRNACUAPyl, a pyrrolysyl-tRNA synthetase (PylRS) mutant was able to site-specifically incorporate 3-methylhistidine, 3-pyridylalanine, 2-furylalanine, and 3-(2-thienyl)alanine into proteins in response to an amber codon. Substitution of His66 in the blue fluorescent protein (BFP) with these histidine analogs created mutant proteins with distinct spectral properties. This work further expands the structural and chem. diversity of unnatural amino acids (UAAs) that can be genetically encoded in prokaryotic and eukaryotic organisms and affords new probes of protein structure and function.
- 273Martin, C.; Zhang, Y. The Diverse Functions of Histone Lysine Methylation. Nat. Rev. Mol. Cell Biol. 2005, 6 (11), 838– 849, DOI: 10.1038/nrm1761273The diverse functions of histone lysine methylationMartin, Cyrus; Zhang, YiNature Reviews Molecular Cell Biology (2005), 6 (11), 838-849CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. Covalent modifications of histone tails play fundamental roles in chromatin structure and function. One such modification, lysine methylation, has important functions in many biol. processes that include heterochromatin formation, X-chromosome inactivation, and transcriptional regulation. Here, the authors summarize recent advances in the understanding of how lysine methylation functions in these diverse biol. processes, and raise questions that need to be addressed in the future.
- 274Niu, W.; Guo, J. Co-translational Installation of Posttranslational Modifications by Non-canonical Amino Acid Mutagenesis. ChemBioChem 2023, 24 (9), e202300039 DOI: 10.1002/cbic.202300039There is no corresponding record for this reference.
- 275Rust, H. L.; Subramanian, V.; West, G. M.; Young, D. D.; Schultz, P. G.; Thompson, P. R. Using Unnatural Amino Acid Mutagenesis To Probe the Regulation of PRMT1. ACS Chem. Biol. 2014, 9 (3), 649– 655, DOI: 10.1021/cb400859z275Using Unnatural Amino Acid Mutagenesis To Probe the Regulation of PRMT1Rust, Heather L.; Subramanian, Venkataraman; West, Graham M.; Young, Douglas D.; Schultz, Peter G.; Thompson, Paul R.ACS Chemical Biology (2014), 9 (3), 649-655CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Protein arginine methyltransferase 1 (PRMT1)-dependent methylation contributes to the onset and progression of numerous diseases (e.g., cancer, heart disease, ALS); however, the regulatory mechanisms that control PRMT1 activity are relatively unexplored. We therefore set out to decipher how phosphorylation regulates PRMT1 activity. Curated mass spectrometry data identified Tyr291, a residue adjacent to the conserved THW loop, as being phosphorylated. Natural and unnatural amino acid mutagenesis, including the incorporation of p-carboxymethyl-L-phenylalanine (pCmF) as a phosphotyrosine mimic, were used to show that Tyr291 phosphorylation alters the substrate specificity of PRMT1. Addnl., p-benzoyl-L-phenylalanine (pBpF) was incorporated at the Tyr291 position, and crosslinking expts. with K562 cell exts. identified several proteins (e.g., hnRNP A1 and hnRNP H3) that bind specifically to this site. Moreover, we also demonstrate that Tyr291 phosphorylation impairs PRMT1's ability to bind and methylate both proteins. In total, these studies demonstrate that Tyr291 phosphorylation alters both PRMT1 substrate specificity and protein-protein interactions.
- 276Neumann, H.; Neumann-Staubitz, P.; Witte, A.; Summerer, D. Epigenetic Chromatin Modification by Amber Suppression Technology. Curr. Opin. Chem. Biol. 2018, 45, 1– 9, DOI: 10.1016/j.cbpa.2018.01.017276Epigenetic chromatin modification by amber suppression technologyNeumann, Heinz; Neumann-Staubitz, Petra; Witte, Anna; Summerer, DanielCurrent Opinion in Chemical Biology (2018), 45 (), 1-9CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)The genetic incorporation of unnatural amino acids (UAAs) into proteins by amber suppression technol. provides unique avenues to study protein structure, function and interactions both in vitro and in living cells and organisms. This approach has been particularly useful for studying mechanisms of epigenetic chromatin regulation, since these extensively involve dynamic changes in structure, complex formation and posttranslational modifications that are difficult to access by traditional approaches. Here, we review recent achievements in this field, emphasizing UAAs that help to unravel protein-protein interactions in cells by photo-crosslinking or that allow the biosynthesis of proteins with defined posttranslational modifications for studying their function and turnover in vitro and in cells.
- 277Wang, Z. A.; Cole, P. A. The Chemical Biology of Reversible Lysine Post-translational Modifications. Cell Chem. Biol. 2020, 27 (8), 953– 969, DOI: 10.1016/j.chembiol.2020.07.002277The Chemical Biology of Reversible Lysine Post-translational ModificationsWang, Zhipeng A.; Cole, Philip A.Cell Chemical Biology (2020), 27 (8), 953-969CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)A review. Lysine (Lys) residues in proteins undergo a wide range of reversible post-translational modifications (PTMs), which can regulate enzyme activities, chromatin structure, protein-protein interactions, protein stability, and cellular localization. Here we discuss the "writers," "erasers," and "readers" of some of the common protein Lys PTMs and summarize examples of their major biol. impacts. We also review chem. biol. approaches, from small-mol. probes to protein chem. technologies, that have helped to delineate Lys PTM functions and show promise for a diverse set of biomedical applications.
- 278Wang, T.; Zhou, Q.; Li, F.; Yu, Y.; Yin, X.; Wang, J. Genetic Incorporation of Nε-Formyllysine, a New Histone Post-translational Modification. ChemBioChem 2015, 16 (10), 1440– 1442, DOI: 10.1002/cbic.201500170There is no corresponding record for this reference.
- 279Cao, L.; Liu, J.; Ghelichkhani, F.; Rozovsky, S.; Wang, L. Genetic Incorporation of ε-N-Benzoyllysine by Engineering Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase. ChemBioChem 2021, 22 (15), 2530– 2534, DOI: 10.1002/cbic.202100218There is no corresponding record for this reference.
- 280Ren, C.; Wu, Q.; Xiao, R.; Ji, Y.; Yang, X.; Zhang, Z.; Qin, H.; Ma, J.-A.; Xuan, W. Expanding the Scope of Genetically Encoded Lysine Post-Translational Modifications with Lactylation, β-Hydroxybutyrylation and Lipoylation. ChemBioChem 2022, 23 (18), e202200302 DOI: 10.1002/cbic.202200302There is no corresponding record for this reference.
- 281Nguyen, D. P.; Garcia Alai, M. M.; Kapadnis, P. B.; Neumann, H.; Chin, J. W. Genetically Encoding Nϵ-Methyl-l-lysine in Recombinant Histones. J. Am. Chem. Soc. 2009, 131 (40), 14194– 14195, DOI: 10.1021/ja906603s281Genetically encoding Nε-methyl-L-lysine in recombinant histonesNguyen, Duy P.; Garcia Alai, Maria M.; Kapadnis, Prashant B.; Neumann, Heinz; Chin, Jason W.Journal of the American Chemical Society (2009), 131 (40), 14194-14195CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Lysine methylation is an important post-translational modification of histone proteins that defines epigenetic status and controls heterochromatin formation, X-chromosome inactivation, genome imprinting, DNA repair, and transcriptional regulation. Despite considerable efforts by chem. biologists to synthesize modified histones for use in deciphering the mol. role of methylation in these phenomena, no general method exists to synthesize proteins bearing quant. site-specific methylation. Here we demonstrate a general method for the quant. installation of Nε-methyl-L-lysine at defined positions in recombinant histones and demonstrate the use of this method for investigating the methylation dependent binding of HP1 to full length histone H3 monomethylated on K9 (H3K9me1). This strategy will find wide application in defining the mol. mechanisms by which histone methylation orchestrates cellular phenomena.
- 282Wang, Z. A.; Liu, W. R. Proteins with Site-Specific Lysine Methylation. Chem. Eur. J. 2017, 23 (49), 11732– 11737, DOI: 10.1002/chem.201701655There is no corresponding record for this reference.
- 283Wang, Y.-S.; Wu, B.; Wang, Z.; Huang, Y.; Wan, W.; Russell, W. K.; Pai, P.-J.; Moe, Y. N.; Russell, D. H.; Liu, W. R. A Genetically Encoded Photocaged Nε-Methyl-L-Lysine. Mol. BioSyst. 2010, 6 (9), 1557– 1560, DOI: 10.1039/c002155eThere is no corresponding record for this reference.
- 284Neumann, H.; Peak-Chew, S. Y.; Chin, J. W. Genetically Encoding Nε-Acetyllysine in Recombinant Proteins. Nat. Chem. Biol. 2008, 4 (4), 232– 234, DOI: 10.1038/nchembio.73284Genetically encoding Nε-acetyllysine in recombinant proteinsNeumann, Heinz; Peak-Chew, Sew Y.; Chin, Jason W.Nature Chemical Biology (2008), 4 (4), 232-234CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Nε-acetylation of lysine is a reversible post-translational modification with a regulatory role that rivals that of phosphorylation in eukaryotes. No general methods exist to synthesize proteins contg. Nε-acetyllysine at defined sites. The site-specific incorporation of Nε-acetyllysine in recombinant proteins produced in Escherichia coli was achieved via the evolution of an orthogonal Nε-acetyllysyl-tRNA synthetase/tRNACUA pair. This strategy should find wide applications in defining the cellular role of this modification.
- 285Gattner, M. J.; Vrabel, M.; Carell, T. Synthesis of ε-N-Propionyl-, ε-N-Butyryl-, and ε-N-Crotonyl-Lysine Containing Histone H3 Using the Pyrrolysine System. Chem. Commun. 2013, 49 (4), 379– 381, DOI: 10.1039/C2CC37836AThere is no corresponding record for this reference.
- 286Wilkins, B. J.; Hahn, L. E.; Heitmüller, S.; Frauendorf, H.; Valerius, O.; Braus, G. H.; Neumann, H. Genetically Encoding Lysine Modifications on Histone H4. ACS Chem. Biol. 2015, 10 (4), 939– 944, DOI: 10.1021/cb501011v286Genetically encoding lysine modifications on histone H4Wilkins, Bryan J.; Hahn, Liljan E.; Heitmueller, Svenja; Frauendorf, Holm; Valerius, Oliver; Braus, Gerhard H.; Neumann, HeinzACS Chemical Biology (2015), 10 (4), 939-944CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Post-translational modifications of proteins are important modulators of protein function. In order to identify the specific consequences of individual modifications, general methods are required for homogeneous prodn. of modified proteins. The direct installation of modified amino acids by genetic code expansion facilitates the prodn. of such proteins independent of the knowledge and availability of the enzymes naturally responsible for the modification. The prodn. of recombinant histone H4 with genetically encoded modifications has proven notoriously difficult in the past. Here, we present a general strategy to produce histone H4 with acetylation, propionylation, butyrylation, and crotonylation on lysine residues. We produce homogeneous histone H4 contg. up to four simultaneous acetylations to analyze the impact of the modifications on chromatin array compaction. Furthermore, we explore the ability of antibodies to discriminate between alternative lysine acylations by incorporating these modifications in recombinant histone H4.
- 287Xiao, H.; Xuan, W.; Shao, S.; Liu, T.; Schultz, P. G. Genetic Incorporation of ε-N-2-Hydroxyisobutyryl-Lysine into Recombinant Histones. ACS Chem. Biol. 2015, 10 (7), 1599– 1603, DOI: 10.1021/cb501055hThere is no corresponding record for this reference.
- 288Kim, C. H.; Kang, M.; Kim, H. J.; Chatterjee, A.; Schultz, P. G. Site-Specific Incorporation of ε-N-Crotonyllysine into Histones. Angew. Chem. Int. Ed. 2012, 51 (29), 7246– 7249, DOI: 10.1002/anie.201203349There is no corresponding record for this reference.
- 289Tian, H.; Yang, J.; Guo, A.-D.; Ran, Y.; Yang, Y.-Z.; Yang, B.; Huang, R.; Liu, H.; Chen, X.-H. Genetically Encoded Benzoyllysines Serve as Versatile Probes for Interrogating Histone Benzoylation and Interactions in Living Cells. ACS Chem. Biol. 2021, 16 (11), 2560– 2569, DOI: 10.1021/acschembio.1c00614There is no corresponding record for this reference.
- 290Fatema, N.; Fan, C. Studying Lysine Acetylation of Citric Acid Cycle Enzymes by Genetic Code Expansion. Mol. Microbiol. 2023, 119 (5), 551– 559, DOI: 10.1111/mmi.15052There is no corresponding record for this reference.
- 291Araujo, J.; Ottinger, S.; Venkat, S.; Gan, Q.; Fan, C. Studying Acetylation of Aconitase Isozymes by Genetic Code Expansion. Front. Chem. 2022, 10, 1, DOI: 10.3389/fchem.2022.862483There is no corresponding record for this reference.
- 292Venkat, S.; Chen, H.; Stahman, A.; Hudson, D.; McGuire, P.; Gan, Q.; Fan, C. Characterizing Lysine Acetylation of Isocitrate Dehydrogenase in Escherichia coli. J. Mol. Biol. 2018, 430 (13), 1901– 1911, DOI: 10.1016/j.jmb.2018.04.031There is no corresponding record for this reference.
- 293Venkat, S.; Gregory, C.; Sturges, J.; Gan, Q.; Fan, C. Studying the Lysine Acetylation of Malate Dehydrogenase. J. Mol. Biol. 2017, 429 (9), 1396– 1405, DOI: 10.1016/j.jmb.2017.03.027There is no corresponding record for this reference.
- 294Venkat, S.; Chen, H.; McGuire, P.; Stahman, A.; Gan, Q.; Fan, C. Characterizing Lysine Acetylation of Escherichia coli Type II Citrate Synthase. FEBS J. 2019, 286 (14), 2799– 2808, DOI: 10.1111/febs.14845There is no corresponding record for this reference.
- 295Wright, D. E.; Altaany, Z.; Bi, Y.; Alperstein, Z.; O’Donoghue, P. Acetylation Regulates Thioredoxin Reductase Oligomerization and Activity. Antioxid. Redox Signal. 2018, 29 (4), 377– 388, DOI: 10.1089/ars.2017.7082There is no corresponding record for this reference.
- 296Rogerson, D. T.; Sachdeva, A.; Wang, K.; Haq, T.; Kazlauskaite, A.; Hancock, S. M.; Huguenin-Dezot, N.; Muqit, M. M. K.; Fry, A. M.; Bayliss, R. Efficient Genetic Encoding of Phosphoserine and its Nonhydrolyzable Analog. Nat. Chem. Biol. 2015, 11 (7), 496– 503, DOI: 10.1038/nchembio.1823296Efficient genetic encoding of phosphoserine and its nonhydrolyzable analogRogerson, Daniel T.; Sachdeva, Amit; Wang, Kaihang; Haq, Tamanna; Kazlauskaite, Agne; Hancock, Susan M.; Huguenin-Dezot, Nicolas; Muqit, Miratul M. K.; Fry, Andrew M.; Bayliss, Richard; Chin, Jason W.Nature Chemical Biology (2015), 11 (7), 496-503CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Serine phosphorylation is a key post-translational modification that regulates diverse biol. processes. Powerful anal. methods have identified thousands of phosphorylation sites, but many of their functions remain to be deciphered. A key to understanding the function of protein phosphorylation is access to phosphorylated proteins, but this is often challenging or impossible. Here we evolve an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair that directs the efficient incorporation of phosphoserine (pSer (1)) into recombinant proteins in Escherichia coli. Moreover, combining the orthogonal pair with a metabolically engineered E. coli enables the site-specific incorporation of a nonhydrolyzable analog of pSer. Our approach enables quant. decoding of the amber stop codon as pSer, and we purify, with yields of several milligrams per L of culture, proteins bearing biol. relevant phosphorylations that were previously challenging or impossible to access-including phosphorylated ubiquitin and the kinase Nek7, which is synthetically activated by a genetically encoded phosphorylation in its activation loop.
- 297Venkat, S.; Sturges, J.; Stahman, A.; Gregory, C.; Gan, Q.; Fan, C. Genetically Incorporating Two Distinct Post-translational Modifications into One Protein Simultaneously. ACS Synth. Biol. 2018, 7 (2), 689– 695, DOI: 10.1021/acssynbio.7b00408297Genetically Incorporating Two Distinct Post-translational Modifications into One Protein SimultaneouslyVenkat, Sumana; Sturges, Jourdan; Stahman, Alleigh; Gregory, Caroline; Gan, Qinglei; Fan, ChenguangACS Synthetic Biology (2018), 7 (2), 689-695CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Post-translational modifications (PTMs) play important roles in regulating a variety of biol. processes. To facilitate PTM studies, the genetic code expansion strategy has been used to cotranslationally incorporate individual PTMs such as acetylation and phosphorylation into proteins at specific sites. However, recent studies demonstrated that PTMs actually work together to regulate protein functions and structures. Thus, simultaneous incorporation of multiple distinct PTMs into one protein is highly desirable. The authors used the genetic incorporation systems of phosphoserine and acetyllysine to install both phosphorylation and acetylation into target proteins simultaneously in Escherichia coli. And this system was used to study the effect of coexisting acetylation and phosphorylation on malate dehydrogenase, demonstrating a practical application of this system in biochem. studies. Furthermore, the authors tested the mutual orthogonality of three widely used genetic incorporation systems, indicating the possibility of incorporating three distinct PTMs into one protein simultaneously.
- 298Zang, J.; Chen, Y.; Liu, C.; Lin, S. Probing the Role of Aurora Kinase A Threonylation with Site-Specific Lysine Threonylation. ACS Chem. Biol. 2023, 18 (4), 674– 678, DOI: 10.1021/acschembio.1c00682There is no corresponding record for this reference.
- 299Wan, N.; Wang, N.; Yu, S.; Zhang, H.; Tang, S.; Wang, D.; Lu, W.; Li, H.; Delafield, D. G.; Kong, Y. Cyclic Immonium Ion of Lactyllysine Reveals Widespread Lactylation in the Human Proteome. Nat. Methods 2022, 19 (7), 854– 864, DOI: 10.1038/s41592-022-01523-1There is no corresponding record for this reference.
- 300Whittaker, J. W. Free Radical Catalysis by Galactose Oxidase. Chem. Rev. 2003, 103 (6), 2347– 2364, DOI: 10.1021/cr020425z300Free Radical Catalysis by Galactose OxidaseWhittaker, James W.Chemical Reviews (Washington, DC, United States) (2003), 103 (6), 2347-2363CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. The unusual two-electron reactivity of the mononuclear copper active site in galactose oxidase has been explained in terms of the direct participation of the protein in the redox chem. of the active site, forming a stable free radical-copper complex in the active enzyme. The copper-free apoprotein is readily oxidized under mild conditions, forming a stable free radical, with distinctive optical absorption and ESR (EPR) spectra. A single free radical species is obsd., implying a unique reactive site in the protein. At X-band (9 GHz) the EPR spectrum exhibits an av. g-value of 2.005 and a complex pattern of fine structure splittings. Isotopic labeling demonstrates that the free radical site in the apoprotein is derived from a tyrosine residue, and it allows the major splittings to be assigned to hyperfine interactions between the unpaired electron and the β hydrogens in the side chain of a perturbed tyrosine residue. ENDOR spectroscopy (also at X-band) yields more refined ests. of the hyperfine coupling consts. and provides evidence for hydrogen bonding to the phenoxy oxygen. This review will focus on research aimed at understanding the nature of the free radical site, the reactivity of the unique metalloradical complex, and the mechanism of free radical catalysis by galactose oxidase.
- 301Polyakov, K. M.; Boyko, K. M.; Tikhonova, T. V.; Slutsky, A.; Antipov, A. N.; Zvyagilskaya, R. A.; Popov, A. N.; Bourenkov, G. P.; Lamzin, V. S.; Popov, V. O. High-Resolution Structural Analysis of a Novel Octaheme Cytochrome c Nitrite Reductase from the Haloalkaliphilic Bacterium Thioalkalivibrio nitratireducens. J. Mol. Biol. 2009, 389 (5), 846– 862, DOI: 10.1016/j.jmb.2009.04.037There is no corresponding record for this reference.
- 302Ye, S.; Wu, X. a.; Wei, L.; Tang, D.; Sun, P.; Bartlam, M.; Rao, Z. An Insight into the Mechanism of Human Cysteine Dioxygenase: key roles of the thioether-bonded tyrosine-cysteine cofactor. J. Biol. Chem. 2007, 282 (5), 3391– 3402, DOI: 10.1074/jbc.M609337200302An Insight into the Mechanism of Human Cysteine Dioxygenase. Key Roles of the Thioether-Bonded Tyrosine-Cysteine CofactorYe, Sheng; Wu, Xiao'ai; Wei, Lei; Tang, Danming; Sun, Ping; Bartlam, Mark; Rao, ZiheJournal of Biological Chemistry (2007), 282 (5), 3391-3402CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Cysteine dioxygenase (CDO) is a non-heme mononuclear iron metalloenzyme that catalyzes the oxidn. of cysteine to cysteine sulfinic acid with addn. of mol. dioxygen. This irreversible oxidative catabolism of cysteine initiates several important metabolic pathways related to diverse sulfurate compds. Cysteine dioxygenase is therefore very important for maintaining the proper hepatic concn. of intracellular free cysteine. Mechanisms for mouse and rat cysteine dioxygenases have recently been reported based on their crystal structures in the absence of substrates, although there is still a lack of direct evidence. Here we report the first crystal structure of human cysteine dioxygenase in complex with its substrate L-cysteine to 2.7Å, together with enzymic activity and metal content assays of several single point mutants. Our results provide an insight into a new mechanism of cysteine thiol dioxygenation catalyzed by cysteine dioxygenase, which is tightly assocd. with a thioether-bonded tyrosine-cysteine cofactor involving Tyr-157 and Cys-93. This cross-linked protein-derived cofactor plays several key roles different from those in galactose oxidase. This report provides a new potential target for therapy of diseases related to human cysteine dioxygenase, including neurodegenerative and autoimmune diseases.
- 303Zhou, Q.; Hu, M.; Zhang, W.; Jiang, L.; Perrett, S.; Zhou, J.; Wang, J. Probing the Function of the Tyr-Cys Cross-Link in Metalloenzymes by the Genetic Incorporation of 3-Methylthiotyrosine. Angew. Chem. Int. Ed. 2013, 52 (4), 1203– 1207, DOI: 10.1002/anie.201207229There is no corresponding record for this reference.
- 304Dominy, J. E.; Hwang, J.; Guo, S.; Hirschberger, L. L.; Zhang, S.; Stipanuk, M. H. Synthesis of Amino Acid Cofactor in Cysteine Dioxygenase Is Regulated by Substrate and Represents a Novel Post-translational Regulation of Activity. J. Biol. Chem. 2008, 283 (18), 12188– 12201, DOI: 10.1074/jbc.M800044200304Synthesis of Amino Acid Cofactor in Cysteine Dioxygenase Is Regulated by Substrate and Represents a Novel Post-translational Regulation of ActivityDominy, John E., Jr.; Hwang, Jesse; Guo, Stephanie; Hirschberger, Lawrence L.; Zhang, Sheng; Stipanuk, Martha H.Journal of Biological Chemistry (2008), 283 (18), 12188-12201CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Cysteine dioxygenase (CDO) catalyzes the conversion of cysteine to cysteinesulfinic acid and is important in the regulation of intracellular cysteine levels in mammals and in the provision of oxidized cysteine metabolites such as sulfate and taurine. Several crystal structure studies of mammalian CDO have shown that there is a cross-linked cofactor present in the active site of the enzyme. The cofactor consists of a thioether bond between the γ-sulfur of residue cysteine 93 and the arom. side chain of residue tyrosine 157. The exact requirements for cofactor synthesis and the contribution of the cofactor to the catalytic activity of the enzyme have yet to be fully described. In this study, therefore, we explored the factors necessary for cofactor biogenesis in vitro and in vivo and examd. what effect cofactor formation had on activity in vitro. Like other cross-linked cofactor-contg. enzymes, formation of the Cys-Tyr cofactor in CDO required a transition metal cofactor (Fe2+) and O2. Unlike other enzymes, however, biogenesis was also strictly dependent upon the presence of substrate. Cofactor formation was also appreciably slower than the rates reported for other enzymes and, indeed, took hundreds of catalytic turnover cycles to occur. In the absence of the Cys-Tyr cofactor, CDO possessed appreciable catalytic activity, suggesting that the cofactor was not essential for catalysis. Nevertheless, at physiol. relevant cysteine concns., cofactor formation increased CDO catalytic efficiency by ∼10-fold. Overall, the regulation of Cys-Tyr cofactor formation in CDO by ambient cysteine levels represents an unusual form of substrate-mediated feed-forward activation of enzyme activity with important physiol. consequences.
- 305Li, J.; Griffith, W. P.; Davis, I.; Shin, I.; Wang, J.; Li, F.; Wang, Y.; Wherritt, D. J.; Liu, A. Cleavage of a Carbon-Fluorine Bond by an Engineered Cysteine Dioxygenase. Nat. Chem. Biol. 2018, 14 (9), 853– 860, DOI: 10.1038/s41589-018-0085-5There is no corresponding record for this reference.
- 306Li, J.; Koto, T.; Davis, I.; Liu, A. Probing the Cys-Tyr Cofactor Biogenesis in Cysteine Dioxygenase by the Genetic Incorporation of Fluorotyrosine. Biochemistry 2019, 58 (17), 2218– 2227, DOI: 10.1021/acs.biochem.9b00006306Probing the Cys-Tyr Cofactor Biogenesis in Cysteine Dioxygenase by the Genetic Incorporation of FluorotyrosineLi, Jiasong; Koto, Teruaki; Davis, Ian; Liu, AiminBiochemistry (2019), 58 (17), 2218-2227CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Cysteine dioxygenase (CDO) is a nonheme iron enzyme that adds two oxygen atoms from dioxygen to the sulfur atom of L-cysteine. Adjacent to the iron site of mammalian CDO, there is a post-translationally generated Cys-Tyr cofactor, whose presence substantially enhances the oxygenase activity. The formation of the Cys-Tyr cofactor in CDO is an autocatalytic process, and it is challenging to study by traditional techniques because the crosslinking reaction is a side, uncoupled, single-turnover oxidn. buried among multiple turnovers of L-cysteine oxygenation. Here, we take advantage of our recent success in obtaining a purely uncross-linked human CDO due to site-specific incorporation of 3,5-difluoro-L-tyrosine (F2-Tyr) at the crosslinking site through the genetic code expansion strategy. Using EPR spectroscopy, we show that nitric oxide (•NO), an oxygen surrogate, similarly binds to uncross-linked F2-Tyr157 CDO as in wild-type human CDO. We detd. X-ray crystal structures of uncross-linked F2-Tyr157 CDO and mature wild-type CDO in complex with both L-cysteine and •NO. These structural data reveal that the active site cysteine (Cys93 in the human enzyme), rather than the generally expected tyrosine (i.e., Tyr157), is well-aligned to be oxidized should the normal oxidn. reaction uncouple. This structure-based understanding is further supported by a computational study with models built on the uncross-linked ternary complex structure. Together, these results strongly suggest that the first target to oxidize during the iron-assisted Cys-Tyr cofactor biogenesis is Cys93. Based on these data, a plausible reaction mechanism implementing a cysteine radical involved in the crosslink formation is proposed.
- 307Chen, L.; Naowarojna, N.; Song, H.; Wang, S.; Wang, J.; Deng, Z.; Zhao, C.; Liu, P. Use of a Tyrosine Analogue To Modulate the Two Activities of a Nonheme Iron Enzyme OvoA in Ovothiol Biosynthesis, Cysteine Oxidation versus Oxidative C-S Bond Formation. J. Am. Chem. Soc. 2018, 140 (13), 4604– 4612, DOI: 10.1021/jacs.7b13628307Use of a Tyrosine Analogue To Modulate the Two Activities of a Nonheme Iron Enzyme OvoA in Ovothiol Biosynthesis, Cysteine Oxidation versus Oxidative C-S Bond FormationChen, Li; Naowarojna, Nathchar; Song, Heng; Wang, Shu; Wang, Jiangyun; Deng, Zixin; Zhao, Changming; Liu, PinghuaJournal of the American Chemical Society (2018), 140 (13), 4604-4612CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Ovothiol is a histidine thiol deriv. The biosynthesis of ovothiol involves an extremely efficient trans-sulfuration strategy. The nonheme iron enzyme OvoA catalyzed oxidative coupling between cysteine and histidine is one of the key steps. Besides catalyzing the oxidative coupling between cysteine and histidine, OvoA also catalyzes the oxidn. of cysteine to cysteine sulfinic acid (cysteine dioxygenase activity). Thus far, very little mechanistic information is available for OvoA-catalysis. In this report, we measured the kinetic isotope effect (KIE) in OvoA-catalysis using the isotopically sensitive branching method. In addn., by replacing an active site tyrosine (Tyr417) with 2-amino-3-(4-hydroxy-3-(methylthio)phenyl)propanoic acid (MtTyr) through the amber suppressor mediated unnatural amino acid incorporation method, the two OvoA activities (oxidative coupling between cysteine and histidine, and cysteine dioxygenase activity) can be modulated. These results suggest that the two OvoA activities branch out from a common intermediate and that the active site tyrosine residue plays some key roles in controlling the partitioning between these two pathways.
- 308Trumpower, B. L.; Gennis, R. B. Energy Transduction By Cytochrome Complexes In Mitochondrial And Bacterial Respiration: The Enzymology of Coupling Electron Transfer Reactions to Transmembrane Proton Translocation. Annu. Rev. Biochem. 1994, 63 (1), 675– 716, DOI: 10.1146/annurev.bi.63.070194.003331There is no corresponding record for this reference.
- 309Kim, E.; Chufán, E. E.; Kamaraj, K.; Karlin, K. D. Synthetic Models for Heme-Copper Oxidases. Chem. Rev. 2004, 104 (2), 1077– 1134, DOI: 10.1021/cr0206162309Synthetic Models for Heme-Copper OxidasesKim, Eunsuk; Chufan, Eduardo E.; Kamaraj, Kaliappan; Karlin, Kenneth D.Chemical Reviews (Washington, DC, United States) (2004), 104 (2), 1077-1133CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. The newest directions in heme-copper oxidase modeling have come from examn. of compds. with reduced heme and copper ion complex 1:1 mixts. or heterobinuclear constructs. Thus, generation and characterization of carbon monoxide adducts of heme and/or copper provide preliminary insights into the binding of this O2 surrogate and allow probing of the heme-copper environment. However, it is dioxygen reactivity that has really led to exciting developments, including biomimetic functional modeling studies using electrochem. approaches and O2 reactivity studies leading to discrete superoxoheme (with copper present) and heme-peroxocopper assemblies. The latter may be directly relevant to an enzyme transient intermediate or may be a precursor to such (i.e., by protonation giving a heme hydroperoxo FeIII-OOH···CuII moiety). It was demonstrated that peroxospectroscopic signatures (and perhaps structures) can be influenced by binucleating ligand superstructure and copper-ligand denticity.
- 310Miner, K. D.; Mukherjee, A.; Gao, Y.-G.; Null, E. L.; Petrik, I. D.; Zhao, X.; Yeung, N.; Robinson, H.; Lu, Y. A Designed Functional Metalloenzyme that Reduces O2 to H2O with Over One Thousand Turnovers. Angew. Chem. Int. Ed. 2012, 51 (23), 5589– 5592, DOI: 10.1002/anie.201201981There is no corresponding record for this reference.
- 311Liu, X.; Yu, Y.; Hu, C.; Zhang, W.; Lu, Y.; Wang, J. Significant Increase of Oxidase Activity through the Genetic Incorporation of a Tyrosine-Histidine Cross-Link in a Myoglobin Model of Heme-Copper Oxidase. Angew. Chem. Int. Ed. 2012, 51 (18), 4312– 4316, DOI: 10.1002/anie.201108756311Significant Increase of Oxidase Activity through the Genetic Incorporation of a Tyrosine-Histidine Cross-Link in a Myoglobin Model of Heme-Copper OxidaseLiu, Xiaohong; Yu, Yang; Hu, Cheng; Zhang, Wei; Lu, Yi; Wang, JiangyunAngewandte Chemie, International Edition (2012), 51 (18), 4312-4316, S4312/1-S4312/6CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)By directly incorporating the unnatural amino acid imiTyr (1) into myoglobins in E. coli in response to the amber codon TAG, we have successfully designed a functional heme copper oxidase (HCO) model imiTyrCuBMb that catalyzes selective and efficient oxygen redn. to water. The HCO model imiTyrCuBMb bearing the Tyr-His cross-link is eightfold more selective with threefold more turnovers than F33YCuBMb, which does not contain the cross-link but harbors His and Tyr residues at the same positions in the same protein. Since the synthesis of imiTyr contains only two steps with 50% overall yield, and mutant proteins bearing imiTyr (1) at any site can be easily obtained and purified in milligram quantities through site-directed mutagenesis and recombinant protein expression, further systematic investigation of the function of the Tyr-His cross-link is now possible. While imiTyrCuBMb exhibits lower enzymic activity (2 02/min) in comparison to native heme-copper oxidase (ca. 300 02/s), it is possible to rapidly improve our HCO model to achieve higher activity through directed evolution and incorporation of unnatural amino acids. Our designed enzyme harbors the unnatural amino acid imiTyr, which is highly analogous to the post-translationally modified tyrosine-histidine ligand found in the CuB site of HCO; this designed enzyme serves as an ideal model for a more detailed understanding of HCOs and allows for potential applications in synthetic biol. and alternative energy.
- 312Yu, Y.; Lv, X.; Li, J.; Zhou, Q.; Cui, C.; Hosseinzadeh, P.; Mukherjee, A.; Nilges, M. J.; Wang, J.; Lu, Y. Defining the Role of Tyrosine and Rational Tuning of Oxidase Activity by Genetic Incorporation of Unnatural Tyrosine Analogs. J. Am. Chem. Soc. 2015, 137 (14), 4594– 4597, DOI: 10.1021/ja5109936312Defining the Role of Tyrosine and Rational Tuning of Oxidase Activity by Genetic Incorporation of Unnatural Tyrosine AnalogsYu, Yang; Lv, Xiaoxuan; Li, Jiasong; Zhou, Qing; Cui, Chang; Hosseinzadeh, Parisa; Mukherjee, Arnab; Nilges, Mark J.; Wang, Jiangyun; Lu, YiJournal of the American Chemical Society (2015), 137 (14), 4594-4597CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)While a conserved tyrosine (Tyr) is found in oxidases, the roles of phenol ring pKa and redn. potential in O2 redn. have not been defined despite many years of research on numerous oxidases and their models. These issues represent major challenges in our understanding of O2 redn. mechanism in bioenergetics. Through genetic incorporation of unnatural amino acid analogs of Tyr, with progressively decreasing pKa of the phenol ring and increasing redn. potential, in the active site of a functional model of oxidase in myoglobin, a linear dependence of both the O2 redn. activity and the fraction of H2O formation with the pKa of the phenol ring has been established. By using these unnatural amino acids as spectroscopic probe, we have provided conclusive evidence for the location of a Tyr radical generated during reaction with H2O2, by the distinctive hyperfine splitting patterns of the halogenated tyrosines and one of its deuterated derivs. incorporated at the 33 position of the protein. These results demonstrate for the first time that enhancing the proton donation ability of the Tyr enhances the oxidase activity, allowing the Tyr analogs to augment enzymic activity beyond that of natural Tyr. 3-Chlorotyrosine (ClTyr), 3,5-difluorotyrosine (F2Tyr), and 2,3,5-trifluorotyrosine.
- 313Yu, Y.; Zhou, Q.; Wang, L.; Liu, X.; Zhang, W.; Hu, M.; Dong, J.; Li, J.; Lv, X.; Ouyang, H. Significant Improvement of Oxidase Activity Through the Genetic Incorporation of a Redox-Active Unnatural Amino Acid. Chem. Sci. 2015, 6 (7), 3881– 3885, DOI: 10.1039/C5SC01126D313Significant improvement of oxidase activity through the genetic incorporation of a redox-active unnatural amino acidYu, Yang; Zhou, Qing; Wang, Li; Liu, Xiaohong; Zhang, Wei; Hu, Meirong; Dong, Jianshu; Li, Jiasong; Lv, Xiaoxuan; Ouyang, Hanlin; Li, Han; Gao, Feng; Gong, Weimin; Lu, Yi; Wang, JiangyunChemical Science (2015), 6 (7), 3881-3885CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)While Nature employs various covalent and noncovalent strategies to modulate tyrosine redox potential and pKa in order to optimize enzyme activities, such approaches have not been systematically applied for the design of functional metalloproteins. Here, through the genetic incorporation of 3-methoxytyrosine (I) into myoglobin, the authors replicated important features of cytochrome c oxidase (CcO) in this small sol. protein, which exhibited selective O2 redn. activity while generating a small amt. of reactive O species (ROS). These results demonstrated that the electron-donating ability of a Tyr residue in the active site is important for CcO function. Moreover, the authors elucidated the structural basis for the genetic incorporation of I into proteins by solving the x-ray structure of I-specific aminoacyl-tRNA synthetase complexed with I.
- 314Rigoldi, F.; Donini, S.; Redaelli, A.; Parisini, E.; Gautieri, A. Review: Engineering of Thermostable Enzymes for Industrial Applications. APL Bioengineering 2018, 2 (1), 011501, DOI: 10.1063/1.4997367There is no corresponding record for this reference.
- 315Sheldon, R. A.; Basso, A.; Brady, D. New Frontiers in Enzyme Immobilisation: Robust Biocatalysts for a Circular Bio-Based Economy. Chem. Soc. Rev. 2021, 50 (10), 5850– 5862, DOI: 10.1039/D1CS00015B315New frontiers in enzyme immobilization: robust biocatalysts for a circular bio-based economySheldon, Roger A.; Basso, Alessandra; Brady, DeanChemical Society Reviews (2021), 50 (10), 5850-5862CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. This tutorial review focuses on recent advances in technologies for enzyme immobilization, enabling their cost-effective use in the bio-based economy and continuous processing in general. The application of enzymes, particularly in aq. media, is generally on a single use, throw-away basis which is neither cost-effective nor compatible with a circular economy concept. This shortcoming can be overcome by immobilizing the enzyme as an insol. recyclable solid, that is as a heterogeneous catalyst.
- 316Baker, P. J.; Montclare, J. K. Enhanced Refoldability and Thermoactivity of Fluorinated Phosphotriesterase. ChemBioChem 2011, 12 (12), 1845– 1848, DOI: 10.1002/cbic.201100221There is no corresponding record for this reference.
- 317Deepankumar, K.; Shon, M.; Nadarajan, S. P.; Shin, G.; Mathew, S.; Ayyadurai, N.; Kim, B.-G.; Choi, S.-H.; Lee, S.-H.; Yun, H. Enhancing Thermostability and Organic Solvent Tolerance of ω-Transaminase through Global Incorporation of Fluorotyrosine. Adv. Synth. Catal. 2014, 356 (5), 993– 998, DOI: 10.1002/adsc.201300706There is no corresponding record for this reference.
- 318Ohtake, K.; Mukai, T.; Iraha, F.; Takahashi, M.; Haruna, K.-i.; Date, M.; Yokoyama, K.; Sakamoto, K. Engineering an Automaturing Transglutaminase with Enhanced Thermostability by Genetic Code Expansion with Two Codon Reassignments. ACS Synth. Biol. 2018, 7 (9), 2170– 2176, DOI: 10.1021/acssynbio.8b00157318Engineering an Automaturing Transglutaminase with Enhanced Thermostability by Genetic Code Expansion with Two Codon ReassignmentsOhtake, Kazumasa; Mukai, Takahito; Iraha, Fumie; Takahashi, Mihoko; Haruna, Ken-ichi; Date, Masayo; Yokoyama, Keiichi; Sakamoto, KensakuACS Synthetic Biology (2018), 7 (9), 2170-2176CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)In the present study, we simultaneously incorporated 2 types of synthetic components into microbial transglutaminase (MTG) from Streptoverticillium mobaraense, to enhance the utility of this industrial enzyme. The 1st amino acid, 3-chloro-L-tyrosine, was incorporated into MTG in response to in-frame UAG codons, to substitute for the 15 Tyr residues sep. Two substitutions at positions 20 and 62 were found to each increase the thermostability of the enzyme, while 7 substitutions (at positions 24, 34, 75, 146, 171, 217, and 310) exhibited neutral effects. Then, these 2 stabilizing chlorinations were combined with one of the neutral ones, and the most stabilized variant was found to contain 3-chlorotyrosines at positions 20, 62, and 171, exhibiting a 5.1-fold longer half-life than the wild-type enzyme at 60°. Next, this MTG variant was further modified by incorporating the α-hydroxy acid analog of Nε-allyloxycarbonyl-L-lysine (AlocKOH), specified by the AGG codon, at the end of the N-terminal inhibitory peptide. We used an Escherichia coli strain previously engineered to have a synthetic genetic code with 2 codon reassignments, for synthesizing MTG variants contg. both 3-chlorotyrosine and AlocKOH. The ester bond, thus incorporated into the main chain, efficiently self-cleaved under alk. conditions (pH 11.0), achieving the autonomous maturation of the thermostabilized MTG. The results suggested that synthetic genetic codes with multiple codon reassignments would be useful for developing the novel designs of enzymes.
- 319Politzer, P.; Murray, J. S. Halogen Bonding: An Interim Discussion. ChemPhysChem 2013, 14 (2), 278– 294, DOI: 10.1002/cphc.201200799319Halogen Bonding: An Interim DiscussionPolitzer, Peter; Murray, Jane S.ChemPhysChem (2013), 14 (2), 278-294CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Halogen bonding is a noncovalent interaction that is receiving rapidly increasing attention because of its significance in biol. systems and its importance in the design of new materials in a variety of areas, for example, electronics, nonlinear optical activity, and pharmaceuticals. The interactions can be understood in terms of electrostatics/polarization and dispersion; they involve a region of pos. electrostatic potential on a covalently bonded halogen and a neg. site, such as the lone pair of a Lewis base. The pos. potential, labeled a σ hole, is on the extension of the covalent bond to the halogen, which accounts for the characteristic near-linearity of halogen bonding. In many instances, the lateral sides of the halogen have neg. electrostatic potentials, allowing it to also interact favorably with pos. sites. In this discussion, after looking at some of the exptl. observations of halogen bonding, we address the origins of σ holes, the factors that govern the magnitudes of their electrostatic potentials, and the properties of the resulting complexes with neg. sites. The relationship of halogen and hydrogen bonding is examd. We also point out that σ-hole interactions are not limited to halogens, but can also involve covalently bonded atoms of Groups IV-VI. Examples of applications in biol./medicinal chem. and in crystal engineering are mentioned, taking note that halogen bonding can be "tuned" to fit various requirements, i.e., strength of interaction, steric factors, and so forth.
- 320Scholfield, M. R.; Ford, M. C.; Carlsson, A.-C. C.; Butta, H.; Mehl, R. A.; Ho, P. S. Structure-Energy Relationships of Halogen Bonds in Proteins. Biochemistry 2017, 56 (22), 2794– 2802, DOI: 10.1021/acs.biochem.7b00022320Structure-Energy Relationships of Halogen Bonds in ProteinsScholfield, Matthew R.; Ford, Melissa Coates; Carlsson, Anna-Carin C.; Butta, Hawera; Mehl, Ryan A.; Ho, P. ShingBiochemistry (2017), 56 (22), 2794-2802CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The structures and stabilities of proteins are defined by a series of weak non-covalent electrostatic, van der Waals, and H-bond (HB) interactions. Here, we designed and engineered halogen bonds (XBs) site-specifically in order to study their structure-energy relations in a model protein, phage T4 lysozyme. The evidence for XBs is the displacement of the arom. side-chain toward an oxygen acceptor, at distances that are at or less than the sums of their resp. van der Waals radii, when the hydroxyl substituent of the wild-type Tyr residue is replaced by iodine. In addn., thermal melting studies showed that the iodine XB rescued the stabilization energy from an otherwise destabilizing substitution (at an equiv. non-interacting site), indicating that the interaction is also present in soln. Quantum chem. calcns. showed that the XB complements an HB at this site and that solvent structure must also be considered in trying to design mol. interactions such as XBs into biol. systems. A Br substitution also showed displacement of the side-chain, but the distances and geometries did not indicate formation of an XB. Thus, we have dissected the contributions from various noncovalent interactions of halogens introduced into proteins, to drive the application of XBs, particularly in biomol. design.
- 321Acevedo-Rocha, C. G.; Hoesl, M. G.; Nehring, S.; Royter, M.; Wolschner, C.; Wiltschi, B.; Antranikian, G.; Budisa, N. Non-Canonical Amino Acids as a Useful Synthetic Biological Tool for Lipase-Catalysed Reactions in Hostile Environments. Catal. Sci. Technol. 2013, 3 (5), 1198– 1201, DOI: 10.1039/c3cy20712aThere is no corresponding record for this reference.
- 322Mendel, D.; Ellman, J. A.; Chang, Z.; Veenstra, D. L.; Kollman, P. A.; Schultz, P. G. Probing Protein Stability with Unnatural Amino Acids. Science 1992, 256 (5065), 1798– 1802, DOI: 10.1126/science.1615324322Probing protein stability with unnatural amino acidsMendel, David; Ellman, Jonathan A.; Chang, Zhiyuh; Veenstra, David L.; Kollman, Peter A.; Schultz, Peter G.Science (Washington, DC, United States) (1992), 256 (5065), 1798-802CODEN: SCIEAS; ISSN:0036-8075.Unnatural amino acid mutagenesis, in combination with mol. modeling and simulation techniques, was used to probe the effect of side chain structure on protein stability. Specific replacements at position 133 in T4 lysozyme included (i) leucine (wt), norvaline, ethyl-glycine, and alanine to measure the cost of stepwise removal of Me groups from the hydrophobic core, (ii) norvaline and O-Me serine to evaluate the effects of side chain solvation, and (iii) leucine, S,S-2-amino-4-methylhexanoic acid, and S-2-amino-3-cyclopentylpropanoic acid to measure the influence of packing d. and side chain conformational entropy on protein stability. All of these factors (hydrophobicity, packing, conformational entropy, and cavity formation) significantly influence protein stability and must be considered when analyzing any structural change to proteins.
- 323Ismail, A. R.; Kashtoh, H.; Baek, K.-H. Temperature-Resistant and Solvent-Tolerant Lipases as Industrial Biocatalysts: Biotechnological Approaches and Applications. Int. J. Biol. Macromol. 2021, 187, 127– 142, DOI: 10.1016/j.ijbiomac.2021.07.101323Temperature-resistant and solvent-tolerant lipases as industrial biocatalysts: Biotechnological approaches and applicationsIsmail, Abdallah R.; Kashtoh, Hamdy; Baek, Kwang-HyunInternational Journal of Biological Macromolecules (2021), 187 (), 127-142CODEN: IJBMDR; ISSN:0141-8130. (Elsevier B.V.)The development of new biocatalytic systems to replace the chem. catalysts, with suitable characteristics in terms of efficiency, stability under high temp. reactions and in the presence of org. solvents, reusability, and eco-friendliness is considered a very important step to move towards the green processes. From this basis, the use of lipase as a catalyst is highly desired for many industrial applications because it offers the reactions in which could be used, stability in harsh conditions, reusability and a greener process. Therefore, the introduction of temp.-resistant and solvent-tolerant lipases have become essential and ideal for industrial applications. Temp.-resistant and solvent-tolerant lipases have been involved in many large-scale applications including biodiesel, detergent, food, pharmaceutical, org. synthesis, biosensing, pulp and paper, textile, animal feed, cosmetics, and leather industry. So, the present review provides a comprehensive overview of the industrial use of lipase. Moreover, special interest in biotechnol. and biochem. techniques for enhancing temp.-resistance and solvent-tolerance of lipases to be suitable for the industrial uses.
- 324Vivek, K.; Sandhia, G. S.; Subramaniyan, S. Extremophilic Lipases for Industrial Applications: A General Review. Biotechnol. Adv. 2022, 60, 108002, DOI: 10.1016/j.biotechadv.2022.108002324Extremophilic lipases for industrial applications: A general reviewVivek, K.; Sandhia, G. S.; Subramaniyan, S.Biotechnology Advances (2022), 60 (), 108002CODEN: BIADDD; ISSN:0734-9750. (Elsevier Inc.)A Review on. With industrialization and development in modern science enzymes and their applications increased widely. There is always a hunt for new proficient enzymes with novel properties to meet specific needs of various industrial sectors. Along with the high efficiency, the green and eco-friendly side of enzymes attracts human attention, as they form a true answer to counter the hazardous and toxic conventional industrial catalyst. Lipases have always earned industrial attention due to the broad range of hydrolytic and synthetic reactions they catalyze. When these catalytic properties get accompanied by features like temp. stability, pH stability, and solvent stability lipases becomes an appropriate tool for use in many industrial processes. Extremophilic lipases offer the same, thermostable: hot and cold active thermophilic and psychrophilic lipases, acid and alkali resistant and active acidophilic and alkaliphilic lipases, and salt tolerant halophilic lipases form excellent biocatalyst for detergent formulations, biofuel synthesis, ester synthesis, food processing, pharmaceuticals, leather, and paper industry. An interesting application of these lipases is in the bioremediation of lipid waste in harsh environments. The review gives a brief account on various extremophilic lipases with emphasis on thermophilic, psychrophilic, halophilic, alkaliphilic, and acidophilic lipases, their sources, biochem. properties, and potential applications in recent decades.
- 325Budisa, N.; Wenger, W.; Wiltschi, B. Residue-Specific Global Fluorination of Candida antarctica Lipase B in Pichia pastoris. Mol. BioSyst. 2010, 6 (9), 1630– 1639, DOI: 10.1039/c002256j325Residue-specific global fluorination of Candida antarctica lipase B in Pichia pastorisBudisa, Nediljko; Wenger, Waltraud; Wiltschi, BirgitMolecular BioSystems (2010), 6 (9), 1630-1639CODEN: MBOIBW; ISSN:1742-206X. (Royal Society of Chemistry)We report the in vivo fluorination of the tryptophan, tyrosine, and phenylalanine residues in a glycosylation-deficient mutant of Candida antarctica lipase B, CalB N74D, expressed in the methylotrophic yeast Pichia pastoris and subsequently segregated into the growth medium. To achieve this, a P. pastoris strain auxotrophic for all three arom. amino acids was supplemented with 5-fluoro-L-tryptophan, meta-fluoro-(DL)-tyrosine, or para-fluoro-L-phenylalanine during expression of CalB N74D. The residue-specific replacement of the canonical amino acids by their fluorinated analogs was confirmed by mass anal. Although global fluorination induced moderate changes in the secondary structure of CalB N74D, the fluorous variant proteins were still active lipases. However, their catalytic activity was lower than that of the non-fluorinated parent protein while their resistance to proteolytic degrdn. by proteinase K remained unchanged. Importantly, we obsd. that the global fluorination prolonged the shelf life of the lipase activity, which is an esp. useful feature for the storage of, e.g., therapeutic proteins. Our study represents the first step on the road to the prodn. of biotechnol. and pharmacol. relevant fluorous proteins in P. pastoris.
- 326Merkel, L.; Schauer, M.; Antranikian, G.; Budisa, N. Parallel Incorporation of Different Fluorinated Amino Acids: On the Way to “Teflon” Proteins. ChemBioChem 2010, 11 (11), 1505– 1507, DOI: 10.1002/cbic.201000295There is no corresponding record for this reference.
- 327Hoesl, M. G.; Acevedo-Rocha, C. G.; Nehring, S.; Royter, M.; Wolschner, C.; Wiltschi, B.; Budisa, N.; Antranikian, G. Lipase Congeners Designed by Genetic Code Engineering. ChemCatChem 2011, 3 (1), 213– 221, DOI: 10.1002/cctc.201000253There is no corresponding record for this reference.
- 328Kelly, S. A.; Pohle, S.; Wharry, S.; Mix, S.; Allen, C. C. R.; Moody, T. S.; Gilmore, B. F. Application of ω-Transaminases in the Pharmaceutical Industry. Chem. Rev. 2018, 118 (1), 349– 367, DOI: 10.1021/acs.chemrev.7b00437328Application of ω-Transaminases in the Pharmaceutical IndustryKelly, Stephen A.; Pohle, Stefan; Wharry, Scott; Mix, Stefan; Allen, Christopher C. R.; Moody, Thomas S.; Gilmore, Brendan F.Chemical Reviews (Washington, DC, United States) (2018), 118 (1), 349-367CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Chiral amines are valuable building blocks for the pharmaceutical industry. ω-TAms have emerged as an exciting option for their synthesis, offering a potential "green alternative" to overcome the drawbacks assocd. with conventional chem. methods. In this review, we explore the application of ω-TAms for pharmaceutical prodn. We discuss the diverse array of reactions available involving ω-TAms and process considerations of their use in both kinetic resoln. and asym. synthesis. With the aid of specific drug intermediates and APIs, we chart the development of ω-TAms using protein engineering and their contribution to elegant one-pot cascades with other enzymes, including carbonyl reductases (CREDs), hydrolases and monoamine oxidases (MAOs), providing a comprehensive overview of their uses, beginning with initial applications through to the present day.
- 329Mathew, S.; Yun, H. ω-Transaminases for the Production of Optically Pure Amines and Unnatural Amino Acids. ACS Catal. 2012, 2 (6), 993– 1001, DOI: 10.1021/cs300116n329ω-Transaminases for the Production of Optically Pure Amines and Unnatural Amino AcidsMathew, Sam; Yun, HyungdonACS Catalysis (2012), 2 (6), 993-1001CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)A review. ω-Transaminases have been increasingly used as efficient biocatalysts due to their ability to produce a wide range of optically pure amine compds. Several approaches have been adopted, including screening, engineering, and development of new techniques in reaction systems for different aspects of the enzymes. This review summarizes the various methodologies and approaches adopted to produce enantiomerically pure amines and unnatural amino acids using ω-transaminases.
- 330Won, Y.; Jeon, H.; Pagar, A. D.; Patil, M. D.; Nadarajan, S. P.; Flood, D. T.; Dawson, P. E.; Yun, H. In vivo Biosynthesis of Tyrosine Analogs and their Concurrent Incorporation into a Residue-Specific Manner for Enzyme Engineering. Chem. Commun. 2019, 55 (100), 15133– 15136, DOI: 10.1039/C9CC08503CThere is no corresponding record for this reference.
- 331Votchitseva, Y. A.; Efremenko, E. N.; Varfolomeyev, S. D. Insertion of an Unnatural Amino Acid into the Protein Structure: Preparation and Properties of 3-Fluorotyrosine-Containing Organophosphate Hydrolase. Russ. Chem. Bull. 2006, 55 (2), 369– 374, DOI: 10.1007/s11172-006-0262-7There is no corresponding record for this reference.
- 332Castro, A. A. d.; Prandi, I. G.; Kuca, K.; Ramalho, T. C. Enzimas Degradantes de Organofosforados: Base Molecular e Perspectivas para Biorremediação Enzimática de Agroquímicos. Ciênc. Agrotec. 2017, 41 (5), 471, DOI: 10.1590/1413-70542017415000417There is no corresponding record for this reference.
- 333Makkar, R. S.; DiNovo, A. A.; Westwater, C.; Schofield, D. A. Enzyme-Mediated Bioremediation of Organophosphates using Stable Yeast Biocatalysts. J. Bioremed. Biodeg. 2013, 4 (182), 2, DOI: 10.4172/2155-6199.1000182There is no corresponding record for this reference.
- 334Holzberger, B.; Marx, A. Replacing 32 Proline Residues by a Noncanonical Amino Acid Results in a Highly Active DNA Polymerase. J. Am. Chem. Soc. 2010, 132 (44), 15708– 15713, DOI: 10.1021/ja106525y334Replacing 32 Proline Residues by a Noncanonical Amino Acid Results in a Highly Active DNA PolymeraseHolzberger, Bastian; Marx, AndreasJournal of the American Chemical Society (2010), 132 (44), 15708-15713CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein engineering may be achieved by rational design, directed evolution-based methods, or computational protein design. Mostly these methods make recourse to the restricted pool of the 20 natural amino acids. With the ability to introduce different new kinds of functionalities into proteins, the use of noncanonical amino acids became a promising new method in protein engineering. Here, we report on the generation of a multifluorinated DNA polymerase. DNA polymerases are highly dynamic enzymes that catalyze DNA synthesis in a template-dependent manner, thereby passing several conformational states during the catalytic cycle. Here, we globally replaced 32 proline residues by the noncanonical imino acid (4R)-fluoroproline in a DNA polymerase of 540 amino acids (KlenTaq DNA polymerase). Interestingly, the substitution level of the proline residues was very efficient (92%). Nonetheless, the introduction of (4R)-fluoroproline into the DNA polymerase resulted in a highly active fluorinated enzyme, which was investigated in primer extension and PCR assays to analyze activity, selectivity, and stability in comparison to the parental enzyme. The DNA polymerase retained fidelity, activity, and sensitivity as the parental wild-type enzyme accompanied by some loss in thermostability. These results demonstrate that a noncanonical amino acid can be used for substitutions of natural counterparts in a highly dynamic enzyme with high mol. wt. without effecting crucial enzyme properties. Furthermore, the employed DNA polymerase represents a promising starting point for directed DNA polymerase evolution with noncanonical amino acids.
- 335Holzberger, B.; Obeid, S.; Welte, W.; Diederichs, K.; Marx, A. Structural Insights into the Potential of 4-Fluoroproline to Modulate Biophysical Properties of Proteins. Chem. Sci. 2012, 3 (10), 2924– 2931, DOI: 10.1039/c2sc20545aThere is no corresponding record for this reference.
- 336Panchenko, T.; Zhu, W. W.; Montclare, J. K. Influence of Global Fluorination on Chloramphenicol Acetyltransferase Activity and Stability. Biotechnol. Bioeng. 2006, 94 (5), 921– 930, DOI: 10.1002/bit.20910336Influence of global fluorination on chloramphenicol acetyltransferase activity and stabilityPanchenko, Tatyana; Zhu, Wan Wen; Montclare, Jin KimBiotechnology and Bioengineering (2006), 94 (5), 921-930CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)Varied levels of fluorinated amino acid have been introduced biosynthetically to test the functional limits of global substitution on enzymic activity and stability. Replacement of all the leucine (LEU) residues in the enzyme chloramphenicol acetyltransferase (CAT) with the analog, 5',5',5'-trifluoroleucine (TFL), results in the maintenance of enzymic activity under ambient temps. as well as an enhancement in secondary structure but loss in stability against heat and denaturants or org. co-solvents. Although catalytic activity of the fully substituted CAT is preserved under std. reaction conditions compared to the wild-type enzyme both in vitro and in vivo, as the incorporation levels increase, a concomitant redn. in thermostability and chemostability is obsd. CD studies reveal that although fluorination greatly improves the secondary structure of CAT, a large structural destabilization upon increased levels of TFL incorporation occurs at elevated temps. These data suggest that enhanced secondary structure afforded by TFL incorporation does not necessarily lead to an improvement in stability.
- 337Yang, C.-Y.; Renfrew, P. D.; Olsen, A. J.; Zhang, M.; Yuvienco, C.; Bonneau, R.; Montclare, J. K. Improved Stability and Half-Life of Fluorinated Phosphotriesterase Using Rosetta. ChemBioChem 2014, 15 (12), 1761– 1764, DOI: 10.1002/cbic.201402062There is no corresponding record for this reference.
- 338Ohtake, K.; Yamaguchi, A.; Mukai, T.; Kashimura, H.; Hirano, N.; Haruki, M.; Kohashi, S.; Yamagishi, K.; Murayama, K.; Tomabechi, Y. Protein Stabilization Utilizing a Redefined Codon. Sci. Rep. 2015, 5 (1), 9762, DOI: 10.1038/srep09762There is no corresponding record for this reference.
- 339Carlsson, A. C.; Scholfield, M. R.; Rowe, R. K.; Ford, M. C.; Alexander, A. T.; Mehl, R. A.; Ho, P. S. Increasing Enzyme Stability and Activity through Hydrogen Bond-Enhanced Halogen Bonds. Biochemistry 2018, 57 (28), 4135– 4147, DOI: 10.1021/acs.biochem.8b00603339Increasing Enzyme Stability and Activity through Hydrogen Bond-Enhanced Halogen BondsCarlsson, Anna-Carin C.; Scholfield, Matthew R.; Rowe, Rhianon K.; Ford, Melissa Coates; Alexander, Austin T.; Mehl, Ryan A.; Ho, P. ShingBiochemistry (2018), 57 (28), 4135-4147CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The construction of more stable proteins is important in biomol. engineering, particularly in the design of biologics based therapeutics. We show here that replacing the tyrosine at position 18 (Y18) of T4 lysozyme with the unnatural amino acid meta-chlorotyrosine (mClY) increases both the thermal stability (raising the melting temp. by ∼1°C and melting enthalpy by 3 kcal/mol) and enzymic activity at elevated temps. (15% higher than the parent enzyme at 40°C) of this classic enzyme. The chlorine of mClY forms a halogen bond (XB) to the carbonyl oxygen of the peptide bond at glycine 28 (G28) in a tight loop near the active site. In this case, the XB potential of the typically weak XB donor Cl is shown from quantum chem. calcns. to be significantly enhanced by polarization via an intramol. hydrogen bond (HB) from the adjacent hydroxyl substituent of the tyrosyl side-chain, resulting in a distinctive synergistic HB enhanced XB (or HeX-B for short) interaction. The larger halogens (bromine and iodine) are not well accommodated within this same loop and, consequently, do not exhibit the effects on protein stability or function assocd. with the HeX-B interaction. Thus, we have for the first time demonstrated that an XB can be engineered to stabilize and increase the activity of an enzyme, with the increased stabilizing potential of the HeX-B further extending the application of halogenated amino acids in the design of more stable protein therapeutics.
- 340Nicholson, H.; Anderson, D. E.; Dao Pin, S.; Matthews, B. W. Analysis of the Interaction Between Charged Side Chains and the Alpha-Helix Dipole Using Designed Thermostable Mutants of Phage T4 Lysozyme. Biochemistry 1991, 30 (41), 9816– 9828, DOI: 10.1021/bi00105a002There is no corresponding record for this reference.
- 341Klink, T. A.; Woycechowsky, K. J.; Taylor, K. M.; Raines, R. T. Contribution of Disulfide Bonds to the Conformational Stability and Catalytic Activity Of Ribonuclease A. Eur. J. Biochem. 2000, 267 (2), 566– 572, DOI: 10.1046/j.1432-1327.2000.01037.x341Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease AKlink, Tony A.; Woycechowsky, Kenneth J.; Taylor, Kimberly M.; Raines, Ronald T.European Journal of Biochemistry (2000), 267 (2), 566-572CODEN: EJBCAI; ISSN:0014-2956. (Blackwell Science Ltd.)Disulfide bonds between the side-chains of Cys residues are the only common crosslinks in proteins. Bovine pancreatic RNase A is a 124-residue enzyme that contains 4 interweaving disulfide bonds (Cys-26-Cys-84, Cys-40-Cys-95, Cys-58-Cys-110, and Cys-65-Cys-72) and catalyzes the cleavage of RNA. Here, the contribution of each disulfide bond to the conformational stability and catalytic activity of RNase A was detd. by using variants in which each cystine residue was replaced independently with a pair of Ala residues. Thermal unfolding expts. monitored by UV spectroscopy and DSC revealed that wild-type RNase A and each disulfide variant unfolded in a 2-state process and that each disulfide bond contributed substantially to conformational stability. The 2 terminal disulfide bonds in the amino acid sequence (Cys-26-Cys-84 and Cys-58-Cys-110) enhanced the stability more than did the 2 embedded ones (Cys-40-Cys-95 and Cys-65-Cys-72). Removing either one of the terminal disulfide bonds liberated a similar no. of residues and had a similar effect on conformational stability, decreasing the midpoint of the thermal transition by almost 40°. The disulfide variants catalyzed the cleavage of poly(cytidylic acid) with values of kcat/Km that were 2- to 40-fold less than that of wild-type RNase A. The 2 embedded disulfide bonds, which were least important to conformational stability, were most important to catalytic activity. These embedded disulfide bonds likely contribute to the proper alignment of residues (such as Lys-41 and Lys-66) that are necessary for efficient catalysis of RNA cleavage.
- 342Yin, X.; Hu, D.; Li, J.-F.; He, Y.; Zhu, T.-D.; Wu, M.-C. Contribution of Disulfide Bridges to the Thermostability of a Type A Feruloyl Esterase from Aspergillus usamii. PLOS ONE 2015, 10 (5), e0126864 DOI: 10.1371/journal.pone.0126864342Contribution of disulfide bridges to the thermostability of a type A feruloyl esterase from Aspergillus usamiiYin, Xin; Hu, Die; Li, Jian-Fang; He, Yao; Zhu, Tian-Di; Wu, Min-ChenPLoS One (2015), 10 (5), e0126864/1-e0126864/16CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)The contribution of disulfide bridges to the thermostability of a type A feruloyl esterase (AuFaeA) from Aspergillus usamii E001 was studied by introducing an extra disulfide bridge or eliminating a native one from the enzyme. MODIP and DbD, two computational tools that can predict the possible disulfide bridges in proteins for thermostability improvement, and mol. dynamics (MD) simulations were used to design the extra disulfide bridge. One residue pair A126-N152 was chosen, and the resp. amino acid residues were mutated to cysteine. The wild-type AuFaeA and its variants were expressed in Pichia pastoris GS115. The temp. optimum of the recombinant (re-) AuFaeAA126C-N152C was increased by 6°C compared to that of re-AuFaeA. The thermal inactivation half-lives of re-AuFaeAA126C-N152C at 55 and 60°C were 188 and 40 min, which were 12.5- and 10-folds longer than those of re-AuFaeA. The catalytic efficiency (kcat/Km) of re-AuFaeAA126C-N152C was similar to that of re-AuFaeA. Addnl., after elimination of each native disulfide bridge in AuFaeA, a great decrease in expression level and at least 10°C decrease in thermal stability of recombinant AuEaeA variants were also obsd.
- 343Gihaz, S.; Bash, Y.; Rush, I.; Shahar, A.; Pazy, Y.; Fishman, A. Bridges to Stability: Engineering Disulfide Bonds Towards Enhanced Lipase Biodiesel Synthesis. ChemCatChem 2020, 12 (1), 181– 192, DOI: 10.1002/cctc.201901369There is no corresponding record for this reference.
- 344Zhou, X.; Xu, Z.; Li, Y.; He, J.; Zhu, H. Improvement of the Stability and Activity of an LPMO Through Rational Disulfide Bonds Design. Front. bioeng. biotechnol. 2022, DOI: 10.3389/fbioe.2021.815990There is no corresponding record for this reference.
- 345Dombkowski, A. A.; Sultana, K. Z.; Craig, D. B. Protein Disulfide Engineering. FEBS Lett. 2014, 588 (2), 206– 212, DOI: 10.1016/j.febslet.2013.11.024345Protein disulfide engineeringDombkowski, Alan A.; Sultana, Kazi Zakia; Craig, Douglas B.FEBS Letters (2014), 588 (2), 206-212CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)A review. Improving the stability of proteins is an important goal in many biomedical and industrial applications. A logical approach is to emulate stabilizing mol. interactions found in nature. Disulfide bonds are covalent interactions that provide substantial stability to many proteins and conform to well-defined geometric conformations, thus making them appealing candidates in protein engineering efforts. Disulfide engineering is the directed design of novel disulfide bonds into target proteins. This important biotechnol. tool has achieved considerable success in a wide range of applications, yet the rules that govern the stabilizing effects of disulfide bonds are not fully characterized. Contrary to expectations, many designed disulfide bonds have resulted in decreased stability of the modified protein. The authors review progress in disulfide engineering, with an emphasis on the issue of stability and computational methods that facilitate engineering efforts.
- 346Liu, T.; Wang, Y.; Luo, X.; Li, J.; Reed, S. A.; Xiao, H.; Young, T. S.; Schultz, P. G. Enhancing Protein Stability with Extended Disulfide Bonds. Proc. Natl. Acad. Sci. U.S.A. 2016, 113 (21), 5910– 5915, DOI: 10.1073/pnas.1605363113346Enhancing protein stability with extended disulfide bondsLiu, Tao; Wang, Yan; Luo, Xiaozhou; Li, Jack; Reed, Sean A.; Xiao, Han; Young, Travis S.; Schultz, Peter G.Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (21), 5910-5915CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Disulfide bonds play an important role in protein folding and stability. However, the crosslinking of sites within proteins by cysteine disulfides has significant distance and dihedral angle constraints. Here we report the genetic encoding of noncanonical amino acids contg. long side-chain thiols that are readily incorporated into both bacterial and mammalian proteins in good yields and with excellent fidelity. These amino acids can pair with cysteines to afford extended disulfide bonds and allow crosslinking of more distant sites and distinct domains of proteins. To demonstrate this notion, we preformed growth-based selection expts. at nonpermissive temps. using a library of random β-lactamase mutants contg. these noncanonical amino acids. A mutant enzyme that is cross-linked by one such extended disulfide bond and is stabilized by ∼9 °C was identified. This result indicates that an expanded set of building blocks beyond the canonical 20 amino acids can lead to proteins with improved properties by unique mechanisms, distinct from those possible through conventional mutagenesis schemes.
- 347Hecky, J.; Müller, K. M. Structural Perturbation and Compensation by Directed Evolution at Physiological Temperature Leads to Thermostabilization of β-Lactamase. Biochemistry 2005, 44 (38), 12640– 12654, DOI: 10.1021/bi0501885347Structural perturbation and compensation by directed evolution at physiological temperature leads to thermostabilization of β-lactamaseHecky, Jochen; Mueller, Kristian M.Biochemistry (2005), 44 (38), 12640-12654CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The choice of protein for use in tech. and medical applications is limited by stability issues, making understanding and engineering of stability key. Here, enzyme destabilization by truncation was combined with directed evolution to create stable variants of TEM-1 β-lactamase (I). I was chosen because of its implication in prodrug activation therapy, pathogen resistance to lactam antibiotics, and reporter enzyme bioassays. Removal of 5 N-terminal residues generated a mutant which did not confer antibiotic resistance at 37°. Accordingly, the half-life time in vitro was only 7 s at 40°. However, 3 cycles comprising random mutagenesis, DNA shuffling, and metabolic selection at 37° yielded mutants providing resistance levels significantly higher than that of wild-type I. These mutants demonstrated increased thermoactivity and thermostability in time-resolved kinetics at various temps. Chem. denaturation revealed improved thermodn. stabilities of a 3-state unfolding pathway exceeding wild-type construct stability. Elongation of one optimized deletion mutant to full length increased its stability even further. Compared to that of wild-type I, the temp. optimum was shifted from 35 to 50°, and the beginning of heat inactivation increased by 20° while full activity at low temps. was maintained. These effects were attributed mainly to 2 independently acting boundary interface residue exchanges (M182T and A224V). Thus, structural perturbation by terminal truncation, evolutionary compensation at physiol. temps., and elongation is an efficient way to analyze and improve thermostability without the need for high-temp. selection, structural information, or homologous proteins.
- 348Brown, N. G.; Pennington, J. M.; Huang, W.; Ayvaz, T.; Palzkill, T. Multiple Global Suppressors of Protein Stability Defects Facilitate the Evolution of Extended-Spectrum TEM β-Lactamases. J. Mol. Biol. 2010, 404 (5), 832– 846, DOI: 10.1016/j.jmb.2010.10.008348Multiple Global Suppressors of Protein Stability Defects Facilitate the Evolution of Extended-Spectrum TEM β-LactamasesBrown, Nicholas G.; Pennington, Jeanine M.; Huang, Wanzhi; Ayvaz, Tulin; Palzkill, TimothyJournal of Molecular Biology (2010), 404 (5), 832-846CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)The introduction of extended-spectrum cephalosporins and β-lactamase inhibitors has driven the evolution of extended-spectrum β-lactamases (ESBLs) that possess the ability to hydrolyze these drugs. The evolved TEM ESBLs from clin. isolates of bacteria often contain substitutions that occur in the active site and alter the catalytic properties of the enzyme to provide an increased hydrolysis of extended-spectrum cephalosporins or an increased resistance to inhibitors. These active-site substitutions often result in a cost in the form of reduced enzyme stability. The evolution of TEM ESBLs is facilitated by mutations that act as global suppressors of protein stability defects in the sense that they allow the enzyme to absorb multiple amino acid changes despite incremental losses in stability assocd. with the substitutions. The best-studied example is the M182T substitution, which corrects protein stability defects and is commonly found in TEM ESBLs or inhibitor-resistant variants from clin. isolates. In this study, a genetic selection for second-site mutations that could partially restore function to a severely destabilized primary mutant enabled the identification of A184V, T265M, R275Q, and N276D, which are known to occur in TEM ESBLs from clin. isolates, as suppressors of TEM-1 protein stability defects. Further characterization demonstrated that these substitutions increased the thermal stability of TEM-1 and were able to correct the stability defects of two different sets of destabilizing mutations. The acquisition of compensatory global suppressors of stability costs assocd. with active-site mutations may be a common mechanism for the evolution of novel protein function.
- 349Moore, E. J.; Zorine, D.; Hansen, W. A.; Khare, S. D.; Fasan, R. Enzyme Stabilization via Computationally Guided Protein Stapling. Proc. Natl. Acad. Sci. U.S.A. 2017, 114 (47), 12472– 12477, DOI: 10.1073/pnas.1708907114349Enzyme stabilization via computationally guided protein staplingMoore, Eric J.; Zorine, Dmitri; Hansen, William A.; Khare, Sagar D.; Fasan, RudiProceedings of the National Academy of Sciences of the United States of America (2017), 114 (47), 12472-12477CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Thermostabilization represents a crit. and often obligatory step toward enhancing the robustness of enzymes for org. synthesis and other applications. While directed evolution methods have provided valuable tools for this purpose, these protocols are laborious and time-consuming and typically require the accumulation of several mutations, potentially at the expense of catalytic function. Here, we report a minimally invasive strategy for enzyme stabilization that relies on the installation of genetically encoded, nonreducible covalent staples in a target protein scaffold using computational design. This methodol. enables the rapid development of myoglobin-based cyclopropanation biocatalysts featuring dramatically enhanced thermostability (ΔTm = +18.0 °C and ΔT50 = +16.0 °C) as well as increased stability against chem. denaturation [ΔCm (GndHCl) = 0.53 M], without altering their catalytic efficiency and stereoselectivity properties. In addn., the stabilized variants offer superior performance and selectivity compared with the parent enzyme in the presence of a high concn. of org. cosolvents, enabling the more efficient cyclopropanation of a water-insol. substrate. This work introduces and validates an approach for protein stabilization which should be applicable to a variety of other proteins and enzymes.
- 350Bi, J.; Jing, X.; Wu, L.; Zhou, X.; Gu, J.; Nie, Y.; Xu, Y. Computational Design of Noncanonical Amino Acid-Based Thioether Staples at N/C-Terminal Domains of Multi-Modular Pullulanase for Thermostabilization in Enzyme Catalysis. Comput. Struct. Biotechnol. J. 2021, 19, 577– 585, DOI: 10.1016/j.csbj.2020.12.043350Computational design of noncanonical amino acid-based thioether staples at N/C-terminal domains of multi-modular pullulanase for thermostabilization in enzyme catalysisBi, Jiahua; Jing, Xiaoran; Wu, Lunjie; Zhou, Xia; Gu, Jie; Nie, Yao; Xu, YanComputational and Structural Biotechnology Journal (2021), 19 (), 577-585CODEN: CSBJAC; ISSN:2001-0370. (Elsevier B.V.)Enzyme thermostabilization is considered a crit. and often obligatory step in biosynthesis, because thermostability is a significant property of enzymes that can be used to evaluate their feasibility for industrial applications. However, conventional strategies for thermostabilizing enzymes generally introduce non-covalent interactions and/or natural covalent bonds caused by natural amino acid substitutions, and the trade-off between the activity and stability of enzymes remains a challenge. Here, we developed a computationally guided strategy for constructing thioether staples by incorporating noncanonical amino acid (ncAA) into the more flexible N/C-terminal domains of the multi-modular pullulanase from Bacillus thermoleovorans (BtPul) to enhance its thermostability. First, potential thioether staples located in the N/C-terminal domains of BtPul were predicted using RosettaMatch. Next, eight variants involving stable thioether staples were precisely predicted using FoldX and Rosetta ddg_monomer. Six pos. variants were obtained, of which T73(O2beY)-171C had a 157% longer half-life at 70 °C and an increase of 7.0 °C in Tm, when compared with the wild-type (WT). T73(O2beY)-171C/T126F/A72R exhibited an even more improved thermostability, with a 211% increase in half-life at 70 °C and a 44% enhancement in enzyme activity compared with the WT, which was attributed to further optimization of the local interaction network. This work introduces and validates an efficient strategy for enhancing the thermostability and activity of multi-modular enzymes.
- 351Iannuzzelli, J. A.; Bacik, J.-P.; Moore, E. J.; Shen, Z.; Irving, E. M.; Vargas, D. A.; Khare, S. D.; Ando, N.; Fasan, R. Tuning Enzyme Thermostability via Computationally Guided Covalent Stapling and Structural Basis of Enhanced Stabilization. Biochemistry 2022, 61 (11), 1041– 1054, DOI: 10.1021/acs.biochem.2c00033351Tuning enzyme thermostability via computationally guided covalent stapling and structural basis of enhanced stabilizationIannuzzelli, Jacob A.; Bacik, John-Paul; Moore, Eric J.; Shen, Zhuofan; Irving, Ellen M.; Vargas, David A.; Khare, Sagar D.; Ando, Nozomi; Fasan, RudiBiochemistry (2022), 61 (11), 1041-1054CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Enhancing the thermostability of enzymes without impacting their catalytic function represents an important yet challenging goal in protein engineering and biocatalysis. We recently introduced a novel method for enzyme thermostabilization that relies on the computationally guided installation of genetically encoded thioether "staples" into a protein via cysteine alkylation with the noncanonical amino acid O-2-bromoethyl tyrosine (O2beY). Here, we demonstrate the functionality of an expanded set of electrophilic amino acids featuring chloroacetamido, acrylamido, and vinylsulfonamido side-chain groups for protein stapling using this strategy. Using a myoglobin-based cyclopropanase as a model enzyme, our studies show that covalent stapling with p-chloroacetamido-phenylalanine (pCaaF) provides higher stapling efficiency and enhanced stability (thermodn. and kinetic) compared to the other stapled variants and the parent protein. Interestingly, mol. simulations of conformational flexibility of the crosslinks show that the pCaaF staple allows fewer energetically feasible conformers than the other staples, and this property may be a broader indicator of stability enhancement. Using this strategy, pCaaF-stapled variants with significantly enhanced stability against thermal denaturation (ΔTm' = +27°C) and temp.-induced heme loss (ΔT50 = +30°C) were obtained while maintaining high levels of catalytic activity and stereoselectivity. Crystallog. analyses of singly and doubly stapled variants provide key insights into the structural basis for stabilization, which includes both direct interactions of the staples with protein residues and indirect interactions through adjacent residues involved in heme binding. This work expands the toolbox of protein stapling strategies available for protein stabilization.
- 352Gur, E.; Biran, D.; Gazit, E.; Ron, E. Z. In vivo Aggregation of a Single Enzyme Limits Growth of Escherichia coli at Elevated Temperatures. Mol. Microbiol. 2002, 46 (5), 1391– 1397, DOI: 10.1046/j.1365-2958.2002.03257.x352In vivo aggregation of a single enzyme limits growth of Escherichia coli at elevated temperaturesGur, Eyal; Biran, Dvora; Gazit, Ehud; Ron, Eliora Z.Molecular Microbiology (2002), 46 (5), 1391-1397CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Science Ltd.)The formation of protein aggregates is assocd. with unfolding and denaturation of proteins. Recent studies have indicated that, in Escherichia coli, cellular proteins tend to aggregate when the bacteria are exposed to thermal stress. Here, we show that the aggregation of one single E. coli cytoplasmic protein limits growth at elevated temps. in minimal media. Homoserine trans-succinylase (HTS), the first enzyme in the methionine biosynthetic pathway, aggregates at temps. higher than 44°C in vitro. Above this temp., we can also observe in vivo aggregation that results in the complete disappearance of the enzyme from the sol. fraction. Moreover, reducing the in vivo level of HTS aggregation enables growth at non-permissive temps. This is the first demonstration of the physiol. role of aggregation of a specific protein in the growth of wild-type bacteria.
- 353Li, J. C.; Liu, T.; Wang, Y.; Mehta, A. P.; Schultz, P. G. Enhancing Protein Stability with Genetically Encoded Noncanonical Amino Acids. J. Am. Chem. Soc. 2018, 140 (47), 15997– 16000, DOI: 10.1021/jacs.8b07157353Enhancing Protein Stability with Genetically Encoded Noncanonical Amino AcidsLi, Jack C.; Liu, Tao; Wang, Yan; Mehta, Angad P.; Schultz, Peter G.Journal of the American Chemical Society (2018), 140 (47), 15997-16000CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The ability to add noncanonical amino acids to the genetic code may allow one to evolve proteins with new or enhanced properties using a larger set of building blocks. To this end, we have been able to select mutant proteins with enhanced thermal properties from a library of Escherichia coli homoserine O-succinyltransferase (metA) mutants contg. randomly incorporated noncanonical amino acids. Here, we showed that substitution of Phe-21 with p-benzoyl-L-phenylalanine (pBzF), increased the melting temp. of E. coli metA by 21°. This dramatic increase in thermostability, arising from a single mutation, likely resulted from a covalent adduct between Cys-90 and the keto group of pBzF that stabilized the dimeric form of the enzyme. These expts. show that an expanded genetic code can provide unique solns. to the evolution of proteins with enhanced properties.
- 354Xuan, W.; Li, J.; Luo, X.; Schultz, P. G. Genetic Incorporation of a Reactive Isothiocyanate Group into Proteins. Angew. Chem. Int. Ed. 2016, 55 (34), 10065– 10068, DOI: 10.1002/anie.201604891354Genetic Incorporation of a Reactive Isothiocyanate Group into ProteinsXuan, Weimin; Li, Jack; Luo, Xiaozhou; Schultz, Peter G.Angewandte Chemie, International Edition (2016), 55 (34), 10065-10068CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Methods for the site-specific modification of proteins are useful for introducing biol. probes into proteins and engineering proteins with novel activities. Herein, the authors genetically encode a novel noncanonical amino acid (ncAA) that contains an aryl isothiocyanate group which can form stable thiourea crosslinks with amines under mild conditions. This ncAA (pNCSF) allows the selective conjugation of proteins to amine-contg. mol. probes through formation of a thiourea bridge. PNCSF was also used to replace a native salt bridge in myoglobin with an intramol. crosslink to a proximal Lys residue, leading to increased thermal stability. Finally, pNCSF can form stable intermol. crosslinks between two interacting proteins.
- 355Li, J. C.; Nastertorabi, F.; Xuan, W.; Han, G. W.; Stevens, R. C.; Schultz, P. G. A Single Reactive Noncanonical Amino Acid Is Able to Dramatically Stabilize Protein Structure. ACS Chem. Biol. 2019, 14 (6), 1150– 1153, DOI: 10.1021/acschembio.9b00002There is no corresponding record for this reference.
- 356Deiters, A.; Cropp, T. A.; Summerer, D.; Mukherji, M.; Schultz, P. G. Site-Specific PEGylation of Proteins Containing Unnatural Amino Acids. Bioorg. Med. Chem. Lett. 2004, 14 (23), 5743– 5745, DOI: 10.1016/j.bmcl.2004.09.059There is no corresponding record for this reference.
- 357Schoffelen, S.; Lambermon, M. H. L.; Eldijk, M. B. v.; Hest, J. C. M. v. Site-Specific Modification of Candida antarctica Lipase B via Residue-Specific Incorporation of a Non-Canonical Amino Acid. Bioconjug. Chem. 2008, 19 (6), 1127– 1131, DOI: 10.1021/bc800019v357Site-Specific Modification of Candida antarctica Lipase B via Residue-Specific Incorporation of a Non-Canonical Amino AcidSchoffelen, Sanne; Lambermon, Mark H. L.; van Eldijk, Mark B.; van Hest, Jan C. M.Bioconjugate Chemistry (2008), 19 (6), 1127-1131CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)In order to modify proteins in a controlled way, new functionalities need to be introduced in a defined manner. One way to accomplish this is by the incorporation of a non-natural amino acid of which the side chain can selectively be reacted to other mols. We have investigated whether the relatively simple method of residue-specific replacement of methionine by azidohomoalanine can be used to achieve monofunctionalization of the model enzyme Candida antarctica lipase B. A protein variant was engineered with one addnl. methionine residue. Due to the high hydrophobicity and low abundance of methionine, this was the only residue out of five that was exposed to the solvent. The use of the CuI-catalyzed [3+2] cycloaddn. under native conditions resulted in a monofunctionalized enzyme which retained hydrolytic activity. The strategy can be considered a convenient tool to modify proteins at a single position as long as one solvent-exposed methionine is available.
- 358van Dongen, S. F. M.; Nallani, M.; Schoffelen, S.; Cornelissen, J. J. L. M.; Nolte, R. J. M.; van Hest, J. C. M. A Block Copolymer for Functionalisation of Polymersome Surfaces. Macromol. Rapid Commun. 2008, 29 (4), 321– 325, DOI: 10.1002/marc.200700765358A block copolymer for functionalisation of polymersome surfacesvan Dongen, Stijn F. M.; Nallani, Madhavan; Schoffelen, Sanne; Cornelissen, Jeroen J. L. M.; Nolte, Roeland J. M.; van Hest, Jan C. M.Macromolecular Rapid Communications (2008), 29 (4), 321-325CODEN: MRCOE3; ISSN:1022-1336. (Wiley-VCH Verlag GmbH & Co. KGaA)A block copolymer was designed to functionalize the surface of polystyrene-based polymersomes via coaggregation. An α,ω-diacetylene-functionalized poly(ethylene glycol) (PEG) was coupled to an azide-terminated polystyrene via a Cu(I)-catalyzed cycloaddn. to produce a PS-b-PEG polymer with an acetylene at its hydrophilic extremity. Incorporation of this 'anchor' compd. in the bilayer of a polymersome places its bio-orthogonal group at the surface of this aggregate. Its accessibility was demonstrated using an azido-functionalized Candida antarctica Lipase B (CalB), which retained its activity while immobilized on the polymersome.
- 359Teeuwen, R. L. M.; van Berkel, S. S.; van Dulmen, T. H. H.; Schoffelen, S.; Meeuwissen, S. A.; Zuilhof, H.; de Wolf, F. A.; van Hest, J. C. M. “Clickable” Elastins: Elastin-Like Polypeptides Functionalized with Azide or Alkyne Groups. Chem. Commun. 2009, (27), 4022– 4024, DOI: 10.1039/b903903aThere is no corresponding record for this reference.
- 360Debets, M. F.; van Berkel, S. S.; Schoffelen, S.; Rutjes, F. P. J. T.; van Hest, J. C. M.; van Delft, F. L. Aza-Dibenzocyclooctynes for Fast and Efficient Enzyme PEGylation via Copper-Free (3 + 2) Cycloaddition. Chem. Commun. 2010, 46 (1), 97– 99, DOI: 10.1039/B917797C360Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3 + 2) cycloadditionDebets, Marjoke F.; van Berkel, Sander S.; Schoffelen, Sanne; Rutjes, Floris P. J. T.; van Hest, Jan C. M.; van Delft, Floris L.Chemical Communications (Cambridge, United Kingdom) (2010), 46 (1), 97-99CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Didehydrodibenzazocines (azadibenzocyclooctynes) I [R = PhCH2OCO, HO2C(CH2)3CO, Me(OCH2CH2)nCH2CH2NHCO(CH2)3CO] are prepd. as potential reagents for copper-free Huisgen dipolar cycloaddns. with azide-labeled proteins to form triazole-substituted protein conjugates. I [R = HO2C(CH2)3CO] is prepd. in nine steps from 2-iodobenzyl alc., 2-ethynylaniline, and 5-methoxy-5-oxopentanoyl chloride. The kinetics of copper-free Huisgen dipolar cycloaddns. of I [R = PhCH2OCO, HO2C(CH2)3CO] with benzyl azide and with (S)-α-azidopropanoic acid are detd.; I react at comparable or larger rates with benzyl azide than other cyclooctyne reagents. Conjugates of I [R = Me(OCH2CH2)nCH2CH2NHCO(CH2)3CO] with Candida antarctica lipase B contg. five azidohomoalanine residues and an azide-substituted horseradish peroxidase are generated, indicating that I [R = Me(OCH2CH2)nCH2CH2NHCO(CH2)3CO] can be used for the PEGylation of azide-labeled proteins.
- 361Wilding, K. M.; Smith, A. K.; Wilkerson, J. W.; Bush, D. B.; Knotts, T. A. I. V.; Bundy, B. C. The Locational Impact of Site-Specific PEGylation: Streamlined Screening with Cell-Free Protein Expression and Coarse-Grain Simulation. ACS Synth. Biol. 2018, 7 (2), 510– 521, DOI: 10.1021/acssynbio.7b00316361The Locational Impact of Site-Specific PEGylation: Streamlined Screening with Cell-Free Protein Expression and Coarse-Grain SimulationWilding, Kristen M.; Smith, Addison K.; Wilkerson, Joshua W.; Bush, Derek B.; Knotts, Thomas A.; Bundy, Bradley C.ACS Synthetic Biology (2018), 7 (2), 510-521CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Although polyethylene glycol (PEG) is commonly used to improve protein stability and therapeutic efficacy, the optimal location for attaching PEG onto proteins is not well understood. Here, we present a cell-free protein synthesis-based screening platform that facilitates site-specific PEGylation and efficient evaluation of PEG attachment efficiency, thermal stability, and activity for different variants of PEGylated T4 lysozyme, including a di-PEGylated variant. We also report developing a computationally efficient coarse-grain simulation model as a potential tool to narrow exptl. screening candidates. We use this simulation method as a novel tool to evaluate the locational impact of PEGylation. Using this screen, we also evaluated the predictive impact of PEGylation site solvent accessibility, conjugation site structure, PEG size, and double PEGylation. Our findings indicate that PEGylation efficiency, protein stability, and protein activity varied considerably with PEGylation site, variations that were not well predicted by common PEGylation guidelines. Overall our results suggest current guidelines are insufficiently predictive, highlighting the need for exptl. and simulation screening systems such as the one presented here.
- 362Wu, J. C. Y.; Hutchings, C. H.; Lindsay, M. J.; Werner, C. J.; Bundy, B. C. Enhanced Enzyme Stability Through Site-Directed Covalent Immobilization. J. Biotechnol. 2015, 193, 83– 90, DOI: 10.1016/j.jbiotec.2014.10.039362Enhanced enzyme stability through site-directed covalent immobilizationWu, Jeffrey Chun Yu; Hutchings, Christopher Hayden; Lindsay, Mark Jeffrey; Werner, Christopher James; Bundy, Bradley CharlesJournal of Biotechnology (2015), 193 (), 83-90CODEN: JBITD4; ISSN:0168-1656. (Elsevier B.V.)Breakthroughs in enzyme immobilization have enabled increased enzyme recovery and reusability, leading to significant decreases in the cost of enzyme use and fueling biocatalysis growth. However, current enzyme immobilization techniques suffer from leaching, enzyme stability, and recoverability and reusability issues. Moreover, these techniques lack the ability to control the orientation of the immobilized enzymes. To det. the impact of orientation on covalently immobilized enzyme activity and stability, the authors applied their PRECISE (Protein Residue-Explicit Covalent Immobilization for Stability Enhancement) system to a model enzyme, phage T4 lysozyme. The PRECISE system uses non-canonical amino acid incorporation and the Huisgen 1,3-dipolar cycloaddn. "click" reaction to enable directed enzyme immobilization at rationally chosen residues throughout an enzyme. Unlike previous site-specific systems, the PRECISE system is a truly covalent immobilization method. Utilizing this system, enzymes immobilized at proximate and distant locations from the active site were tested for activity and stability under denaturing conditions. The results demonstrated that orientation control of covalently immobilized enzymes could provide activity and stability benefits exceeding that of traditional random covalent immobilization techniques. PRECISE immobilized enzymes were 50 and 73% more active than randomly immobilized enzymes after harsh freeze-thaw and chem. denaturant treatments.
- 363Basso, A.; Serban, S. Industrial Applications of Immobilized Enzymes─A Review. Mol. Catal. 2019, 479, 110607, DOI: 10.1016/j.mcat.2019.110607363Industrial applications of immobilized enzymes-A reviewBasso, Alessandra; Serban, SimonaMolecular Catalysis (2019), 479 (), 110607CODEN: MCOADH ISSN:. (Elsevier B.V.)A review. The use of immobilized enzymes is now a routine process for the manuf. of many industrial products in the pharmaceutical, chem. and food industry. Some enzymes, such as lipases, are naturally robust and efficient, can be used for the prodn. of many different mols. and have a wide range of industrial applications thanks to their broad selectivity. As an example, lipase from Candida antarctica (CalB) has been used by BASF to produce chiral compds., such as the herbicide Dimethenamide-P, which was previously made chem. The use of the immobilized enzyme has provided significant advantages over a chem. process, such as the possibility to use equimolar concn. of substrates, obtain an enantiomeric excess > 99%, use relatively low temps. (< 60 °C) in org. solvent, obtain a single enantiomer instead of the racemate as in the chem. process and use a column configuration that allows dramatic increases in productivity. This process would not have been possible without the use of an immobilized enzyme, since it runs in org. solvent [1]. Some more specific enzymes, like transaminases, have required protein engineering to become suitable for applications in prodn. of APIs (Active Pharmaceutical Ingredients) in conditions which are extreme for a wild type enzyme. The process developed by Merck for sitagliptin manuf. is a good example of challenging enzyme engineering applied to API manuf. The previous process of sitagliptin involved hydrogenation of enamine at high pressure using a rhodium-based chiral catalyst. By developing an engineered transaminase, the enzymic process was able to convert 200 g/l of prositagliptin in the final product, with e.e. >99.5% and using an immobilized enzyme in the presence of DMSO as a cosolvent [2]. For all enzymes, the possibility to be immobilized and used in a heterogeneous form brings important industrial and environmental advantages, such as simplified downstream processing or continuous process operations. Here, we present a series of large-scale applications of immobilized enzymes with benefits for the food, chem., pharmaceutical, cosmetics and medical device industries, some of which have been scarcely reported on previously. In general, all enzymic reactions can benefit from the immobilization, however, the final choice to use them in immobilized form depends on the economic evaluation of costs assocd. with their use vs. benefits obtained in the process. It can be concluded that the benefits are rather significant, since the use of immobilized enzymes in industry is increasing.
- 364Hernandez, K.; Fernandez-Lafuente, R. Control of Protein Immobilization: Coupling Immobilization and Site-Directed Mutagenesis to Improve Biocatalyst or Biosensor Performance. Enzyme Microb. Technol. 2011, 48 (2), 107– 122, DOI: 10.1016/j.enzmictec.2010.10.003364Control of protein immobilization: Coupling immobilization and site-directed mutagenesis to improve biocatalyst or biosensor performanceHernandez, Karel; Fernandez-Lafuente, RobertoEnzyme and Microbial Technology (2011), 48 (2), 107-122CODEN: EMTED2; ISSN:0141-0229. (Elsevier B.V.)A review. Mutagenesis and immobilization are usually considered to be unrelated techniques with potential applications to improve protein properties. However, there are several reports showing that the use of site-directed mutagenesis to improve enzyme properties directly, but also how enzymes are immobilized on a support, can be a powerful tool to improve the properties of immobilized biomols. for use as biosensors or biocatalysts. Std. immobilizations are not fully random processes, but the protein orientation may be difficult to alter. Initially, most efforts using this idea were addressed towards controlling the orientation of the enzyme on the immobilization support, in many cases to facilitate electron transfer from the support to the enzyme in redox biosensors. Usually, Cys residues are used to directly immobilize the protein on a support that contains disulfide groups or that is made from gold. There are also some examples using His in the target areas of the protein and using supports modified with immobilized metal chelates and other tags (e.g., using immobilized antibodies). Furthermore, site-directed mutagenesis to control immobilization is useful for improving the activity, the stability and even the selectivity of the immobilized protein, for example, via site-directed rigidification of selected areas of the protein. Initially, only Cys and disulfide supports were employed, but other supports with higher potential to give multipoint covalent attachment are being employed (e.g., glyoxyl or epoxy-disulfide supports). The advances in support design and the deeper knowledge of the mechanisms of enzyme-support interactions have permitted exploration of the possibilities of the coupled use of site-directed mutagenesis and immobilization in a new way. This paper intends to review some of the advances and possibilities that these coupled strategies permit.
- 365Guan, D.; Kurra, Y.; Liu, W.; Chen, Z. A Click Chemistry Approach to Site-Specific Immobilization of a Small Laccase Enables Efficient Direct Electron Transfer in a Biocathode. Chem. Commun. 2015, 51 (13), 2522– 2525, DOI: 10.1039/C4CC09179E365A click chemistry approach to site-specific immobilization of a small laccase enables efficient direct electron transfer in a biocathodeGuan, Dongli; Kurra, Yadagiri; Liu, Wenshe; Chen, ZhileiChemical Communications (Cambridge, United Kingdom) (2015), 51 (13), 2522-2525CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Controlled orientation of a small laccase on a multi-walled carbon nanotube (MWCNT) electrode was achieved via copper-free click chem.-mediated immobilization. Modification of the enzyme was limited to only the tethering site and involved the genetic incorporation of the unnatural amino acid 4-azido-L-phenylalanine (AzF). This approach enabled efficient direct electron transfer (DET).
- 366Lim, S. I.; Mizuta, Y.; Takasu, A.; Kim, Y. H.; Kwon, I. Site-Specific Bioconjugation of a Murine Dihydrofolate Reductase Enzyme by Copper(I)-Catalyzed Azide-Alkyne Cycloaddition with Retained Activity. PLOS ONE 2014, 9 (6), e98403 DOI: 10.1371/journal.pone.0098403366Site-specific bioconjugation of a murine dihydrofolate reductase enzyme by copper(I)-catalyzed azide-alkyne cycloaddition with retained activityLim, Sung In; Mizuta, Yukina; Takasu, Akinori; Kim, Yong Hwan; Kwon, InchanPLoS One (2014), 9 (6), e98403/1-e98403/10, 10 pp.CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Cu(I)-catalyzed azide-alkyne cycloaddn. (CuAAC) is an efficient reaction linking an azido and an alkynyl group in the presence of copper catalyst. Incorporation of a non-natural amino acid (NAA) contg. either an azido or an alkynyl group into a protein allows site-specific bioconjugation in mild conditions via CuAAC. Despite its great potential, bioconjugation of an enzyme has been hampered by several issues including low yield, poor soly. of a ligand, and protein structural/functional perturbation by CuAAC components. In the present study, we incorporated an alkyne-bearing NAA into an enzyme, murine dihydrofolate reductase (mDHFR), in high cell d. cultivation of Escherichia coli, and performed CuAAC conjugation with fluorescent azide dyes to evaluate enzyme compatibility of various CuAAC conditions comprising combination of com. available Cu(I)-chelating ligands and reductants. The condensed culture improves the protein yield 19-fold based on the same amt. of non-natural amino acid, and the enzyme incubation under the optimized reaction condition did not lead to any activity loss but allowed a fast and high-yield bioconjugation. Using the established conditions, a biotin-azide spacer was efficiently conjugated to mDHFR with retained activity leading to the site-specific immobilization of the biotin-conjugated mDHFR on a streptavidin-coated plate. These results demonstrate that the combination of reactive non-natural amino acid incorporation and the optimized CuAAC can be used to bioconjugate enzymes with retained enzymic activity.
- 367Wang, A.; Du, F.; Pei, X.; Chen, C.; Wu, S. G.; Zheng, Y. Rational Immobilization of Lipase by Combining the Structure Analysis and Unnatural Amino Acid Insertion. J. Mol. Catal. B Enzym. 2016, 132, 54– 60, DOI: 10.1016/j.molcatb.2016.06.015367Rational immobilization of lipase by combining the structure analysis and unnatural amino acid insertionWang, Anming; Du, Fangchuan; Pei, Xiaolin; Chen, Canyu; Wu, Stephen Gang; Zheng, YuguoJournal of Molecular Catalysis B: Enzymatic (2016), 132 (), 54-60CODEN: JMCEF8; ISSN:1381-1177. (Elsevier B.V.)Improving the conventional covalent immobilization of enzyme and avoiding random covalent linkage to protect enzyme's active sites from unwanted covalent linkage at the mean time are the fundamental topics for enzyme immobilization. In this study, unnatural amino acid was introduced into a recombinant lipase and applied for the rational and smart covalent enzyme immobilization. In the first step, Tyr50, 137, 243, 274, and 355 of lipase were replaced with AzPhe unnatural amino acid based on the anal. of enzyme structure. Then, these novel recombinant lipases were coupled to support using strain-promoted azide-alkyne cycloaddn. (SPAAC), resp. Subsequently, both the effect of the immobilization site and the thermo-stability of immobilized lipases were also examd. The relative activities of the immobilized AzPhe-Lip243 and AzPhe-Lip274 were enhanced to 121.33% and 137.06%, resp., presenting 6.0 and 6.8 fold higher than those of the lipase traditionally immobilized using glutaraldehyde (IM-Lip-GA). In addn., all the immobilized lipases presented better specific activity except for AzPhe-Lip355, whose immobilization site was close to its active site. The rational immobilized lipases also presented better thermo-stability than those by traditionally immobilization method (glutaraldehyde). To sum up, with the aid of protein structure anal., unnatural amino acid can be rationally inserted into enzyme sequence to inform and direct the covalent enzyme immobilization. This method can be further developed for one-step enzyme purifn. and immobilization and applied to a broad scope of enzymes.
- 368Li, H.; Yin, Y.; Wang, A.; Li, N.; Wang, R.; Zhang, J.; Chen, X.; Pei, X.; Xie, T. Stable Immobilization of Aldehyde Ketone Reductase Mutants Containing Nonstandard Amino Acids on an Epoxy Resin via Strain-Promoted Alkyne-Azide Cycloaddition. RSC Adv. 2020, 10 (5), 2624– 2633, DOI: 10.1039/C9RA09067C368Stable immobilization of aldehyde ketone reductase mutants containing nonstandard amino acids on an epoxy resin via strain-promoted alkyne-azide cycloadditionLi, Huimin; Yin, Youcheng; Wang, Anming; Li, Ningning; Wang, Ru; Zhang, Jing; Chen, Xinxin; Pei, Xiaolin; Xie, TianRSC Advances (2020), 10 (5), 2624-2633CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)To avoid random chem. linkage and achieve precisely directed immobilization, mutant enzymes were obtained and immobilized using an incorporated reactive nonstandard amino acid (NSAA). For this purpose, aldehyde ketone reductase (AKR) was used as a model enzyme, and 110Y, 114Y, 143Y, 162Q and 189Q were each replaced with p-azido-L-phenylalanine (pAzF). Then, the mutant AKR was coupled to the functionalized support by strain-promoted alkyne-azide cycloaddn. (SPAAC). The effects of the incorporation no. and site of NSAAs on the loading and thermal stability of the immobilized AKR were examd. The results show that the mutant enzymes presented better specific activity than the wild type, except for AKR-110Y, and AKR-114Y showed 1.16-fold higher activity than the wild type. Moreover, the half-life (t1/2) of the five-point immobilized AKR reached 106 h and 45 h, 13 and 7 times higher than that of the free enzyme at 30 °C and 60 °C, resp. Comparison of these three types of enzymes shows that multi-point immobilization provides improved loading and thermal stability and facilitates one-step purifn. We expect this platform to facilitate a fundamental understanding of precisely oriented and controllable covalent immobilization and enable bio-manufg. paradigms for fine chems. and pharmaceuticals.
- 369Umeda, A.; Thibodeaux, G. N.; Zhu, J.; Lee, Y.; Zhang, Z. J. Site-specific Protein Cross-Linking with Genetically Incorporated 3,4-Dihydroxy-L-Phenylalanine. ChemBioChem 2009, 10 (8), 1302– 1304, DOI: 10.1002/cbic.200900127There is no corresponding record for this reference.
- 370Deepankumar, K.; Nadarajan, S. P.; Mathew, S.; Lee, S.-G.; Yoo, T. H.; Hong, E. Y.; Kim, B.-G.; Yun, H. Engineering Transaminase for Stability Enhancement and Site-Specific Immobilization through Multiple Noncanonical Amino Acids Incorporation. ChemCatChem 2015, 7 (3), 417– 421, DOI: 10.1002/cctc.201402882370Engineering Transaminase for Stability Enhancement and Site-Specific Immobilization through Multiple Noncanonical Amino Acids IncorporationDeepankumar, Kanagavel; Nadarajan, Saravanan Prabhu; Mathew, Sam; Lee, Sun-Gu; Yoo, Tae Hyeon; Hong, Eun Young; Kim, Byung-Gee; Yun, HyungdonChemCatChem (2015), 7 (3), 417-421CODEN: CHEMK3; ISSN:1867-3880. (Wiley-VCH Verlag GmbH & Co. KGaA)In general, conventional enzyme engineering utilizes 20 canonical amino acids to alter and improve the functional properties of proteins such as stability, and activity. In this study, we utilized the noncanonical amino acid (NCAA) incorporation technique to enhance the functional properties of ω-transaminase (ω-TA). Herein, we enhanced the stability of ω-TA by residue-specific incorporation of (4R)-fluoroproline [(4R)-FP] and successfully immobilized onto chitosan or polystyrene (PS) beads with site-specifically incorporated L-3,4-dihydroxyphenylalanine (DOPA) moiety. The immobilization of ω-TAdopa and ω-TAdp[(4R)-FP] onto PS beads showed excellent reusability for 10 cycles in the kinetic resoln. of chiral amines. Compared to the ω-TAdopa, the ω-TAdp[(4R)-FP] immobilized onto PS beads exerted more stability that can serve as suitable biocatalyst for the asym. synthesis of chiral amines.
- 371Bednar, R. M.; Golbek, T. W.; Kean, K. M.; Brown, W. J.; Jana, S.; Baio, J. E.; Karplus, P. A.; Mehl, R. A. Immobilization of Proteins with Controlled Load and Orientation. ACS Appl. Mater. Interfaces 2019, 11 (40), 36391– 36398, DOI: 10.1021/acsami.9b12746371Immobilization of Proteins with Controlled Load and OrientationBednar, Riley M.; Golbek, Thaddeus W.; Kean, Kelsey M.; Brown, Wesley J.; Jana, Subhashis; Baio, Joe E.; Karplus, P. Andrew; Mehl, Ryan A.ACS Applied Materials & Interfaces (2019), 11 (40), 36391-36398CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)Biomaterials based on immobilized proteins are key elements of many biomedical and industrial technologies. However, applications are limited by an inability to precisely construct materials of high homogeneity and defined content. The authors present here a general "protein-limited immobilization" strategy by combining the rapid, bioorthogonal, and biocompatible properties of a tetrazine-strained-trans-cyclooctene reaction with genetic code expansion to site-specifically place the tetrazine into a protein. For the first time, the authors use this strategy to immobilize defined amts. of oriented proteins onto beads and flat surfaces in under five minutes at sub-micromolar concns. without compromising activity. This approach opens the door to generating and studying diverse protein-based biomaterials that are much more precisely defined and characterized, providing a greater ability to engineer properties across a wide range of applications.
- 372Blizzard, R. J.; Backus, D. R.; Brown, W.; Bazewicz, C. G.; Li, Y.; Mehl, R. A. Ideal Bioorthogonal Reactions Using A Site-Specifically Encoded Tetrazine Amino Acid. J. Am. Chem. Soc. 2015, 137 (32), 10044– 10047, DOI: 10.1021/jacs.5b03275372Ideal Bioorthogonal Reactions Using A Site-Specifically Encoded Tetrazine Amino AcidBlizzard, Robert J.; Backus, Dakota R.; Brown, Wes; Bazewicz, Christopher G.; Li, Yi; Mehl, Ryan A.Journal of the American Chemical Society (2015), 137 (32), 10044-10047CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Bioorthogonal reactions for labeling biomols. in live cells have been limited by slow reaction rates or low component selectivity and stability. Ideal bioorthogonal reactions with high reaction rates, high selectivity, and high stability would allow for stoichiometric labeling of biomols. in minutes and eliminate the need to wash out excess labeling reagent. Currently, no general method exists for controlled stoichiometric or substoichiometric labeling of proteins in live cells. To overcome this limitation, the authors developed a significantly improved tetrazine-contg. amino acid (Tet-v2.0, I) and genetically encoded I with an evolved aminoacyl-tRNA synthetase/tRNA(CUA) pair. The authors demonstrated in cellulo that protein contg. I reacts selectively with cyclopropane-fused trans-cyclooctene (sTCO) with a bimol. rate const. of 72,500 ± 1660 M-1 s-1 without reacting with other cellular components. This bioorthogonal ligation of I-protein reacts in cellulo with substoichiometric amts. of sTCO-label fast enough to remove the labeling reagent from media in minutes, thereby eliminating the need to wash out label. This ideal bioorthogonal reaction will enable the monitoring of a larger window of cellular processes in real time.
- 373Switzer, H. J.; Howard, C. A.; Halonski, J. F.; Peairs, E. M.; Smith, N.; Zamecnik, M. P.; Verma, S.; Young, D. D. Employing Non-Canonical Amino Acids Towards the Immobilization of a Hyperthermophilic Enzyme to Increase Protein Stability. RSC Adv. 2023, 13 (13), 8496– 8501, DOI: 10.1039/D3RA00392BThere is no corresponding record for this reference.
- 374Ray, S.; Chand, S.; Zhang, Y.; Nussbaum, S.; Rajeshwar, K.; Perera, R. Implications of Active Site Orientation in Myoglobin for Direct Electron Transfer and Electrocatalysis Based on Monolayer and Multilayer Covalent Immobilization on Gold Electrodes. Electrochim. Acta 2013, 99, 85– 93, DOI: 10.1016/j.electacta.2013.03.080374Implications of active site orientation in myoglobin for direct electron transfer and electrocatalysis based on monolayer and multilayer covalent immobilization on gold electrodesRay, Sriparna; Chand, Subhash; Zhang, Yanbo; Nussbaum, Sherry; Rajeshwar, Krishnan; Perera, RoshanElectrochimica Acta (2013), 99 (), 85-93CODEN: ELCAAV; ISSN:0013-4686. (Elsevier Ltd.)Cyclic voltammetry (CV) and at. force microscopy (AFM) were used to study the importance of active site orientation of an immobilized protein for direct electron transfer (DET) and electrocatalysis. While the bioconjugated wild-type myoglobin (WT Mb) was immobilized on the modified Au electrode surface in a random multilayered fashion, the Ser3 replaced with unnatural amino acid, 3-amino-L-tyrosine, (NH2Tyr) mutant Mb was immobilized via a Diels-Alder reaction specific to NH2Tyr residue to form a homogeneous monolayer. Electrochem. calcns. for the no. of surface exposed redox-active mols. on the electrode surface (Γ) and heterogeneous rate const. for DET were 1.29 × 10-10 mol cm-2; 2.3 s-1 for the WT Mb and 1.54 × 10-10 mol cm-2; 1.3 s-1 for the S3NH2Tyr Mb mutant, resp. Electro-catalytic conversion of thioanisole to sulfoxide products showed similar turnover frequencies (TOF) around 1.9 × 103 s-1 (with 87% conversion) for the WT Mb, and 1.5 × 103 s-1 for the mutant S3NH2Tyr Mb (with 81% conversion). Site-directed single monolayer immobilization affords almost the same no. of surface exposed Mb active sites as the random multilayer immobilization strategy, though the latter contains a greater no. of protein mols. on the electrode surface, as obsd. from the AFM data.
- 375Xia, L.; Han, M.-J.; Zhou, L.; Huang, A.; Yang, Z.; Wang, T.; Li, F.; Yu, L.; Tian, C.; Zang, Z. S-Click Reaction for Isotropic Orientation of Oxidases on Electrodes to Promote Electron Transfer at Low Potentials. Angew. Chem. Int. Ed. 2019, 58 (46), 16480– 16484, DOI: 10.1002/anie.201909203There is no corresponding record for this reference.
- 376Pan, Y.; Li, G.; Liu, R.; Guo, J.; Liu, Y.; Liu, M.; Zhang, X.; Chi, L.; Xu, K.; Wu, R. Unnatural Activities and Mechanistic Insights of Cytochrome P450 PikC Gained from Site-Specific Mutagenesis by Non-Canonical Amino Acids. Nature Commun. 2023, 14 (1), 1669, DOI: 10.1038/s41467-023-37288-0There is no corresponding record for this reference.
- 377Kolev, J. N.; Zaengle, J. M.; Ravikumar, R.; Fasan, R. Enhancing the Efficiency and Regioselectivity of P450 Oxidation Catalysts by Unnatural Amino Acid Mutagenesis. ChemBioChem 2014, 15 (7), 1001– 1010, DOI: 10.1002/cbic.201400060377Enhancing the Efficiency and Regioselectivity of P450 Oxidation Catalysts by Unnatural Amino Acid MutagenesisKolev, Joshua N.; Zaengle, Jacqueline M.; Ravikumar, Rajesh; Fasan, RudiChemBioChem (2014), 15 (7), 1001-1010CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)The development of effective strategies for modulating the reactivity and selectivity of cytochrome P 450 enzymes represents a key step toward expediting the use of these biocatalysts for synthetic applications. We have investigated the potential of unnatural amino acid mutagenesis to aid efforts in this direction. Four unnatural amino acids with diverse arom. side chains were incorporated at 11 active-site positions of a substrate-promiscuous CYP102A1 variant. The resulting "uP450s" were then tested for their catalytic activity and regioselectivity in the oxidn. of two representative substrates: a small-mol. drug and a natural product. Large shifts in regioselectivity resulted from these single mutations, and in particular, for para-acetyl-Phe substitutions at positions close to the heme cofactor. Screening this mini library of uP 450s enabled us to identify P 450 catalysts for the selective hydroxylation of four aliph. positions in the target substrates, including a C(Sp3)-H site not oxidized by the parent enzyme. Furthermore, we discovered a general activity-enhancing effect of active-site substitutions involving the unnatural amino acid para-amino-Phe, which resulted in P 450 catalysts capable of supporting the highest total turnover no. reported to date on a complex mol. (34 650). The functional changes induced by the unnatural amino acids could not be reproduced by any of the 20 natural amino acids. This study thus demonstrates that unnatural amino acid mutagenesis constitutes a promising new strategy for improving the catalytic activity and regioselectivity of P 450 oxidn. catalysts.
- 378Ma, H.; Yang, X.; Lu, Z.; Liu, N.; Chen, Y. The “Gate Keeper” Role of Trp222 Determines the Enantiopreference of Diketoreductase toward 2-Chloro-1-Phenylethanone. PLOS ONE 2014, 9 (7), e103792 DOI: 10.1371/journal.pone.0103792There is no corresponding record for this reference.
- 379Yu, Z.; Yu, H.; Tang, H.; Wang, Z.; Wu, J.; Yang, L.; Xu, G. Site-specifically Incorporated Non-Canonical Amino Acids into Pseudomonas alcaligenes Lipase to Hydrolyze L-menthol Propionate among the Eight Isomers. ChemCatChem 2021, 13 (11), 2691– 2701, DOI: 10.1002/cctc.202100358379Site-specifically Incorporated Non-Canonical Amino Acids into Pseudomonas alcaligenes Lipase to Hydrolyze L-menthol Propionate among the Eight IsomersYu, Zhonglang; Yu, Haoran; Tang, Haibin; Wang, Zhe; Wu, Jianping; Yang, Lirong; Xu, GangChemCatChem (2021), 13 (11), 2691-2701CODEN: CHEMK3; ISSN:1867-3880. (Wiley-VCH Verlag GmbH & Co. KGaA)It remains a challenge to improve the diastereopreference of enzymes when there are multiple chiral centers in the substrate, mainly because the limited understanding of mechanism detg. diastereoselectivity. Compared with natural amino acids, non-canonical amino acids (ncAAs) provide side chains with wider range of functional groups and genetically encoded ncAAs have been applied in probing the complex enzyme mechanisms, improving catalytic activity, and even designing enzymes with new catalytic mechanisms. Here, the ncAAs were site-specifically incorporated into a lipase (PaL) produced by Pseudomonas alcaligenes to explore its diastereopreference mechanism. Menthol propionate has three chiral centers, eight isomers in total. Mol. dynamics (MD) simulations were first applied to analyze the interactions between the active sites of PaL and the target substrate L-menthol propionate. Furthermore, the four ncAAs (o-bromophenylalanine, o-chlorophenylalanine, p-cyanophenylalanine and p-aminophenylalanine) were substituted for 9 amino acids sites that potentially influenced three chiral centers and several variants with significant improvement in the diastereopreference were obtained. The diastereomer selectivity of beat variant at Ala253 was 100% higher than that of the wild-type. A linear relationship was found between vol., flexibility of the active center and diastereoselectivity.
- 380Drienovská, I.; Gajdoš, M.; Kindler, A.; Takhtehchian, M.; Darnhofer, B.; Birner-Gruenberger, R.; Dörr, M.; Bornscheuer, U. T.; Kourist, R. Folding Assessment of Incorporation of Noncanonical Amino Acids Facilitates Expansion of Functional-Group Diversity for Enzyme Engineering. Chem. Eur. J. 2020, 26 (54), 12338– 12342, DOI: 10.1002/chem.202002077There is no corresponding record for this reference.
- 381Green, A. P.; Hayashi, T.; Mittl, P. R. E.; Hilvert, D. A Chemically Programmed Proximal Ligand Enhances the Catalytic Properties of a Heme Enzyme. J. Am. Chem. Soc. 2016, 138 (35), 11344– 11352, DOI: 10.1021/jacs.6b07029381A chemically programmed proximal ligand enhances the catalytic properties of a heme enzymeGreen, Anthony P.; Hayashi, Takahiro; Mittl, Peer R. E.; Hilvert, DonaldJournal of the American Chemical Society (2016), 138 (35), 11344-11352CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Enzymes rely on complex interactions between precisely positioned active site residues as a mechanism to compensate for the limited functionality contained within the genetic code. Heme enzymes provide a striking example of this complexity, whereby the electronic properties of reactive ferryl intermediates are finely tuned through H-bonding interactions between proximal ligands and neighboring amino acids. Here, the authors show that introduction of a chem. programmed proximal Nδ-methylhistidine (NMH) ligand into an engineered ascorbate peroxidase (APX2) overcomes the reliance on the conserved Asp-His H-bonding interaction, leading to a catalytically modified enzyme (APX2 NMH), which is able to achieve a significantly higher no. of turnovers compared with APX2 without compromising catalytic efficiency. Structural, spectroscopic, and kinetic characterization of APX2 NMH and several active site variants provided valuable insights into the role of the Asp-His-Fe triad of heme peroxidases. More significantly, simplification of catalytic mechanisms through the incorporation of chem. optimized ligands may facilitate efforts to create and evolve new active site heme environments within proteins.
- 382Sharp, K. H.; Mewies, M.; Moody, P. C. E.; Raven, E. L. Crystal Structure of the Ascorbate Peroxidase-Ascorbate Complex. Nat. Struct. Biol. 2003, 10 (4), 303– 307, DOI: 10.1038/nsb913There is no corresponding record for this reference.
- 383Vojtechovský, J.; Chu, K.; Berendzen, J.; Sweet, R. M.; Schlichting, I. Crystal Structures of Myoglobin-Ligand Complexes at Near-Atomic Resolution. Biophys. J. 1999, 77 (4), 2153– 2174, DOI: 10.1016/S0006-3495(99)77056-6There is no corresponding record for this reference.
- 384Pott, M.; Hayashi, T.; Mori, T.; Mittl, P. R. E.; Green, A. P.; Hilvert, D. A Noncanonical Proximal Heme Ligand Affords an Efficient Peroxidase in a Globin Fold. J. Am. Chem. Soc. 2018, 140 (4), 1535– 1543, DOI: 10.1021/jacs.7b12621384A Noncanonical Proximal Heme Ligand Affords an Efficient Peroxidase in a Globin FoldPott, Moritz; Hayashi, Takahiro; Mori, Takahiro; Mittl, Peer R. E.; Green, Anthony P.; Hilvert, DonaldJournal of the American Chemical Society (2018), 140 (4), 1535-1543CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Expanding the range of genetically encoded metal coordination environments accessible within tunable protein scaffolds presents excellent opportunities for the creation of metalloenzymes with augmented properties and novel activities. Here, we demonstrate that installation of a noncanonical Nδ-Me histidine (NMH) as the proximal heme ligand in the oxygen binding protein myoglobin (Mb) leads to substantial increases in heme redox potential and promiscuous peroxidase activity. Structural characterization of this catalytically modified myoglobin variant (Mb NMH) revealed significant changes in the proximal pocket, including alterations to hydrogen-bonding interactions involving the prosthetic porphyrin cofactor. Further optimization of Mb NMH via a combination of rational modification and several rounds of lab. evolution afforded efficient peroxidase biocatalysts within a globin fold, with activities comparable to those displayed by nature's peroxidases.
- 385Matsuo, T.; Fukumoto, K.; Watanabe, T.; Hayashi, T. Precise Design of Artificial Cofactors for Enhancing Peroxidase Activity of Myoglobin: Myoglobin Mutant H64D Reconstituted with a ″Single-Winged Cofactor″ Is Equivalent to Native Horseradish Peroxidase in Oxidation Activity. Chem. Asian J. 2011, 6 (9), 2491– 2499, DOI: 10.1002/asia.201100107There is no corresponding record for this reference.
- 386Hayashi, T.; Tinzl, M.; Mori, T.; Krengel, U.; Proppe, J.; Soetbeer, J.; Klose, D.; Jeschke, G.; Reiher, M.; Hilvert, D. Capture and Characterization of a Reactive Haem-Carbenoid Complex in an Artificial Metalloenzyme. Nat. Catal. 2018, 1 (8), 578– 584, DOI: 10.1038/s41929-018-0105-6There is no corresponding record for this reference.
- 387Pott, M.; Tinzl, M.; Hayashi, T.; Ota, Y.; Dunkelmann, D. L.; Mittl, P. R. E.; Hilvert, D. Noncanonical Heme Ligands Steer Carbene Transfer Reactivity in an Artificial Metalloenzyme. Angew. Chem. Int. Ed 2021, 60, 15063– 15068, DOI: 10.1002/anie.202103437387Noncanonical Heme Ligands Steer Carbene Transfer Reactivity in an Artificial MetalloenzymePott, Moritz; Tinzl, Matthias; Hayashi, Takahiro; Ota, Yusuke; Dunkelmann, Daniel; Mittl, Peer R. E.; Hilvert, DonaldAngewandte Chemie, International Edition (2021), 60 (27), 15063-15068CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Changing the primary metal coordination sphere is a powerful strategy for tuning metalloprotein properties. Here we used amber stop codon suppression with engineered pyrrolysyl-tRNA synthetases, including two newly evolved enzymes, to replace the proximal histidine in myoglobin with Nδ-methylhistidine, 5-thiazoylalanine, 4-thiazoylalanine and 3-(3-thienyl)alanine. In addn. to tuning the heme redox potential over a >200 mV range, these noncanonical ligands modulate the protein's carbene transfer activity with Et diazoacetate. Variants with increased redn. potential proved superior for cyclopropanation and N-H insertion, whereas variants with reduced Eo values gave higher S-H insertion activity. Given the functional importance of histidine in many enzymes, these genetically encoded analogs could be valuable tools for probing mechanism and enabling new chemistries.
- 388Carminati, D. M.; Fasan, R. Stereoselective Cyclopropanation of Electron-Deficient Olefins with a Cofactor Redesigned Carbene Transferase Featuring Radical Reactivity. ACS Catal. 2019, 9 (10), 9683– 9697, DOI: 10.1021/acscatal.9b02272388Stereoselective Cyclopropanation of Electron-Deficient Olefins with a Cofactor Redesigned Carbene Transferase Featuring Radical ReactivityCarminati, Daniela M.; Fasan, RudiACS Catalysis (2019), 9 (10), 9683-9697CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Engineered myoglobins and other hemoproteins have recently emerged as promising catalysts for asym. olefin cyclopropanation reactions via carbene-transfer chem. Despite this progress, the transformation of electron-poor alkenes has proven to be very challenging using these systems. Here, we describe the design of a myoglobin-based carbene transferase incorporating a non-native iron-porphyrin cofactor and axial ligand, as an efficient catalyst for the asym. cyclopropanation of electron-deficient alkenes. Using this metalloenzyme, a broad range of both electron-rich and electron-deficient alkenes are cyclopropanated with high efficiency and high diastereo- and enantioselectivity (up to >99% de and ee). Mechanistic studies revealed that the expanded reaction scope of this carbene transferase is dependent upon the acquisition of metallocarbene radical reactivity as a result of the reconfigured coordination environment around the metal center. The radical-based reactivity of this system diverges from the electrophilic reactivity of myoglobin and most of the known organometallic carbene-transfer catalysts. This work showcases the value of cofactor redesign toward tuning and expanding the reactivity of metalloproteins in abiol. reactions, and it provides a biocatalytic soln. to the asym. cyclopropanation of electron-deficient alkenes. The metallocarbene radical reactivity exhibited by this biocatalyst is anticipated to prove useful in the context of a variety of other synthetic transformations.
- 389Moore, E. J.; Fasan, R. Effect of Proximal Ligand Substitutions on the Carbene and Nitrene Transferase Activity of Myoglobin. Tetrahedron 2019, 75 (16), 2357– 2363, DOI: 10.1016/j.tet.2019.03.009389Effect of proximal ligand substitutions on the carbene and nitrene transferase activity of myoglobinMoore, Eric J.; Fasan, RudiTetrahedron (2019), 75 (16), 2357-2363CODEN: TETRAB; ISSN:0040-4020. (Elsevier Ltd.)Engineered myoglobins (Mbs) were recently shown to be effective catalysts for abiol. carbene and nitrene transfer reactions. Here, we investigated the impact of substituting the conserved heme-coordinating histidine residue with both proteinogenic (Cys, Ser, Tyr, Asp) and non-proteinogenic Lewis basic amino acids (3-(3'-pyridyl)-alanine, p-aminophenylalanine, and β-(3-thienyl)-alanine), on the reactivity of this metalloprotein toward these abiotic transformations. These studies showed that mutation of the proximal histidine residue with both natural and non-natural amino acids result in stable myoglobin variants that can function as both carbene and nitrene transferases. In addn., substitution of the proximal histidine with an aspartate residue led to a myoglobin-based catalyst capable of promoting stereoselective olefin cyclopropanation under nonreducing conditions. Overall, these studies demonstrate that proximal ligand substitution provides a promising strategy to tune the reactivity of myoglobin-based carbene and nitrene transfer catalysts and provide a first, proof-of-principle demonstration of the viability of pyridine-, thiophene-, and aniline-based unnatural amino acids for metalloprotein engineering.
- 390Gan, F.; Liu, R.; Wang, F.; Schultz, P. G. Functional Replacement of Histidine in Proteins To Generate Noncanonical Amino Acid Dependent Organisms. J. Am. Chem. Soc. 2018, 140 (11), 3829– 3832, DOI: 10.1021/jacs.7b13452390Functional replacement of histidine in proteins to generate noncanonical amino acid dependent organismsGan, Fei; Liu, Renhe; Wang, Feng; Schultz, Peter G.Journal of the American Chemical Society (2018), 140 (11), 3829-3832CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Simple strategies to produce organisms whose growth is strictly dependent on the presence of a noncanonical amino acid are useful for the generation of live vaccines and the biol. containment of recombinant organisms. To this end, we report an approach based on genetically replacing key histidine (His) residues in essential proteins with functional His analogs. We demonstrate that 3-methyl-L-histidine (MeH) functionally substitutes for a key metal binding ligand, H264, in the zinc-contg. metalloenzyme mannose-6-phosphate isomerase (ManA). An evolved variant, Opt5, harboring both N262S and H264MeH substitutions exhibited comparable activities to wild type ManA. An engineered Escherichia coli strain contg. the ManA variant Opt5 was strictly dependent on MeH for growth with an extremely low reversion rate. This straightforward strategy should be applicable to other metallo- or nonmetalloproteins that contain essential His residues.
- 391Chand, S.; Ray, S.; Yadav, P.; Samanta, S.; Pierce, B. S.; Perera, R. Abiological Catalysis by Myoglobin Mutant with a Genetically Incorporated Unnatural Amino Acid. Biochem. J. 2021, 478 (9), 1795– 1808, DOI: 10.1042/BCJ20210091391Abiological catalysis by myoglobin mutant with a genetically incorporated unnatural amino acidChand, Subhash; Ray, Sriparna; Yadav, Poonam; Samanta, Susruta; Pierce, Brad S.; Perera, RoshanBiochemical Journal (2021), 478 (9), 1795-1808CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)To inculcate biocatalytic activity in the oxygen-storage protein myoglobin (Mb), a genetically engineered myoglobin mutant H64DOPA (DOPA = L-3,4-dihydroxyphenylalanine) has been created. Incorporation of unnatural amino acids has already demonstrated their ability to accomplish many non-natural functions in proteins efficiently. Herein, the presence of redox-active DOPA residue in the active site of mutant Mb presumably stabilizes the compd. I in the catalytic oxidn. process by participating in an addnl. hydrogen bonding (H-bonding) as compared to the WT Mb. Specifically, a general acid-base catalytic pathway was achieved due to the availability of the hydroxyl moieties of DOPA. The redn. potential values of WT (E° = -260 mV) and mutant Mb (E° = -300 mV), w.r.t. Ag/AgCl ref. electrode, in the presence of hydrogen peroxide, indicated an addnl. H-bonding in the mutant protein, which is responsible for the peroxidase activity of the mutant Mb. We obsd. that in the presence of 5 mM H2O2, H64DOPA Mb oxidizes thioanisole and benzaldehyde with a 10 and 54 folds higher rate, resp., as opposed to WT Mb. Based on spectroscopic, kinetic, and electrochem. studies, we deduce that DOPA residue, when present within the distal pocket of mutant Mb, alone serves the role of His/Arg-pair of peroxidases.
- 392Jackson, J. C.; Duffy, S. P.; Hess, K. R.; Mehl, R. A. Improving Nature’s Enzyme Active Site with Genetically Encoded Unnatural Amino Acids. J. Am. Chem. Soc. 2006, 128 (34), 11124– 11127, DOI: 10.1021/ja061099y392Improving Nature's enzyme active site with genetically encoded unnatural amino acidsJackson, Jennifer C.; Duffy, Sean P.; Hess, Kenneth R.; Mehl, Ryan A.Journal of the American Chemical Society (2006), 128 (34), 11124-11127CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The ability to site-specifically incorporate a diverse set of unnatural amino acids (>30) into proteins and quickly add new structures of interest has recently changed our approach to protein use and study. One important question yet unaddressed with unnatural amino acids (UAAs) is whether they can improve the activity of an enzyme beyond that available from the natural 20 amino acids. Herein, we report the >30-fold improvement of prodrug activator nitroreductase activity with an UAA over that of the native active site and a >2.3-fold improvement over the best possible natural amino acid. Because immense structural and electrostatic diversity at a single location can be sampled very quickly, UAAs can be implemented to improve enzyme active sites and tune a site to multiple substrates.
- 393Grove, J. I.; Lovering, A. L.; Guise, C.; Race, P. R.; Wrighton, C. J.; White, S. A.; Hyde, E. I.; Searle, P. F. Generation of Escherichia coli Nitroreductase Mutants Conferring Improved Cell Sensitization to the Prodrug CB19541. Cancer Res. 2003, 63 (17), 5532– 5537There is no corresponding record for this reference.
- 394Ugwumba, I. N.; Ozawa, K.; Xu, Z.-Q.; Ely, F.; Foo, J.-L.; Herlt, A. J.; Coppin, C.; Brown, S.; Taylor, M. C.; Ollis, D. L. Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino Acids. J. Am. Chem. Soc. 2011, 133 (2), 326– 333, DOI: 10.1021/ja106416g394Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino AcidsUgwumba, Isaac N.; Ozawa, Kiyoshi; Xu, Zhi-Qiang; Ely, Fernanda; Foo, Jee-Loon; Herlt, Anthony J.; Coppin, Chris; Brown, Sue; Taylor, Matthew C.; Ollis, David L.; Mander, Lewis N.; Schenk, Gerhard; Dixon, Nicholas E.; Otting, Gottfried; Oakeshott, John G.; Jackson, Colin J.Journal of the American Chemical Society (2011), 133 (2), 326-333CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and to probe the role of peripheral active site residues in nonchem. steps of the catalytic cycle (substrate binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with unnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino acids, as well as leucine, phenylalanine, and tryptophan. Kinetic anal. suggests that the 7-hydroxyl group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product release step of substrate turnover as a result of its electrostatic repulsion of the neg. charged 4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to the difficulty in improving this native activity through screening hundreds of thousands of mutants with natural amino acids. These results demonstrate that designer amino acids provide easy access to new and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.
- 395Pagar, A. D.; Jeon, H.; Khobragade, T. P.; Sarak, S.; Giri, P.; Lim, S.; Yoo, T. H.; Ko, B. J.; Yun, H. Non-Canonical Amino Acid-Based Engineering of (R)-Amine Transaminase. Front. Chem. 2022, DOI: 10.3389/fchem.2022.839636There is no corresponding record for this reference.
- 396Savile, C. K.; Janey, J. M.; Mundorff, E. C.; Moore, J. C.; Tam, S.; Jarvis, W. R.; Colbeck, J. C.; Krebber, A.; Fleitz, F. J.; Brands, J. Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin Manufacture. Science 2010, 329 (5989), 305– 309, DOI: 10.1126/science.1188934396Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin ManufactureSavile, Christopher K.; Janey, Jacob M.; Mundorff, Emily C.; Moore, Jeffrey C.; Tam, Sarena; Jarvis, William R.; Colbeck, Jeffrey C.; Krebber, Anke; Fleitz, Fred J.; Brands, Jos; Devine, Paul N.; Huisman, Gjalt W.; Hughes, Gregory J.Science (Washington, DC, United States) (2010), 329 (5989), 305-309CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Pharmaceutical synthesis can benefit greatly from the selectivity gains assocd. with enzymic catalysis. Here, we report an efficient biocatalytic process to replace a recently implemented rhodium-catalyzed asym. enamine hydrogenation for the large-scale manuf. of the antidiabetic compd. sitagliptin. Starting from an enzyme that had the catalytic machinery to perform the desired chem. but lacked any activity toward the prositagliptin ketone, we applied a substrate walking, modeling, and mutation approach to create a transaminase with marginal activity for the synthesis of the chiral amine; this variant was then further engineered via directed evolution for practical application in a manufg. setting. The resultant biocatalysts showed broad applicability toward the synthesis of chiral amines that previously were accessible only via resoln. This work underscores the maturation of biocatalysis to enable efficient, economical, and environmentally benign processes for the manuf. of pharmaceuticals.
- 397Wilkinson, H. C.; Dalby, P. A. Fine-Tuning the Activity and Stability of an Evolved Enzyme Active-Site Through Noncanonical Amino-Acids. FEBS J. 2021, 288 (6), 1935– 1955, DOI: 10.1111/febs.15560There is no corresponding record for this reference.
- 398Parsons, J. F.; Xiao, G.; Gilliland, G. L.; Armstrong, R. N. Enzymes Harboring Unnatural Amino Acids: Mechanistic and Structural Analysis of the Enhanced Catalytic Activity of a Glutathione Transferase Containing 5-Fluorotryptophan. Biochemistry 1998, 37 (18), 6286– 6294, DOI: 10.1021/bi980219e398Enzymes Harboring Unnatural Amino Acids: Mechanistic and Structural Analysis of the Enhanced Catalytic Activity of a Glutathione Transferase Containing 5-FluorotryptophanParsons, James F.; Xiao, Gaoyi; Gilliland, Gary L.; Armstrong, Richard N.Biochemistry (1998), 37 (18), 6286-6294CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The catalytic characteristics and structure of the M1-1 isoenzyme of rat glutathione (GSH) transferase in which all four tryptophan residues in each monomer are replaced with 5-fluorotryptophan are described. The fluorine-for-hydrogen substitution does not change the interaction of the enzyme with GSH even though two tryptophan residues (Trp7 and Trp45) are involved in direct hydrogen-bonding interactions with the substrate. The rate consts. for assocn. and dissocn. of the peptide, measured by stopped-flow spectrometry, remain unchanged by the unnatural amino acid. The 5-FTrp-substituted enzyme exhibits a kcat of 73 s-1 as compared to 18 s-1 for the native enzyme toward 1-chloro-2,4-dinitrobenzene. That the increase in the turnover no. is due to an enhanced rate of product release in the mutant is confirmed by the kinetics of the approach to equil. for binding of the product. The crystal structure of the 5-FTrp-contg. enzyme was solved at a resoln. of 2.0 Å by difference Fourier techniques. The structure reveals local conformational changes in the structural elements that define the approach to the active site which are attributed to steric interactions of the fluorine atoms assocd. with 5-FTrp146 and 5-FTrp214 in domain II. These changes appear to result in the enhanced rate of product release. This structure represents the first of a protein substituted with 5-fluorotryptophan.
- 399Dominguez, M. A., Jr; Thornton, K. C.; Melendez, M. G.; Dupureur, C. M. Differential Effects of Isomeric Incorporation of Fluorophenylalanines into PvuII Endonuclease. Proteins 2001, 45 (1), 55– 61, DOI: 10.1002/prot.1123There is no corresponding record for this reference.
- 400Hoesl, M. G.; Budisa, N. Expanding and Engineering the Genetic Code in a Single Expression Experiment. ChemBioChem 2011, 12, 552– 555, DOI: 10.1002/cbic.201000586400Expanding and Engineering the Genetic Code in a Single Expression ExperimentHoesl, Michael G.; Budisa, NediljkoChemBioChem (2011), 12 (4), 552-555CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)The concept of expanded and engineered genetic code was exptl. verified by simultaneous insertions of p-benzoyl-phenylalanine at UAG stop codons together with the global replacements Met→norleucine in TTL or Pro→cis-4-fluoroproline in EGFP model proteins. In this way, residue-specific, sense codon reassignment ("code engineering") was combined with position-specific stop-codon suppression ("code expansion") in a single in vivo expression expt.
- 401Cirino, P. C.; Tang, Y.; Takahashi, K.; Tirrell, D. A.; Arnold, F. H. Global Incorporation of Norleucine in Place of Methionine in Cytochrome P450 BM-3 Heme Domain Increases Peroxygenase Activity. Biotechnol. Bioeng. 2003, 83 (6), 729– 734, DOI: 10.1002/bit.10718There is no corresponding record for this reference.
- 402Xiao, H.; Nasertorabi, F.; Choi, S.-h.; Han, G. W.; Reed, S. A.; Stevens, R. C.; Schultz, P. G. Exploring the Potential Impact of an Expanded Genetic Code on Protein Function. Proc. Natl. Acad. Sci. U.S.A. 2015, 112 (22), 6961– 6966, DOI: 10.1073/pnas.1507741112There is no corresponding record for this reference.
- 403Young, T. S.; Ahmad, I.; Yin, J. A.; Schultz, P. G. An Enhanced System for Unnatural Amino Acid Mutagenesis in E. coli. J. Mol. Biol. 2010, 395, 361– 374, DOI: 10.1016/j.jmb.2009.10.030403An enhanced system for unnatural amino acid mutagenesis in E. coliYoung, Travis S.; Ahmad, Insha; Yin, Jun A.; Schultz, Peter G.Journal of Molecular Biology (2010), 395 (2), 361-374CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)We report a new vector, pEVOL, for the incorporation of unnatural amino acids into proteins in Escherichia coli using evolved Methanocaldococcus jannaschii aminoacyl-tRNA synthetase(s) (aaRS)/suppressor tRNA pairs. This new system affords higher yields of mutant proteins through the use of both constitutive and inducible promoters to drive the transcription of two copies of the M. jannaschii aaRS gene. Yields were further increased by coupling the dual-aaRS promoter system with a newly optimized suppressor tRNACUA opt in a single-vector construct. The optimized suppressor tRNACUA opt afforded increased plasmid stability compared with previously reported vectors for unnatural amino acid mutagenesis. To demonstrate the utility of this new system, we introduced 14 mutant aaRS into pEVOL and compared their ability to insert unnatural amino acids in response to three independent amber nonsense codons in sperm whale myoglobin or green fluorescent protein. When cultured in rich media in shake flasks, pEVOL was capable of producing more than 100 mg/L mutant GroEL protein. The versatility, increased yields, and increased stability of the pEVOL vector will further facilitate the expression of proteins with unnatural amino acids.
- 404Schoffelen, S.; Beekwilder, J.; Debets, M. F.; Bosch, D.; Hest, J. C. M. v. Construction of a Multifunctional Enzyme Complex via the Strain-Promoted Azide-Alkyne Cycloaddition. Bioconjug. Chem. 2013, 24 (6), 987– 996, DOI: 10.1021/bc400021j404Construction of a Multifunctional Enzyme Complex via the Strain-Promoted Azide-Alkyne CycloadditionSchoffelen, Sanne; Beekwilder, Jules; Debets, Marjoke F.; Bosch, Dirk; Hest, Jan C. M. vanBioconjugate Chemistry (2013), 24 (6), 987-996CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)Inspired by the multienzyme complexes occurring in nature, enzymes have been brought together in vitro as well. We report a co-localization strategy milder than nonspecific crosslinking, and free of any scaffold and affinity tags. Using non-natural amino acid incorporation, two heterobifunctional linkers, and the strain-promoted azide-alkyne cycloaddn. as conjugation reaction, three metabolic enzymes are linked together in a controlled manner. Conjugate formation was demonstrated by size-exclusion chromatog. and gel electrophoresis. The multienzyme complexes were further characterized by native mass spectrometry. It was shown that the complexes catalyzed the three-step biosynthesis of piceid in vitro with comparable kinetic behavior to the uncoupled enzymes. The approach is envisioned to have high potential for various biotechnol. applications, in which multiple biocatalysts collaborate at low concns., in which diffusion may be limited and/or side-reactions are prone to occur.
- 405Lim, S. I.; Cho, J.; Kwon, I. Double Clicking for Site-Specific Coupling of Multiple Enzymes. Chem. Commun. 2015, 51 (71), 13607– 13610, DOI: 10.1039/C5CC04611DThere is no corresponding record for this reference.
- 406Lim, S. I.; Yang, B.; Jung, Y.; Cha, J.; Cho, J.; Choi, E.-S.; Kim, Y. H.; Kwon, I. Controlled Orientation of Active Sites in a Nanostructured Multienzyme Complex. Sci. Rep. 2016, 6 (1), 39587, DOI: 10.1038/srep39587406Controlled Orientation of Active Sites in a Nanostructured Multienzyme ComplexLim, Sung In; Yang, Byungseop; Jung, Younghan; Cha, Jaehyun; Cho, Jinhwan; Choi, Eun-Sil; Kim, Yong Hwan; Kwon, InchanScientific Reports (2016), 6 (), 39587CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)Multistep cascade reactions in nature maximize reaction efficiency by co-assembling related enzymes. Such organization facilitates the processing of intermediates by downstream enzymes. Previously, the studies on multienzyme nanocomplexes assembled on DNA scaffolds demonstrated that closer interenzyme distance enhances the overall reaction efficiency. However, it remains unknown how the active site orientation controlled at nanoscale can have an effect on multienzyme reaction. Here, we show that controlled alignment of active sites promotes the multienzyme reaction efficiency. By genetic incorporation of a non-natural amino acid and two compatible bioorthogonal chemistries, we conjugated mannitol dehydrogenase to formate dehydrogenase with the defined active site arrangement with the residue-level accuracy. The study revealed that the multienzyme complex with the active sites directed towards each other exhibits four-fold higher relative efficiency enhancement in the cascade reaction and produces 60% more D-mannitol than the other complex with active sites directed away from each other.
- 407Ha, J. M.; Jeon, S. T.; Yoon, H. J.; Lee, H. H. Formate Dehydrogenase. PDB 2014, DOI: 10.2210/pdb3WR5/pdbThere is no corresponding record for this reference.
- 408Kavanagh, K. L.; Klimacek, M.; Nidetzky, B.; Wilson, D. K. Crystal Structure of Pseudomonas fluorescens Mannitol 2-Dehydrogenase Binary and Ternary Complexes: Specificity And Catalytic Mechanism. J. Biol. Chem. 2002, 277 (45), 43433– 43442, DOI: 10.1074/jbc.M206914200There is no corresponding record for this reference.
- 409Li, J.; Jia, S.; Chen, P. R. Diels-Alder Reaction-Triggered Bioorthogonal Protein Decaging in Living Cells. Nat. Chem. Biol. 2014, 10 (12), 1003– 1005, DOI: 10.1038/nchembio.1656409Diels-Alder reaction-triggered bioorthogonal protein decaging in living cellsLi, Jie; Jia, Shang; Chen, Peng R.Nature Chemical Biology (2014), 10 (12), 1003-1005CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Small mols. that specifically activate an intracellular protein of interest are highly desirable. A generally applicable strategy, however, remains elusive. Herein we describe a small mol.-triggered bioorthogonal protein decaging technique that relies on the inverse electron-demand Diels-Alder reaction for eliminating a chem. caged protein side chain within living cells. This method permits the efficient activation of a given protein (for example, an enzyme) in its native cellular context within minutes.
- 410Li, J.; Yu, J.; Zhao, J.; Wang, J.; Zheng, S.; Lin, S.; Chen, L.; Yang, M.; Jia, S.; Zhang, X. Palladium-Triggered Deprotection Chemistry for Protein Activation in Living Cells. Nat. Chem. 2014, 6 (4), 352– 361, DOI: 10.1038/nchem.1887410Palladium-triggered deprotection chemistry for protein activation in living cellsLi, Jie; Yu, Juntao; Zhao, Jingyi; Wang, Jie; Zheng, Siqi; Lin, Shixian; Chen, Long; Yang, Maiyun; Jia, Shang; Zhang, Xiaoyu; Chen, Peng R.Nature Chemistry (2014), 6 (4), 352-361CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)Employing small mols. or chem. reagents to modulate the function of an intracellular protein, particularly in a gain-of-function fashion, remains a challenge. In contrast to inhibitor-based loss-of-function approaches, methods based on a gain of function enable specific signalling pathways to be activated inside a cell. Here we report a chem. rescue strategy that uses a palladium-mediated deprotection reaction to activate a protein within living cells. We identify biocompatible and efficient palladium catalysts that cleave the propargyl carbamate group of a protected lysine analog to generate a free lysine. The lysine analog can be genetically and site-specifically incorporated into a protein, which enables control over the reaction site. This deprotection strategy is shown to work with a range of different cell lines and proteins. We further applied this biocompatible protection group/catalyst pair for caging and subsequent release of a crucial lysine residue in a bacterial Type III effector protein within host cells, which reveals details of its virulence mechanism.
- 411Wang, J.; Zheng, S.; Liu, Y.; Zhang, Z.; Lin, Z.; Li, J.; Zhang, G.; Wang, X.; Li, J.; Chen, P. R. Palladium-Triggered Chemical Rescue of Intracellular Proteins via Genetically Encoded Allene-Caged Tyrosine. J. Am. Chem. Soc. 2016, 138 (46), 15118– 15121, DOI: 10.1021/jacs.6b08933411Palladium-Triggered Chemical Rescue of Intracellular Proteins via Genetically Encoded Allene-Caged TyrosineWang, Jie; Zheng, Siqi; Liu, Yanjun; Zhang, Zhaoyue; Lin, Zhi; Li, Jiaofeng; Zhang, Gong; Wang, Xin; Li, Jie; Chen, Peng R.Journal of the American Chemical Society (2016), 138 (46), 15118-15121CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Chem. de-caging has emerged as an attractive strategy for gain-of-function study of proteins via small-mol. reagents. The previously reported chem. de-caging reactions have been largely centered on liberating the side chain of lysine on a given protein. Herein, the authors developed an allene-based caging moiety and the corresponding palladium de-caging reagents for chem. rescue of tyrosine (Tyr) activity on intracellular proteins. This bioorthogonal de-caging pair has been successfully applied to unmask enzymic Tyr sites (e.g., Y671 on Taq polymerase and Y728 on Anthrax lethal factor) as well as the posttranslational Tyr modification site (Y416 on Src kinase) in vitro and in living cells. The strategy provides a general platform for chem. rescue of Tyr-dependent protein activity inside cells.
- 412Georgianna, W. E.; Lusic, H.; McIver, A. L.; Deiters, A. Photocleavable Polyethylene Glycol for the Light-Regulation of Protein Function. Bioconjug. Chem. 2010, 21 (8), 1404– 1407, DOI: 10.1021/bc100084n412Photocleavable Polyethylene Glycol for the Light-Regulation of Protein FunctionGeorgianna, Wesleigh E.; Lusic, Hrvoje; McIver, Andrew L.; Deiters, AlexanderBioconjugate Chemistry (2010), 21 (8), 1404-1407CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)PEGylation is commonly employed to enhance the pharmacokinetic properties of proteins, but it can interfere with natural protein function. Protein activity can thus be abrogated through PEGylation, and a controllable means to remove the polyethylene glycol (PEG) group from the protein is desirable. As such, light affords a unique control over biomols. through the application of photosensitive groups. Herein, the authors report the synthesis of a photocleavable PEG reagent (PhotoPEG) and its application to the light-regulation of enzyme activity.
- 413Wu, N.; Deiters, A.; Cropp, T. A.; King, D.; Schultz, P. G. A Genetically Encoded Photocaged Amino Acid. J. Am. Chem. Soc. 2004, 126 (44), 14306– 14307, DOI: 10.1021/ja040175z413A Genetically Encoded Photocaged Amino AcidWu, Ning; Deiters, Alexander; Cropp, T. Ashton; King, David; Schultz, Peter G.Journal of the American Chemical Society (2004), 126 (44), 14306-14307CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We have developed a second orthogonal tRNA/synthetase pair for use in yeast based on the Escherichia coli tRNALeu/leucyl tRNA-synthetase pair. Using a novel genetic selection, we have identified a series of synthetase mutants that selectively charge the amber suppressor tRNA with the α-aminocaprylic acid, O-methyltyrosine and o-nitrobenzyl cysteine (photocaged amino acid) allowing them to be incorporated into proteins in yeast in response to the amber nonsense codon, TAG. Biosynthesis and photoactivation of photocaged cysteine-contg. superoxide dismutase and caspase-3 is demonstrated.
- 414Deiters, A.; Groff, D.; Ryu, Y.; Xie, J.; Schultz, P. G. A Genetically Encoded Photocaged Tyrosine. Angew. Chem. Int. Ed. 2006, 45 (17), 2728– 2731, DOI: 10.1002/anie.200600264414A genetically encoded photocaged tyrosineDeiters, Alexander; Groff, Dan; Ryu, Youngha; Xie, Jianming; Schultz, Peter G.Angewandte Chemie, International Edition (2006), 45 (17), 2728-2731CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A photocaged tyrosine was genetically encoded in Escherichia coli in response to the amber codon TAG. Substitution of Tyr503 in the active site of β-galactosidase allowed photoactivation of this enzyme in vitro or directly in bacteria with 360-nm light. This method should allow photoregulation of the activity of a variety of biol. processes including transcription, signal transduction, and cellular trafficking.
- 415Chou, C.; Young, D. D.; Deiters, A. A Light-Activated DNA Polymerase. Angew. Chem. Int. Ed. 2009, 48 (32), 5950– 5953, DOI: 10.1002/anie.200901115There is no corresponding record for this reference.
- 416Chou, C.; Young, D. D.; Deiters, A. Photocaged T7 RNA Polymerase for the Light Activation of Transcription and Gene Function in Pro- and Eukaryotic Cells. ChemBioChem 2010, 11 (7), 972– 977, DOI: 10.1002/cbic.201000041416Photocaged T7 RNA Polymerase for the Light Activation of Transcription and Gene Function in Pro- and Eukaryotic CellsChou, Chungjung; Young, Douglas D.; Deiters, AlexanderChemBioChem (2010), 11 (7), 972-977CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)A light-activatable bacteriophage T7 RNA polymerase (T7RNAP) has been generated through the site-specific introduction of a photocaged tyrosine residue at the crucial position Tyr639 within the active site of the enzyme. The photocaged tyrosine disrupts polymerase activity by blocking the incoming nucleotide from reaching the active site of the enzyme. However, a brief irradn. with nonphototoxic UV light of 365 nm removes the ortho-nitrobenzyl caging group from Tyr639 and restores the RNA polymerase activity of T7RNAP. The complete orthogonality of T7RNAP to all endogenous RNA polymerases in pro- and eukaryotic systems allowed for the photochem. activation of gene expression in bacterial and mammalian cells. Specifically, E. coli cells were engineered to produce photocaged T7RNAP in the presence of a GFP reporter gene under the control of a T7 promoter. UV irradn. of these cells led to the spatiotemporal activation of GFP expression. In an analogous fashion, caged T7RNAP was transfected into human embryonic kidney (HEK293T) cells. Irradn. with UV light led to the activation of T7RNAP, thereby inducing RNA polymn. and expression of a luciferase reporter gene in tissue culture. The ability to achieve spatiotemporal regulation of orthogonal RNA synthesis enables the precise dissection and manipulation of a wide range of cellular events, including gene function.
- 417Chou, C.; Deiters, A. Light-Activated Gene Editing with a Photocaged Zinc-Finger Nuclease. Angew. Chem. Int. Ed. 2011, 50 (30), 6839, DOI: 10.1002/anie.201101157There is no corresponding record for this reference.
- 418Gautier, A.; Nguyen, D. P.; Lusic, H.; An, W.; Deiters, A.; Chin, J. W. Genetically Encoded Photocontrol of Protein Localization in Mammalian Cells. J. Am. Chem. Soc. 2010, 132 (12), 4086– 4088, DOI: 10.1021/ja910688s418Genetically Encoded Photocontrol of Protein Localization in Mammalian CellsGautier, Arnaud; Nguyen, Duy P.; Lusic, Hrvoje; An, Wenlin; Deiters, Alexander; Chin, Jason W.Journal of the American Chemical Society (2010), 132 (12), 4086-4088CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Precise photochem. control of protein function can be achieved through the site-specific introduction of caging groups. Chem. and enzymic methods, including in vitro translation and chem. ligation, have been used to photocage proteins in vitro. These methods have been extended to allow the introduction of caged proteins into cells by permeabilization or microinjection, but cellular delivery remains challenging. Since lysine residues are key determinants for nuclear localization sequences, the target of key post-translational modifications (including ubiquitination, methylation, and acetylation), and key residues in many important enzyme active sites, we were interested in photocaging lysine to control protein localization, post-translational modification, and enzymic activity. Photochem. control of these important functions mediated by lysine residues in proteins has not previously been demonstrated in living cells. Here we synthesized 1 and evolved a pyrrolysyl-tRNA synthetase/tRNA pair to genetically encode the incorporation of this amino acid in response to an amber codon in mammalian cells. To exemplify the utility of this amino acid, we caged the nuclear localization sequences (NLSs) of nucleoplasmin and the tumor suppressor p53 in human cells, thus mislocalizing the proteins in the cytosol. We triggered protein nuclear import with a pulse of light, allowing us to directly quantify the kinetics of nuclear import.
- 419Gautier, A.; Deiters, A.; Chin, J. W. Light-Activated Kinases Enable Temporal Dissection of Signaling Networks in Living Cells. J. Am. Chem. Soc. 2011, 133 (7), 2124– 2127, DOI: 10.1021/ja1109979419Light-activated kinases enable temporal dissection of signaling networks in living cellsGautier, Arnaud; Deiters, Alexander; Chin, Jason W.Journal of the American Chemical Society (2011), 133 (7), 2124-2127CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We report a general strategy for creating protein kinases in mammalian cells that are poised for very rapid activation by light. By photoactivating a caged version of MEK1, we demonstrate the specific, rapid, and receptor independent activation of an artificial subnetwork within the Raf/MEK/ERK pathway. Time-lapse microscopy allowed us to precisely characterize the kinetics of elementary steps in the signaling cascade and provided insight into adaptive feedback and rate-detg. processes in the pathway.
- 420Hemphill, J.; Chou, C.; Chin, J. W.; Deiters, A. Genetically Encoded Light-Activated Transcription for Spatiotemporal Control of Gene Expression and Gene Silencing in Mammalian Cells. J. Am. Chem. Soc. 2013, 135 (36), 13433– 13439, DOI: 10.1021/ja4051026420Genetically encoded light-activated transcription for spatiotemporal control of gene expression and gene silencing in mammalian cellsHemphill, James; Chou, Chungjung; Chin, Jason W.; Deiters, AlexanderJournal of the American Chemical Society (2013), 135 (36), 13433-13439CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Photocaging provides a method to spatially and temporally control biol. function and gene expression with high resoln. Proteins can be photochem. controlled through the site-specific installation of caging groups on amino acid side chains that are essential for protein function. The photocaging of a synthetic gene network using unnatural amino acid mutagenesis in mammalian cells was demonstrated with an engineered bacteriophage RNA polymerase. A caged T7 RNA polymerase was expressed in cells with an expanded genetic code and used in the photochem. activation of genes under control of an orthogonal T7 promoter, demonstrating tight spatial and temporal control. The synthetic gene expression system was validated with two reporter genes (luciferase and EGFP) and applied to the light-triggered transcription of short hairpin RNA constructs for the induction of RNA interference.
- 421Hemphill, J.; Borchardt, E. K.; Brown, K.; Asokan, A.; Deiters, A. Optical Control of CRISPR/Cas9 Gene Editing. J. Am. Chem. Soc. 2015, 137 (17), 5642– 5645, DOI: 10.1021/ja512664v421Optical control of CRISPR/Cas9 gene editingHemphill, James; Borchardt, Erin K.; Brown, Kalyn; Asokan, Aravind; Deiters, AlexanderJournal of the American Chemical Society (2015), 137 (17), 5642-5645CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The CRISPR/Cas9 system has emerged as an important tool in biomedical research for a wide range of applications, with significant potential for genome engineering and gene therapy. In order to achieve conditional control of the CRISPR/Cas9 system, a genetically encoded light-activated Cas9 was engineered through the site-specific installation of a caged lysine amino acid. Several potential lysine residues were identified as viable caging sites that can be modified to optically control Cas9 function, as demonstrated through optical activation and deactivation of both exogenous and endogenous gene function.
- 422Nguyen, D. P.; Mahesh, M.; Elsässer, S. J.; Hancock, S. M.; Uttamapinant, C.; Chin, J. W. Genetic Encoding of Photocaged Cysteine Allows Photoactivation of TEV Protease in Live Mammalian Cells. J. Am. Chem. Soc. 2014, 136 (6), 2240– 2243, DOI: 10.1021/ja412191m422Genetic Encoding of Photocaged Cysteine Allows Photoactivation of TEV Protease in Live Mammalian CellsNguyen, Duy P.; Mahesh, Mohan; Elsasser, Simon J.; Hancock, Susan M.; Uttamapinant, Chayasith; Chin, Jason W.Journal of the American Chemical Society (2014), 136 (6), 2240-2243CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The authors demonstrate the evolution of the PylRS/tRNACUA pair for genetically encoding photocaged cysteine (I). By characterizing the incorporation in Escherichia coli and mammalian cells, and the photodeprotection process in vitro and in mammalian cells, the authors establish conditions for rapid efficient photodeprotection to reveal native proteins in live cells. They demonstrate the utility of this approach by rapidly activating TEV protease following illumination of single cells.
- 423Uprety, R.; Luo, J.; Liu, J.; Naro, Y.; Samanta, S.; Deiters, A. Genetic Encoding of Caged Cysteine and Caged Homocysteine in Bacterial and Mammalian Cells. ChemBioChem 2014, 15 (12), 1793– 1799, DOI: 10.1002/cbic.201400073There is no corresponding record for this reference.
- 424Yang, X.; Zhao, L.; Wang, Y.; Ji, Y.; Su, X. C.; Ma, J. A.; Xuan, W. Constructing Photoactivatable Protein with Genetically Encoded Photocaged Glutamic Acid. Angew. Chem. Int. Ed. 2023, 135 (40), e202308472 DOI: 10.1002/ange.202308472There is no corresponding record for this reference.
- 425Givens, R. S.; Weber, J. F.; Jung, A. H.; Park, C.-H. New Photoprotecting Groups: Desyl and p-Hydroxyphenacyl Phosphate and Carboxylate Esters. In Methods in enzymology; Elsevier, 1998; Vol. 291, pp 1– 29. DOI: 10.1016/s0076-6879(98)91004-7 .There is no corresponding record for this reference.
- 426Mangubat-Medina, A. E.; Ball, Z. T. Triggering Biological Processes: Methods and Applications of Photocaged Peptides and Proteins. Chem. Soc. Rev. 2021, 50 (18), 10403– 10421, DOI: 10.1039/D0CS01434F426Triggering biological processes: methods and applications of photocaged peptides and proteinsMangubat-Medina, Alicia E.; Ball, Zachary T.Chemical Society Reviews (2021), 50 (18), 10403-10421CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. There has been a significant push in recent years to deploy fundamental knowledge and methods of photochem. toward biol. ends. Photoreactive groups have enabled chemists to activate biol. function using the concept of photocaging. By granting spatiotemporal control over protein activation, these photocaging methods are fundamental in understanding biol. processes. Peptides and proteins are an important group of photocaging targets that present conceptual and tech. challenges, requiring precise chemoselectivity in complex polyfunctional environments. This review focuses on recent advances in photocaging techniques and methodologies, as well as their use in living systems. Photocaging methods include genetic and chem. approaches that require a deep understanding of structure-function relationships based on subtle changes in primary structure. Successful implementation of these ideas can shed light on important spatiotemporal aspects of living systems.
- 427Grasso, K. T.; Singha Roy, S. J.; Osgood, A. O.; Yeo, M. J. R.; Soni, C.; Hillenbrand, C. M.; Ficaretta, E. D.; Chatterjee, A. A Facile Platform to Engineer Escherichia coli Tyrosyl-tRNA Synthetase Adds New Chemistries to the Eukaryotic Genetic Code, Including a Phosphotyrosine Mimic. ACS Cent. Sci. 2022, 8 (4), 483– 492, DOI: 10.1021/acscentsci.1c01465There is no corresponding record for this reference.
- 428Edwards, W. F.; Young, D. D.; Deiters, A. Light-Activated Cre Recombinase as a Tool for the Spatial and Temporal Control of Gene Function in Mammalian Cells. ACS Chem. Biol. 2009, 4 (6), 441– 445, DOI: 10.1021/cb900041s428Light-activated Cre recombinase as a tool for the spatial and temporal control of gene function in mammalian cellsEdwards, Wesleigh F.; Young, Douglas D.; Deiters, AlexanderACS Chemical Biology (2009), 4 (6), 441-445CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Cre recombinase catalyzes DNA exchange between two conserved lox recognition sites. The enzyme has extensive biol. application, from basic cloning to engineering knock-out and knock-in organisms. Widespread use of Cre is due to its simplicity and effectiveness, but the enzyme and the recombination event remain difficult to control with high precision. To obtain such control we report the installation of a light-responsive o-nitrobenzyl caging group directly in the catalytic site of Cre, inhibiting its activity. Prior to irradn., caged Cre is completely inactive, as demonstrated both in vitro and in mammalian cell culture. Exposure to non-damaging UVA light removes the caging group and restores recombinase activity. Tight spatio-temporal control over DNA recombination is thereby achieved.
- 429Le Provost, F.; Lillico, S.; Passet, B.; Young, R.; Whitelaw, B.; Vilotte, J.-L. Zinc Finger Nuclease Technology Heralds a New Era in Mammalian Transgenesis. Trends Biotechnol. 2010, 28 (3), 134– 141, DOI: 10.1016/j.tibtech.2009.11.007There is no corresponding record for this reference.
- 430Porteus, M. H. Mammalian Gene Targeting with Designed Zinc Finger Nucleases. Molecular Therapy 2006, 13 (2), 438– 446, DOI: 10.1016/j.ymthe.2005.08.003There is no corresponding record for this reference.
- 431Porteus, M. H.; Carroll, D. Gene Targeting Using Zinc Finger Nucleases. Nat. Biotechnol. 2005, 23 (8), 967– 973, DOI: 10.1038/nbt1125There is no corresponding record for this reference.
- 432Urnov, F. D.; Rebar, E. J.; Holmes, M. C.; Zhang, H. S.; Gregory, P. D. Genome Editing with Engineered Zinc Finger Nucleases. Nat. Rev. Genet. 2010, 11 (9), 636– 646, DOI: 10.1038/nrg2842432Genome editing with engineered zinc finger nucleasesUrnov, Fyodor D.; Rebar, Edward J.; Holmes, Michael C.; Zhang, H. Steve; Gregory, Philip D.Nature Reviews Genetics (2010), 11 (9), 636-646CODEN: NRGAAM; ISSN:1471-0056. (Nature Publishing Group)A review. Zinc finger nucleases (ZFNs) are versatile tools for making precise modifications to genomes, and their use is now established in a range of model systems. ZFNs are also showing potential in human gene therapy, and several clin. trials are underway. Reverse genetics in model organisms such as Drosophila melanogaster, Arabidopsis thaliana, zebrafish and rats, efficient genome engineering in human embryonic stem and induced pluripotent stem cells, targeted integration in crop plants, and HIV resistance in immune cells - this broad range of outcomes has resulted from the application of the same core technol.: targeted genome cleavage by engineered, sequence-specific zinc finger nucleases followed by gene modification during subsequent repair. Such 'genome editing' is now established in human cells and a no. of model organisms, thus opening the door to a range of new exptl. and therapeutic possibilities.
- 433Rémy, S.; Tesson, L.; Ménoret, S.; Usal, C.; Scharenberg, A. M.; Anegon, I. Zinc-Finger Nucleases: A Powerful Tool for Genetic Engineering of Animals. Transgenic Res. 2010, 19, 363– 371, DOI: 10.1007/s11248-009-9323-7There is no corresponding record for this reference.
- 434Li, Y.; Korolev, S.; Waksman, G. Crystal Structures of Open and Closed Forms of Binary and Ternary Complexes of the Large Fragment of Thermus aquaticus DNA Polymerase I: Structural Basis for Nucleotide Incorporation. EMBO J. 1998, 17 (24), 7514– 7525, DOI: 10.1093/emboj/17.24.7514434Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporationLi, Ying; Korolev, Sergey; Waksman, GabrielEMBO Journal (1998), 17 (24), 7514-7525CODEN: EMJODG; ISSN:0261-4189. (Oxford University Press)The crystal structures of two ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I (Klentaq1) with a primer/template DNA and dideoxycytidine triphosphate, and that of a binary complex of the same enzyme with a primer/template DNA, were detd. to a resoln. of 2.3, 2.3 and 2.5 Å, resp. One ternary complex structure differs markedly from the two other structures by a large reorientation of the tip of the fingers domain. This structure, designated 'closed', represents the ternary polymerase complex caught in the act of incorporating a nucleotide. In the two other structures, the tip of the fingers domain is rotated outward by 46° ('open') in an orientation similar to that of the apo form of Klentaq1. These structures provide the first direct evidence in DNA polymerase I enzymes of a large conformational change responsible for assembling an active ternary complex.
- 435Suzuki, M.; Baskin, D.; Hood, L.; Loeb, L. A. Random Mutagenesis of Thermus aquaticus DNA Polymerase I: Concordance of Immutable Sites in vivo with the Crystal Structure. Proc. Natl. Acad. Sci. U.S.A. 1996, 93 (18), 9670– 9675, DOI: 10.1073/pnas.93.18.9670There is no corresponding record for this reference.
- 436Paul, N.; Shum, J.; Le, T. Hot Start PCR. RT-PCR Protocols, Second ed.; 2010; pp 301– 318. DOI: 10.1007/978-1-60761-629-0There is no corresponding record for this reference.
- 437Cramer, P. Common Structural Features of Nucleic Acid Polymerases. Bioessays 2002, 24 (8), 724– 729, DOI: 10.1002/bies.10127There is no corresponding record for this reference.
- 438Banghart, M. R.; Mourot, A.; Fortin, D. L.; Yao, J. Z.; Kramer, R. H.; Trauner, D. Photochromic Blockers of Voltage-Gated Potassium Channels. Angew. Chem. Int. Ed. 2009, 48 (48), 9097– 9101, DOI: 10.1002/anie.200904504438Photochromic Blockers of Voltage-Gated Potassium ChannelsBanghart, Matthew R.; Mourot, Alexandre; Fortin, Doris L.; Yao, Jennifer Z.; Kramer, Richard H.; Trauner, DirkAngewandte Chemie, International Edition (2009), 48 (48), 9097-9101, S9097/1-S9097/14CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Photochromic blockers of voltage-gated potassium channels were prepd., with potential to be used as tools in neurobiol. and possibly in therapy of vision disorders.
- 439Beharry, A. A.; Wong, L.; Tropepe, V.; Woolley, G. A. Fluorescence Imaging of Azobenzene Photoswitching in vivo. Angew. Chem. Int. Ed. 2011, 50 (6), 1325– 1327, DOI: 10.1002/anie.201006506There is no corresponding record for this reference.
- 440Bléger, D.; Schwarz, J.; Brouwer, A. M.; Hecht, S. o-Fluoroazobenzenes as Readily Synthesized Photoswitches Offering Nearly Quantitative Two-Way Isomerization with Visible Light. J. Am. Chem. Soc. 2012, 134 (51), 20597– 20600, DOI: 10.1021/ja310323y440o-Fluoroazobenzenes as Readily Synthesized Photoswitches Offering Nearly Quantitative Two-Way Isomerization with Visible LightBleger, David; Schwarz, Jutta; Brouwer, Albert M.; Hecht, StefanJournal of the American Chemical Society (2012), 134 (51), 20597-20600CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Azobenzene functionalized with ortho-fluorine atoms has a lower energy of the n-orbital of the Z-isomer, resulting in a sepn. of the E and Z isomers' n→π* absorption bands. Introducing para-substituents allows for further tuning of the absorption spectra of o-fluoroazobenzenes. In particular, electron-withdrawing ester groups give rise to a 50 nm sepn. of the n→π* transitions. Green and blue light can therefore be used to induce E→Z and Z→E isomerizations, resp. The o-fluoroazobenzene scaffold is readily synthesized and can be inserted into larger structures via its aryl termini. These new azobenzene derivs. can be switched in both ways with high photoconversions, and their Z-isomers display a remarkably long thermal half-life.
- 441Bonardi, F.; London, G.; Nouwen, N.; Feringa, B. L.; Driessen, A. J. Light-Induced Control of Protein Translocation by the SecYEG Complex. Angew. Chem. Int. Ed. 2010, 49 (40), 7234– 7238, DOI: 10.1002/anie.201002243441Light-induced control of protein translocation by the secYEG complexBonardi, Francesco; London, Gabor; Nouwen, Nico; Feringa, Ben L.; Driessen, Arnold J. M.Angewandte Chemie, International Edition (2010), 49 (40), 7234-7238, S7234/1-S7234/13CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)An organochem. photoswitch was introduced into two transmembrane segments that comprise the lateral gate of the bacterial-membrane-embedded protein-conducting pore. Reversible switching of the azobenzene between the trans and cis configurations by irradn. with visible and UV light enforced the opening and closure of the protein-conducting pore.
- 442Gorostiza, P.; Isacoff, E. Y. Nanoengineering Ion Channels for Optical Control. Physiology 2008, 23 (5), 238– 247, DOI: 10.1152/physiol.00018.2008442Nanoengineering ion channels for optical controlGorostiza Pau; Isacoff Ehud YPhysiology (Bethesda, Md.) (2008), 23 (), 238-47 ISSN:1548-9213.Chemical modification with photoisomerizable tethered ligands endows proteins with sensitivity to light. These optically actuated proteins are revolutionizing research in biology by making it possible to manipulate biological processes noninvasively and with unprecedented spatiotemporal resolution.
- 443Knie, C.; Utecht, M.; Zhao, F.; Kulla, H.; Kovalenko, S.; Brouwer, A. M.; Saalfrank, P.; Hecht, S.; Bléger, D. ortho-Fluoroazobenzenes: Visible Light Switches with Very Long-Lived Z Isomers. Chem. Eur. J. 2014, 20 (50), 16492– 16501, DOI: 10.1002/chem.201404649There is no corresponding record for this reference.
- 444Liang, X.; Mochizuki, T.; Asanuma, H. A Supra-Photoswitch Involving Sandwiched DNA Base Pairs and Azobenzenes for Light-Driven Nanostructures and Nanodevices. Small 2009, 5 (15), 1761– 1768, DOI: 10.1002/smll.200900223444A Supra-photoswitch Involving Sandwiched DNA Base Pairs and Azobenzenes for Light-Driven Nanostructures and NanodevicesLiang, Xingguo; Mochizuki, Toshio; Asanuma, HiroyukiSmall (2009), 5 (15), 1761-1768CODEN: SMALBC; ISSN:1613-6810. (Wiley-VCH Verlag GmbH & Co. KGaA)A supra-photoswitch is designed for complete ON/OFF switching of DNA hybridization by light irradn. for the purpose of using DNA as a material for building nanostructures. Azobenzenes, attached to D-threoninols that function as scaffolds, are introduced into each DNA strand after every two natural nucleotides (in the form (NNX)n where N and X represent the natural nucleotide and the azobenzene moiety, resp.). Hybridization of these two modified strands forms a supra-photoswitch consisting of alternating natural base pairs and azobenzene moieties. In this newly designed sequence, each base pair is sandwiched between two azobenzene moieties and all the azobenzene moieties are sepd. by base pairs. When the duplex is irradiated by visible light, the azobenzene moieties take the trans form and this duplex is surprisingly stable compared to the corresponding native duplex composed of only natural oligonucleotides. On the other hand, when the azobenzene moieties are isomerized to the cis form by UV light irradn., the duplex is completely dissocd. Based on this design, a DNA hairpin structure is synthesized that should be closed by visible light irradn. and opened by UV light irradn. at the level of a single mol. Indeed, perfect ON/OFF photoregulation is attained. This is a promising strategy for the design of supra-photoswitches such as photoresponsive sticky ends on DNA nanodevices and other nanostructures.
- 445Samanta, S.; Beharry, A. A.; Sadovski, O.; McCormick, T. M.; Babalhavaeji, A.; Tropepe, V.; Woolley, G. A. Photoswitching Azo Compounds in vivo with Red Light. J. Am. Chem. Soc. 2013, 135 (26), 9777– 9784, DOI: 10.1021/ja402220t445Photoswitching Azo Compounds in Vivo with Red LightSamanta, Subhas; Beharry, Andrew A.; Sadovski, Oleg; McCormick, Theresa M.; Babalhavaeji, Amirhossein; Tropepe, Vince; Woolley, G. AndrewJournal of the American Chemical Society (2013), 135 (26), 9777-9784CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The photoisomerization of azobenzenes provides a general means for the photocontrol of mol. structure and function. For applications in vivo, however, the wavelength of irradn. required for trans-to-cis isomerization of azobenzenes is crit. since UV and most visible wavelengths are strongly scattered by cells and tissues. We report here that azobenzene compds. in which all four positions ortho to the azo group are substituted with bulky electron-rich substituents can be effectively isomerized with red light (630-660 nm), a wavelength range that is orders of magnitude more penetrating through tissue than other parts of the visible spectrum. When the ortho substituent is chloro, the compds. also exhibit stability to redn. by glutathione, enabling their use in intracellular environments in vivo.
- 446Schierling, B.; Noel, A.-J.; Wende, W.; Hien, L. T.; Volkov, E.; Kubareva, E.; Oretskaya, T.; Kokkinidis, M.; Rompp, A.; Spengler, B.; Pingoud, A. Controlling the Enzymatic Activity of a Restriction Enzyme by Light. Proc. Natl. Acad. Sci. U.S.A. 2010, 107 (4), 1361– 1366, DOI: 10.1073/pnas.0909444107There is no corresponding record for this reference.
- 447Zhang, F.; Zarrine-Afsar, A.; Al-Abdul-Wahid, M. S.; Prosser, R. S.; Davidson, A. R.; Woolley, G. A. Structure-Based Approach to the Photocontrol of Protein Folding. J. Am. Chem. Soc. 2009, 131 (6), 2283– 2289, DOI: 10.1021/ja807938v447Structure-Based Approach to the Photocontrol of Protein FoldingZhang, Fuzhong; Zarrine-Afsar, Arash; Al-Abdul-Wahid, M. Sameer; Prosser, R. Scott; Davidson, Alan R.; Woolley, G. AndrewJournal of the American Chemical Society (2009), 131 (6), 2283-2289CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Photoswitchable proteins offer exciting prospects for remote control of biochem. processes. We propose a general approach to the design of photoswitchable proteins based on the introduction of a photoswitchable intramol. cross-linker. We chose, as a model, a FynSH3 domain for which the free energy of folding is less than the energy available from photoisomerization of the cross-linker. Taking the exptl. detd. structure of the folded protein as a starting point, mutations were made to introduce pairs of Cys residues so that the distance between Cys sulfur atoms matches the ideal length of the cis form, but not the trans form, of the cross-linker. When the trans cross-linker was introduced into this L3C-L29C-T47AFynSH3 mutant, the protein was destabilized so that folded and unfolded forms coexisted. Irradn. of the cross-linker to produce the cis isomer recovered the folded, active state of the protein. This work shows that structure-based introduction of switchable cross-linkers is a feasible approach for photocontrol of folding/unfolding of globular proteins.
- 448Bose, M.; Groff, D.; Xie, J.; Brustad, E.; Schultz, P. G. The Incorporation of a Photoisomerizable Amino Acid into Proteins in E. c oli. J. Am. Chem. Soc. 2006, 128 (2), 388– 389, DOI: 10.1021/ja055467u448The incorporation of a photoisomerizable amino acid into proteins in E. coliBose, Mohua; Groff, Dan; Xie, Jianming; Brustad, Eric; Schultz, Peter G.Journal of the American Chemical Society (2006), 128 (2), 388-389CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)An orthogonal aminoacyl tRNA synthetase/tRNA pair has been evolved that allows the incorporation of the photoisomerizable amino acid phenylalanine-4'-azobenzene (AzoPhe) into proteins in Escherichia coli in response to the amber nonsense codon. Further, we show that AzoPhe can be used to photoregulate the binding affinity of catabolite activator protein to its promoter. The ability to selectively incorporate AzoPhe into proteins at defined sites should make it possible to regulate a variety of biol. processes with light, including enzyme, receptor, and ion channel activity.
- 449Hoppmann, C.; Lacey, V. K.; Louie, G. V.; Wei, J.; Noel, J. P.; Wang, L. Genetically Encoding Photoswitchable Click Amino Acids in Escherichia coli and Mammalian Cells. Angew. Chem. Int. Ed. 2014, 53 (15), 3932– 3936, DOI: 10.1002/anie.201400001449Genetically Encoding Photoswitchable Click Amino Acids in Escherichia coli and Mammalian CellsHoppmann, Christian; Lacey, Vanessa K.; Louie, Gordon V.; Wei, Jing; Noel, Joseph P.; Wang, LeiAngewandte Chemie, International Edition (2014), 53 (15), 3932-3936CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The ability to reversibly control protein structure and function with light would offer high spatiotemporal resoln. for investigating biol. processes. To confer photoresponsiveness on general proteins, we genetically incorporated a set of photoswitchable click amino acids (PSCaas), which contain both a reversible photoswitch and an addnl. click functional group for further modifications. Orthogonal tRNA-synthetases were evolved to genetically encode PSCaas bearing azobenzene with an alkene, keto, or benzyl chloride group in E. coli and in mammalian cells. After incorporation into calmodulin, the benzyl chloride PSCaa spontaneously generated a covalent protein bridge by reacting with a nearby cysteine residue through proximity-enabled bioreactivity. The resultant azobenzene bridge isomerized in response to light, thereby changing the conformation of calmodulin. These genetically encodable PSCaas will prove valuable for engineering photoswitchable bridges into proteins for reversible optogenetic regulation.
- 450Hoppmann, C.; Maslennikov, I.; Choe, S.; Wang, L. In situ Formation of an Azo Bridge on Proteins Controllable by Visible Light. J. Am. Chem. Soc. 2015, 137 (35), 11218– 11221, DOI: 10.1021/jacs.5b06234450In Situ Formation of an Azo Bridge on Proteins Controllable by Visible LightHoppmann, Christian; Maslennikov, Innokentiy; Choe, Senyon; Wang, LeiJournal of the American Chemical Society (2015), 137 (35), 11218-11221CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Optical modulation of proteins provides superior spatiotemporal resoln. for understanding biol. processes, and photoswitches built on light-sensitive proteins have been significantly advancing neuronal and cellular studies. Small mol. photoswitches could complement protein-based switches by mitigating potential interference and affording high specificity for modulation sites. However, genetic encodability and responsiveness to nonultraviolet light, two desired properties possessed by protein photoswitches, are challenging to be engineered into small mol. photoswitches. Here the authors developed a small mol. photoswitch that can be genetically installed onto proteins in situ and controlled by visible light. A pentafluoro azobenzene-based photoswitchable click amino acid (F-PSCaa) was designed to isomerize in response to visible light. After genetic incorporation into proteins via the expansion of the genetic code, F-PSCaa reacts with a nearby cysteine within the protein generating an azo bridge in situ. The resultant bridge is switchable by visible light and allows conformation and binding of CaM to be regulated by such light. This photoswitch should prove valuable in optobiol. for its minimal interference, site flexibility, genetic encodability, and response to the more biocompatible visible light.
- 451John, A. A.; Ramil, C. P.; Tian, Y.; Cheng, G.; Lin, Q. Synthesis and Site-Specific Incorporation of Red-Shifted Azobenzene Amino Acids into Proteins. Org. Lett. 2015, 17 (24), 6258– 6261, DOI: 10.1021/acs.orglett.5b03268451Synthesis and Site-Specific Incorporation of Red-Shifted Azobenzene Amino Acids into ProteinsJohn, Alford A.; Ramil, Carlo P.; Tian, Yulin; Cheng, Gang; Lin, QingOrganic Letters (2015), 17 (24), 6258-6261CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)A series of red-shifted azobenzene amino acids were synthesized in moderate-to-excellent yields via a two-step procedure in which tyrosine derivs. were first oxidized to the corresponding quinonoidal spirolactones followed by ceric ammonium nitrate-catalyzed azo formation with the substituted phenylhydrazines. The resulting azobenzene-alanine derivs. exhibited efficient trans/cis photoswitching upon irradn. with a blue (448 nm) or green (530 nm) LED light. Moreover, nine superfolder green fluorescent protein (sfGFP) mutants carrying the azobenzene-alanine analogs were expressed in E. coli in good yields via amber codon suppression with an orthogonal tRNA/PylRS pair, and one of the mutants showed durable photoswitching with the LED light.
- 452Klippenstein, V.; Hoppmann, C.; Ye, S.; Wang, L.; Paoletti, P. Optocontrol of Glutamate Receptor Activity by Single Side-Chain Photoisomerization. Elife 2017, 6, e25808 DOI: 10.7554/eLife.25808452Optocontrol of glutamate receptor activity by single side-chain photoisomerizationKlippenstein, Viktoria; Hoppmann, Christian; Ye, Shixin; Wang, Lei; Paoletti, PierreeLife (2017), 6 (), e25808/1-e25808/29CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)Engineering light-sensitivity into proteins has wide ranging applications in mol. studies and neuroscience. Commonly used tethered photoswitchable ligands, however, require solvent-accessible protein labeling, face structural constrains, and are bulky. Here, we designed a set of optocontrollable NMDA receptors by directly incorporating single photoswitchable amino acids (PSAAs) providing genetic encodability, reversibility, and site tolerance. We identified several positions within the multi-domain receptor endowing robust photomodulation. PSAA photoisomerization at the GluN1 clamshell hinge is sufficient to control glycine sensitivity and activation efficacy. Strikingly, in the pore domain, flipping of a M3 residue within a conserved transmembrane cavity impacts both gating and permeation properties. Our study demonstrates the first detection of mol. rearrangements in real-time due to the reversible light-switching of single amino acid side-chains, adding a dynamic dimension to protein site-directed mutagenesis. This novel approach to interrogate neuronal protein function has general applicability in the fast expanding field of optopharmacol.
- 453Kneuttinger, A. C.; Straub, K.; Bittner, P.; Simeth, N. A.; Bruckmann, A.; Busch, F.; Rajendran, C.; Hupfeld, E.; Wysocki, V. H.; Horinek, D. Light Regulation of Enzyme Allostery through Photo-responsive Unnatural Amino Acids. Cell Chem. Biol. 2019, 26 (11), 1501– 1514, DOI: 10.1016/j.chembiol.2019.08.006453Light Regulation of Enzyme Allostery through Photo-responsive Unnatural Amino AcidsKneuttinger, Andrea C.; Straub, Kristina; Bittner, Philipp; Simeth, Nadja A.; Bruckmann, Astrid; Busch, Florian; Rajendran, Chitra; Hupfeld, Enrico; Wysocki, Vicki H.; Horinek, Dominik; Koenig, Burkhard; Merkl, Rainer; Sterner, ReinhardCell Chemical Biology (2019), 26 (11), 1501-1514.e9CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)Imidazole glycerol phosphate synthase (ImGPS) is an allosteric bienzyme complex in which substrate binding to the synthase subunit HisF stimulates the glutaminase subunit HisH. To control this stimulation with light, we have incorporated the photo-responsive unnatural amino acids phenylalanine-4'-azobenzene (AzoF), o-nitropiperonyl-O-tyrosine (NPY), and methyl-o-nitropiperonyllysine (mNPK) at strategic positions of HisF. The light-mediated isomerization of AzoF at position 55 (fS55AzoFE ↔ fS55AzoFZ) resulted in a reversible 10-fold regulation of HisH activity. The light-mediated decaging of NPY at position 39 (fY39NPY → fY39) and of mNPK at position 99 (fK99mNPK → fK99) led to a 4- to 6-fold increase of HisH activity. Mol. dynamics simulations explained how the unnatural amino acids interfere with the allosteric machinery of ImGPS and revealed addnl. aspects of HisH stimulation in wild-type ImGPS. Our findings show that unnatural amino acids can be used as a powerful tool for the spatiotemporal control of a central metabolic enzyme complex by light.
- 454Luo, J.; Samanta, S.; Convertino, M.; Dokholyan, N. V.; Deiters, A. Reversible and Tunable Photoswitching of Protein Function through Genetic Encoding of Azobenzene Amino Acids in Mammalian Cells. ChemBioChem 2018, 19 (20), 2178– 2185, DOI: 10.1002/cbic.201800226454Reversible and Tunable Photoswitching of Protein Function through Genetic Encoding of Azobenzene Amino Acids in Mammalian CellsLuo, Ji; Samanta, Subhas; Convertino, Marino; Dokholyan, Nikolay V.; Deiters, AlexanderChemBioChem (2018), 19 (20), 2178-2185CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)The genetic encoding of three different azobenzene phenylalanines with different photochem. properties was achieved in human cells by using an engineered pyrrolysyl tRNA/tRNA synthetase pair. In order to demonstrate reversible light control of protein function, azobenzenes were site-specifically introduced into firefly luciferase. Computational strategies were applied to guide the selection of potential photoswitchable sites that lead to a reversibly controlled luciferase enzyme. In addn., the new azobenzene analogs provide enhanced thermal stability, high photoconversion, and responsiveness to visible light. These small-mol. photoswitches can reversibly photocontrol protein function with excellent spatiotemporal resoln., and preferred sites for incorporation can be computationally detd., thus providing a new tool for investigating biol. processes.
- 455Kneuttinger, A. C.; Winter, M.; Simeth, N. A.; Heyn, K.; Merkl, R.; König, B.; Sterner, R. Artificial Light Regulation of an Allosteric Bienzyme Complex by a Photosensitive Ligand. ChemBioChem 2018, 19 (16), 1750– 1757, DOI: 10.1002/cbic.201800219455Artificial Light Regulation of an Allosteric Bienzyme Complex by a Photosensitive LigandKneuttinger, Andrea C.; Winter, Martin; Simeth, Nadja A.; Heyn, Kristina; Merkl, Rainer; Koenig, Burkhard; Sterner, ReinhardChemBioChem (2018), 19 (16), 1750-1757CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)The artificial regulation of proteins by light is an emerging subdiscipline of synthetic biol. Here, we used this concept to photocontrol both catalysis and allostery within the heterodimeric enzyme complex imidazole glycerol phosphate synthase (ImGP-S). ImGP-S consists of the cyclase subunit HisF and the glutaminase subunit HisH, which is allosterically stimulated by substrate binding to HisF. We show that a light-sensitive diarylethene (1,2-dithienylethene, DTE)-based competitive inhibitor in its ring-open state binds with low micromolar affinity to the cyclase subunit and displaces its substrate from the active site. As a consequence, catalysis by HisF and allosteric stimulation of HisH are impaired. Following UV-light irradn., the DTE ligand adopts its ring-closed state and loses affinity for HisF, restoring activity and allostery. Our approach allows for the switching of ImGP-S activity and allostery during catalysis and appears to be generally applicable for the light regulation of other multienzyme complexes.
- 456Kneuttinger, A. C.; Rajendran, C.; Simeth, N. A.; Bruckmann, A.; König, B.; Sterner, R. Significance of the Protein Interface Configuration for Allostery in Imidazole Glycerol Phosphate Synthase. Biochemistry 2020, 59 (29), 2729– 2742, DOI: 10.1021/acs.biochem.0c00332456Significance of the Protein Interface Configuration for Allostery in Imidazole Glycerol Phosphate SynthaseKneuttinger, Andrea C.; Rajendran, Chitra; Simeth, Nadja A.; Bruckmann, Astrid; Koenig, Burkhard; Sterner, ReinhardBiochemistry (2020), 59 (29), 2729-2742CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Imidazole glycerol phosphate synthase (ImGPS) from Thermotoga maritima is a model enzyme for studying allostery. The ImGPS complex consists of the cyclase subunit HisF and the glutaminase subunit HisH whose activity is stimulated by substrate binding to HisF in a V-type manner. To investigate the significance of a putative closing hinge motion at the cyclase:glutaminase interface for HisH activity, we replaced residue W123 in HisH with the light-switchable unnatural amino acid phenylalanine-4'-azobenzene (AzoF). Crystal structure anal. employing angle, buried surface area, and distance measurements showed that incorporation of AzoF at this position causes a closing of the interface by ~ 18 ± 3%. This slightly different interface configuration results in a much higher catalytic efficiency in unstimulated HisH due to an elevated turnover no. Moreover, the catalytic efficiency of HisH when stimulated by binding of a substrate to HisF was also significantly increased by AzoF incorporation. This was caused by a K-type stimulation that led to a decrease in the apparent dissocn. const. for its substrate, glutamine. In addn., AzoF improved the apparent binding of a substrate analog at the HisF active site. Remarkably, light-induced isomerization of AzoF considerably enhanced these effects. In conclusion, our findings confirm that signal transduction from HisF to HisH in ImGPS involves the closing of the cyclase:glutaminase subunit interface and that incorporation of AzoF at a hinge position reinforces this catalytically relevant conformational change.
- 457Zubi, Y. S.; Seki, K.; Li, Y.; Hunt, A. C.; Liu, B.; Roux, B.; Jewett, M. C.; Lewis, J. C. Metal-Responsive Regulation of Enzyme Catalysis Using Genetically Encoded Chemical Switches. Nature Commun. 2022, 13 (1), 1864, DOI: 10.1038/s41467-022-29239-yThere is no corresponding record for this reference.
- 458Yang, H.; Swartz, A. M.; Park, H. J.; Srivastava, P.; Ellis-Guardiola, K.; Upp, D. M.; Lee, G.; Belsare, K.; Gu, Y.; Zhang, C. Evolving Artificial Metalloenzymes via Random Mutagenesis. Nat. Chem. 2018, 10 (3), 318– 324, DOI: 10.1038/nchem.2927458Evolving artificial metalloenzymes via random mutagenesisYang, Hao; Swartz, Alan M.; Park, Hyun June; Srivastava, Poonam; Ellis-Guardiola, Ken; Upp, David M.; Lee, Gihoon; Belsare, Ketaki; Gu, Yifan; Zhang, Chen; Moellering, Raymond E.; Lewis, Jared C.Nature Chemistry (2018), 10 (3), 318-324CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Random mutagenesis has the potential to optimize the efficiency and selectivity of protein catalysts without requiring detailed knowledge of protein structure; however, introducing synthetic metal cofactors complicates the expression and screening of enzyme libraries, and activity arising from free cofactor must be eliminated. Here we report an efficient platform to create and screen libraries of artificial metalloenzymes (ArMs) via random mutagenesis, which we use to evolve highly selective dirhodium cyclopropanases. Error-prone PCR and combinatorial codon mutagenesis enabled multiplexed anal. of random mutations, including at sites distal to the putative ArM active site that are difficult to identify using targeted mutagenesis approaches. Variants that exhibited significantly improved selectivity for each of the cyclopropane product enantiomers were identified, and higher activity than previously reported ArM cyclopropanases obtained via targeted mutagenesis was also obsd. This improved selectivity carried over to other dirhodium-catalyzed transformations, including N-H, S-H and Si-H insertion, demonstrating that ArMs evolved for one reaction can serve as starting points to evolve catalysts for others.
- 459Leveson-Gower, R. B.; Zhou, Z.; Drienovská, I.; Roelfes, G. Unlocking Iminium Catalysis in Artificial Enzymes to Create a Friedel-Crafts Alkylase. ACS Catal. 2021, 11 (12), 6763– 6770, DOI: 10.1021/acscatal.1c00996459Unlocking Iminium Catalysis in Artificial Enzymes to Create a Friedel-Crafts AlkylaseLeveson-Gower, Reuben B.; Zhou, Zhi; Drienovska, Ivana; Roelfes, GerardACS Catalysis (2021), 11 (12), 6763-6770CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)The construction and engineering of artificial enzymes consisting of abiol. catalytic moieties incorporated into protein scaffolds is a promising strategy to realize non-natural mechanisms in biocatalysis. Here, incorporation of the noncanonical amino acid para-aminophenylalanine (pAF) into the nonenzymic protein scaffold LmrR creates a proficient and stereoselective artificial enzyme (LmrR_pAF) for the vinylogous Friedel-Crafts alkylation between α,β-unsatd. aldehydes and indoles. PAF acts as a catalytic residue, activating enal substrates toward conjugate addn. via the formation of intermediate iminium ion species, while the protein scaffold provides rate acceleration and stereoinduction. Improved LmrR_pAF variants were identified by low-throughput directed evolution advised by alanine-scanning to obtain a triple mutant that provided higher yields and enantioselectivities for a range of aliph. enals and substituted indoles. Anal. of Michaelis-Menten kinetics of LmrR_pAF and evolved mutants reveals that different activities emerge via evolutionary pathways that diverge from one another and specialize catalytic reactivity. Translating this iminium-based catalytic mechanism into an enzymic context will enable many more biocatalytic transformations inspired by organocatalysis.
- 460Mayer, C.; Dulson, C.; Reddem, E.; Thunnissen, A.-M. W. H.; Roelfes, G. Directed Evolution of a Designer Enzyme Featuring an Unnatural Catalytic Amino Acid. Angew. Chem. Int. Ed. 2019, 58 (7), 2083– 2087, DOI: 10.1002/anie.201813499460Directed Evolution of a Designer Enzyme Featuring an Unnatural Catalytic Amino AcidMayer, Clemens; Dulson, Christopher; Reddem, Eswar; Thunnissen, Andy-Mark W. H.; Roelfes, GerardAngewandte Chemie, International Edition (2019), 58 (7), 2083-2087CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The impressive rate accelerations that enzymes display in nature often result from boosting the inherent catalytic activities of side chains by their precise positioning inside a protein binding pocket. Such fine-tuning is also possible for catalytic unnatural amino acids. Specifically, the directed evolution of a recently described designer enzyme, which utilizes an aniline side chain to promote a model hydrazone formation reaction, is reported. Consecutive rounds of directed evolution identified several mutations in the promiscuous binding pocket, in which the unnatural amino acid is embedded in the starting catalyst. When combined, these mutations boost the turnover frequency (kcat) of the designer enzyme by almost 100-fold. This results from strengthening the catalytic contribution of the unnatural amino acid, as the engineered designer enzymes outperform variants, in which the aniline side chain is replaced with a catalytically inactive tyrosine residue, by more than 200-fold.
- 461Kaes, C.; Katz, A.; Hosseini, M. W. Bipyridine: The Most Widely Used Ligand. A Review of Molecules Comprising at Least Two 2,2‘-Bipyridine Units. Chem. Rev. 2000, 100 (10), 3553– 3590, DOI: 10.1021/cr990376z461Bipyridine: The Most Widely Used Ligand. A Review of Molecules Comprising at Least Two 2,2'-Bipyridine UnitsKaes, Christian; Katz, Alexander; Hosseini, Mir WaisChemical Reviews (Washington, D. C.) (2000), 100 (10), 3553-3590CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review with 256 refs. which presents an overview of the most explored chelate system in coordination chem.
- 462Xie, J.; Liu, W.; Schultz, P. G. A Genetically Encoded Bidentate, Metal-Binding Amino Acid. Angew. Chem. Int. Ed. 2007, 46 (48), 9239– 9242, DOI: 10.1002/anie.200703397462A genetically encoded bidentate, metal-binding amino acidXie, Jianming; Liu, Wenshen; Schultz, Peter G.Angewandte Chemie, International Edition (2007), 46 (48), 9239-9242CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)To facilitate the design of metalloproteins, the bidentate, metal-binding amino acid bipyridylalanine (BpyAla) was genetically encoded in E. coli in response to the amber nonsense codon with high fidelity and yield. The incorporation of BpyAla requires a BpyAla-specific aminoacyl-tRNA synthetase, which was evolved in a stepwise fashion. The structural basis of selective recognition of BpyAla by this synthetase was also detd.
- 463Lee, H. S.; Schultz, P. G. Biosynthesis of a Site-Specific DNA Cleaving Protein. J. Am. Chem. Soc. 2008, 130 (40), 13194– 13195, DOI: 10.1021/ja804653f463Biosynthesis of a Site-Specific DNA Cleaving ProteinLee, Hyun Soo; Schultz, Peter G.Journal of the American Chemical Society (2008), 130 (40), 13194-13195CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)An E. coli catabolite activator protein (CAP) has been converted into a sequence-specific DNA cleaving protein by genetically introducing (2,2'-bipyridin-5-yl)alanine (Bpy-Ala) into the protein. The mutant CAP (CAP-K26Bpy-Ala) showed comparable binding affinity to CAP-WT for the consensus operator sequence. In the presence of Cu(II) and 3-mercaptopropionic acid, CAP-K26Bpy-Ala cleaves double-stranded DNA with high sequence specificity. This method should provide a useful tool for mapping the mol. details of protein-nucleic acid interactions.
- 464Roelfes, G. LmrR: A Privileged Scaffold for Artificial Metalloenzymes. Acc. Chem. Res. 2019, 52 (3), 545– 556, DOI: 10.1021/acs.accounts.9b00004464LmrR: A Privileged Scaffold for Artificial MetalloenzymesRoelfes, GerardAccounts of Chemical Research (2019), 52 (3), 545-556CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. The biotechnol. revolution has made it possible to create enzymes for many reactions by directed evolution. However, because of the immense no. of possibilities, the availability of enzymes that possess a basal level of the desired catalytic activity is a prerequisite for success. For new-to-nature reactions, artificial metalloenzymes (ARMs), which are rationally designed hybrids of proteins and catalytically active transition-metal complexes, can be such a starting point. This Account details our efforts toward the creation of ARMs for the catalysis of new-to-nature reactions. Key to our approach is the notion that the binding of substrates, i.e., effective molarity, is a key component to achieving large accelerations in catalysis. For this reason, our designs are based on the multidrug resistance regulator LmrR, a dimeric transcription factor with a large, hydrophobic binding pocket at its dimer interface. In this pocket, there are two tryptophan moieties, which are important for promiscuous binding of planar hydrophobic conjugated compds. by π-stacking. The catalytic machinery is introduced either by the covalent linkage of a catalytically active metal complex or via the ligand or supramol. assembly, taking advantage of the two central tryptophan moieties for noncovalent binding of transition-metal complexes. Designs based on the chem. modification of LmrR were successful in catalysis, but this approach proved too laborious to be practical. Therefore, expanded genetic code methodologies were used to introduce metal binding unnatural amino acids during LmrR biosynthesis in vivo. These ARMs have been successfully applied in Cu(II) catalyzed Friedel-Crafts alkylation of indoles. The extension to MDRs from the TetR family resulted in ARMs capable of providing the opposite enantiomer of the Friedel-Crafts product. We have employed a computationally assisted redesign of these ARMs to create a more active and selective artificial hydratase, introducing a glutamate as a general base at a judicious position so it can activate and direct the incoming water nucleophile. A supramolecularly assembled ARM from LmrR and copper(II)-phenanthroline was successful in Friedel-Crafts alkylation reactions, giving rise to up to 94% ee. Also, hemin was bound, resulting in an artificial heme enzyme for enantioselective cyclopropanation reactions. The importance of structural dynamics of LmrR was suggested by computational studies, which showed that the pore can open up to allow access of substrates to the catalytic iron center, which, according to the crystal structure, is deeply buried inside the protein. Finally, the assembly approaches were combined to introduce both a catalytic and a regulatory domain, resulting in an ARM that was specifically activated in the presence of Fe(II) salts but not Zn(II) salts. Our work demonstrates that LmrR is a privileged scaffold for ARM design: It allows for multiple assembly methods and even combinations of these, it can be applied in a variety of different catalytic reactions, and it shows significant structural dynamics that contribute to achieving the desired catalytic activity. Moreover, both the creation via expanded genetic code methods as well as the supramol. assembly make LmrR-based ARMs highly suitable for achieving the ultimate goal of the integration of ARMs in biosynthetic pathways in vivo to create a hybrid metab.
- 465Drienovská, I.; Rioz-Martínez, A.; Draksharapu, A.; Roelfes, G. Novel Artificial Metalloenzymes by in vivo Incorporation of Metal-Binding Unnatural Amino Acids. Chem. Sci. 2015, 6 (1), 770– 776, DOI: 10.1039/C4SC01525H465Novel artificial metalloenzymes by in vivo incorporation of metal-binding unnatural amino acidsDrienovska, Ivana; Rioz-Martinez, Ana; Draksharapu, Apparao; Roelfes, GerardChemical Science (2015), 6 (1), 770-776CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)Artificial metalloenzymes have emerged as an attractive new approach to enantioselective catalysis. Herein, we introduce a novel strategy for prepn. of artificial metalloenzymes utilizing amber stop codon suppression methodol. for the in vivo incorporation of metal-binding unnatural amino acids. The resulting artificial metalloenzymes were applied in catalytic asym. Friedel-Crafts alkylation reactions and up to 83% ee for the product was achieved.
- 466Drienovská, I.; Alonso-Cotchico, L.; Vidossich, P.; Lledós, A.; Maréchal, J.-D.; Roelfes, G. Design of an Enantioselective Artificial Metallo-Hydratase Enzyme Containing an Unnatural Metal-Binding Amino Acid. Chem. Sci. 2017, 8 (10), 7228– 7235, DOI: 10.1039/C7SC03477FThere is no corresponding record for this reference.
- 467Ségaud, N.; Drienovská, I.; Chen, J.; Browne, W. R.; Roelfes, G. Artificial Metalloproteins for Binding and Stabilization of a Semiquinone Radical. Inorg. Chem. 2017, 56 (21), 13293– 13299, DOI: 10.1021/acs.inorgchem.7b02073There is no corresponding record for this reference.
- 468Bersellini, M.; Roelfes, G. Multidrug Resistance Regulators (MDRs) as Scaffolds for the Design of Artificial Metalloenzymes. Org. Biomol. Chem. 2017, 15 (14), 3069– 3073, DOI: 10.1039/C7OB00390K468Multidrug resistance regulators (MDRs) as scaffolds for the design of artificial metalloenzymesBersellini, Manuela; Roelfes, GerardOrganic & Biomolecular Chemistry (2017), 15 (14), 3069-3073CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)The choice of protein scaffolds is an important element in the design of artificial metalloenzymes. Here, we introduced multidrug resistance regulators (MDRs) from the TetR family as a viable class of protein scaffolds for artificial metalloenzyme design. The in vivo incorporation of the metal-binding amino acid, (2,2-bipyridin-5yl)alanine (BpyA), by stop codon suppression methods was used to create artificial metalloenzymes from 3 members of the TetR family of MDRs: QacR, CgmR, and RamR. Excellent results were achieved with QacR Y123BpyA in the Cu2+-catalyzed enantioselective vinylogous Friedel-Crafts alkylation reaction with ee's up to 94% of the opposite enantiomer that was achieved with other mutants and the previously reported LmrR-based artificial metalloenzymes.
- 469Jung, S.-M.; Yang, M.; Song, W. J. Symmetry-Adapted Synthesis of Dicopper Oxidases with Divergent Dioxygen Reactivity. Inorg. Chem. 2022, 61 (31), 12433– 12441, DOI: 10.1021/acs.inorgchem.2c01898There is no corresponding record for this reference.
- 470Drienovská, I.; Scheele, R. A.; Gutiérrez de Souza, C.; Roelfes, G. A Hydroxyquinoline-Based Unnatural Amino Acid for the Design of Novel Artificial Metalloenzymes. ChemBioChem 2020, 21 (21), 3077– 3081, DOI: 10.1002/cbic.202000306470A Hydroxyquinoline-Based Unnatural Amino Acid for the Design of Novel Artificial MetalloenzymesDrienovska, Ivana; Scheele, Remkes A.; Gutierrez de Souza, Cora; Roelfes, GerardChemBioChem (2020), 21 (21), 3077-3081CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)We have examd. the potential of the noncanonical amino acid (8-hydroxyquinolin-3-yl)alanine (HQAla) for the design of artificial metalloenzymes. HQAla, a versatile chelator of late transition metals, was introduced into the lactococcal multidrug-resistance regulator (LmrR) by stop codon suppression methodol. LmrR_HQAla was shown to complex efficiently with three different metal ions, CuII, ZnII and RhIII to form unique artificial metalloenzymes. The catalytic potential of the CuII-bound LmrR_HQAla enzyme was shown through its ability to catalyze asym. Friedel-Craft alkylation and water addn., whereas the ZnII-coupled enzyme was shown to mimic natural Zn hydrolase activity.
- 471Stein, A.; Liang, A. D.; Sahin, R.; Ward, T. R. Incorporation of Metal-Chelating Unnatural Amino Acids into Halotag for Allylic Deamination. J. Organomet. Chem. 2022, 962, 122272, DOI: 10.1016/j.jorganchem.2022.122272There is no corresponding record for this reference.
- 472Los, G. V.; Encell, L. P.; McDougall, M. G.; Hartzell, D. D.; Karassina, N.; Zimprich, C.; Wood, M. G.; Learish, R.; Ohana, R. F.; Urh, M. HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis. ACS Chem. Biol. 2008, 3 (6), 373– 382, DOI: 10.1021/cb800025k472HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein AnalysisLos, Georgyi V.; Encell, Lance P.; McDougall, Mark G.; Hartzell, Danette D.; Karassina, Natasha; Zimprich, Chad; Wood, Monika G.; Learish, Randy; Ohana, Rachel Friedman; Urh, Marjeta; Simpson, Dan; Mendez, Jacqui; Zimmerman, Kris; Otto, Paul; Vidugiris, Gediminas; Zhu, Ji; Darzins, Aldis; Klaubert, Dieter H.; Bulleit, Robert F.; Wood, Keith V.ACS Chemical Biology (2008), 3 (6), 373-382CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)We have designed a modular protein tagging system that allows different functionalities to be linked onto a single genetic fusion, either in soln., in living cells, or in chem. fixed cells. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful mols., such as fluorescent dyes, affinity handles, or solid surfaces. Covalent bond formation between the protein tag and the chloroalkane linker is highly specific, occurs rapidly under physiol. conditions, and is essentially irreversible. We demonstrate the utility of this system for cellular imaging and protein immobilization by analyzing multiple mol. processes assocd. with NF-κB-mediated cellular physiol., including imaging of subcellular protein translocation and capture of protein-protein and protein-DNA complexes.
- 473Coquière, D.; Bos, J.; Beld, J.; Roelfes, G. Enantioselective Artificial Metalloenzymes Based on a Bovine Pancreatic Polypeptide Scaffold. Angew. Chem. Int. Ed. 2009, 48 (28), 5159– 5162, DOI: 10.1002/anie.200901134There is no corresponding record for this reference.
- 474Madoori, P. K.; Agustiandari, H.; Driessen, A. J. M.; Thunnissen, A. M. W. H. Structure of the Transcriptional Regulator LmrR and Its Mechanism of Multidrug Recognition. EMBO J. 2009, 28 (2), 156– 166, DOI: 10.1038/emboj.2008.263474Structure of the transcriptional regulator LmrR and its mechanism of multidrug recognitionMadoori, Pramod Kumar; Agustiandari, Herfita; Driessen, Arnold J. M.; Thunnissen, Andy-Mark W. H.EMBO Journal (2009), 28 (2), 156-166CODEN: EMJODG; ISSN:0261-4189. (Nature Publishing Group)LmrR is a PadR-related transcriptional repressor that regulates the prodn. of LmrCD, a major multidrug ABC transporter in Lactococcus lactis. Transcriptional regulation is presumed to follow a drug-sensitive induction mechanism involving the direct binding of transporter ligands to LmrR. Here, we present crystal structures of LmrR in an apo state and in two drug-bound states complexed with Hoechst 33342 and daunomycin. LmrR shows a common topol. contg. a typical β-winged helix-turn-helix domain with an addnl. C-terminal helix involved in dimerization. Its dimeric organization is highly unusual with a flat-shaped hydrophobic pore at the dimer center serving as a multidrug-binding site. The drugs bind in a similar manner with their arom. rings sandwiched in between the indole groups of two dimer-related tryptophan residues. Multidrug recognition is facilitated by conformational plasticity and the absence of drug-specific hydrogen bonds. Combined analyses using site-directed mutagenesis, fluorescence-based drug binding and protein-DNA gel shift assays reveal an allosteric coupling between the multidrug- and DNA-binding sites of LmrR that most likely has a function in the induction mechanism.
- 475Yu, Y.; Hu, C.; Xia, L.; Wang, J. Artificial Metalloenzyme Design with Unnatural Amino Acids and Non-Native Cofactors. ACS Catal. 2018, 8 (3), 1851– 1863, DOI: 10.1021/acscatal.7b03754475Artificial Metalloenzyme Design with Unnatural Amino Acids and Non-Native CofactorsYu, Yang; Hu, Cheng; Xia, Lin; Wang, JiangyunACS Catalysis (2018), 8 (3), 1851-1863CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)A review. There are 20 proteinogenic amino acids and a limited no. of cofactors naturally available to build enzymes. Genetic codon expansion enables us to incorporate more than 200 unnatural amino acids into proteins using cell translation machinery, greatly expanding structures available to protein chemists. Such tools enable scientists to mimic the active site of an enzyme to tune enzymic activity, anchor cofactors, and immobilize enzymes on electrode surfaces. Non-native cofactors can be incorporated into the protein through covalent or noncovalent interactions, expanding the reaction scope of existing enzymes. The review discusses strategies to incorporate unnatural amino acids and non-native cofactors and their applications in tuning and expanding enzymic activities of artificial metalloenzymes.
- 476Yang, H.; Srivastava, P.; Zhang, C.; Lewis, J. C. A General Method for Artificial Metalloenzyme Formation through Strain-Promoted Azide-Alkyne Cycloaddition. ChemBioChem 2014, 15 (2), 223– 227, DOI: 10.1002/cbic.201300661There is no corresponding record for this reference.
- 477Srivastava, P.; Yang, H.; Ellis-Guardiola, K.; Lewis, J. C. Engineering a Dirhodium Artificial Metalloenzyme for Selective Olefin Cyclopropanation. Nature Commun. 2015, 6 (1), 7789, DOI: 10.1038/ncomms8789There is no corresponding record for this reference.
- 478Upp, D. M.; Huang, R.; Li, Y.; Bultman, M. J.; Roux, B.; Lewis, J. C. Engineering Dirhodium Artificial Metalloenzymes for Diazo Coupling Cascade Reactions. Angew. Chem. Int. Ed. 2021, 60 (44), 23672– 23677, DOI: 10.1002/anie.202107982478Engineering Dirhodium Artificial Metalloenzymes for Diazo Coupling Cascade ReactionsUpp, David M.; Huang, Rui; Li, Ying; Bultman, Max J.; Roux, Benoit; Lewis, Jared C.Angewandte Chemie, International Edition (2021), 60 (44), 23672-23677CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Artificial metalloenzymes (ArMs) are commonly used to control the stereoselectivity of catalytic reactions, but controlling chemoselectivity remains challenging. In this study, we engineer a dirhodium ArM to catalyze diazo cross-coupling to form an alkene that, in a one-pot cascade reaction, is reduced to an alkane with high enantioselectivity (typically >99% ee) by an alkene reductase. The numerous protein and small mol. components required for the cascade reaction had minimal effect on ArM catalysis. Directed evolution of the ArM led to improved yields and E/Z selectivities for a variety of substrates, which translated to cascade reaction yields. MD simulations of ArM variants were used to understand the structural role of the cofactor on ArM conformational dynamics. These results highlight the ability of ArMs to control both catalyst stereoselectivity and chemoselectivity to enable reactions in complex media that would otherwise lead to undesired side reactions.
- 479Ellis-Guardiola, K.; Rui, H.; Beckner, R. L.; Srivastava, P.; Sukumar, N.; Roux, B.; Lewis, J. C. Crystal Structure and Conformational Dynamics of Pyrococcus furiosus Prolyl Oligopeptidase. Biochemistry 2019, 58 (12), 1616– 1626, DOI: 10.1021/acs.biochem.9b00031There is no corresponding record for this reference.
- 480Brady, L.; Brzozowski, A. M.; Derewenda, Z. S.; Dodson, E.; Dodson, G.; Tolley, S.; Turkenburg, J. P.; Christiansen, L.; Huge-Jensen, B.; Norskov, L. A Serine Protease Triad Forms the Catalytic Centre of a Triacylglycerol Lipase. Nature 1990, 343 (6260), 767– 770, DOI: 10.1038/343767a0480A serine protease triad forms the catalytic center of a triacylglycerol lipaseBrady, Leo; Brzozowski, Andrzej M.; Derewenda, Zygmunt S.; Dodson, Eleanor; Dodson, Guy; Tolley, Shirley; Turkenburg, Johan P.; Christiansen, Lars; Huge-Jensen, Birgitte; et al.Nature (London, United Kingdom) (1990), 343 (6260), 767-70CODEN: NATUAS; ISSN:0028-0836.The x-ray structure of the Mucor miehei triglyceride lipase, is reported and the at. model obtained at 3.1 Å resoln. and refined to 1.9 Å resoln. is described. It reveals a serine...histidine...aspartate trypsin-like catalytic triad with an active serine buried under a short helical fragment of a long surface loop.
- 481Buller, A. R.; Townsend, C. A. Intrinsic Evolutionary Constraints on Protease Structure, Enzyme Acylation, and the Identity of the Catalytic Triad. Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (8), E653-E661 DOI: 10.1073/pnas.1221050110There is no corresponding record for this reference.
- 482Smith, A. J. T.; Müller, R.; Toscano, M. D.; Kast, P.; Hellinga, H. W.; Hilvert, D.; Houk, K. N. Structural Reorganization and Preorganization in Enzyme Active Sites: Comparisons of Experimental and Theoretically Ideal Active Site Geometries in the Multistep Serine Esterase Reaction Cycle. J. Am. Chem. Soc. 2008, 130 (46), 15361– 15373, DOI: 10.1021/ja803213p482Structural Reorganization and Preorganization in Enzyme Active Sites: Comparisons of Experimental and Theoretically Ideal Active Site Geometries in the Multistep Serine Esterase Reaction CycleSmith, Adam J. T.; Muller, Roger; Toscano, Miguel D.; Kast, Peter; Hellinga, Homme W.; Hilvert, Donald; Houk, K. N.Journal of the American Chemical Society (2008), 130 (46), 15361-15373CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Many enzymes catalyze reactions with multiple chem. steps, requiring the stabilization of multiple transition states during catalysis. Such enzymes must strike a balance between the conformational reorganization required to stabilize multiple transition states of a reaction and the confines of a preorganized active site in the polypeptide tertiary structure. Here we investigate the compromise between structural reorganization during the catalytic process and preorganization of the active site for a multistep enzyme-catalyzed reaction, the hydrolysis of esters by the Ser-His-Asp/Glu catalytic triad. Quantum mech. transition states were used to generate ensembles of geometries that can catalyze each individual step in the mechanism. These geometries are compared to each other by superpositions of catalytic atoms to find "consensus" geometries that can catalyze all steps with minimal rearrangement. These consensus geometries are found to be excellent matches for the natural active site. Preorganization is therefore found to be the major defining characteristic of the active site, and reorganizational motions often proposed to promote catalysis have been minimized. The variability of enzyme active sites obsd. by x-ray crystallog. was also investigated empirically. A catalog of geometrical parameters relating active site residues to each other and to bound inhibitors was collected from a set of crystal structures. The crystal-structure-derived values were then compared to the ranges found in quantum mech. optimized structures along the entire reaction coordinate. The empirical ranges are found to encompass the theor. ranges when thermal fluctuations are taken into account. Therefore, the active sites are preorganized to a geometry that can be objectively and quant. defined as minimizing conformational reorganization while maintaining optimal transition state stabilization for every step during catalysis. The results provide a useful guiding principle for de novo design of enzymes with multistep mechanisms.
- 483Burton, A. J.; Thomson, A. R.; Dawson, W. M.; Brady, R. L.; Woolfson, D. N. Installing Hydrolytic Activity into a Completely De Novo Protein Framework. Nat. Chem. 2016, 8 (9), 837– 844, DOI: 10.1038/nchem.2555483Installing hydrolytic activity into a completely de novo protein frameworkBurton, Antony J.; Thomson, Andrew R.; Dawson, William M.; Brady, R. Leo; Woolfson, Derek N.Nature Chemistry (2016), 8 (9), 837-844CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)The design of enzyme-like catalysts tests the understanding of sequence-to-structure/function relations in proteins. Here, the authors installed hydrolytic activity predictably into a completely de novo and thermostable α-helical barrel, which comprised 7 helixes arranged around an accessible channel. The authors showed that the lumen of the barrel accepted 21 mutations to functional polar residues. The resulting variant, which had Cys-His-Glu triads on each helix, hydrolyzed p-nitrophenyl acetate with catalytic efficiencies that matched the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the 1st report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of this system also allowed the facile incorporation of unnatural side-chains to improve activity and probe the catalytic mechanism. Such a predictable and robust construction of truly de novo biocatalysts holds promise for applications in chem. and biochem. synthesis.
- 484Rajagopalan, S.; Wang, C.; Yu, K.; Kuzin, A. P.; Richter, F.; Lew, S.; Miklos, A. E.; Matthews, M. L.; Seetharaman, J.; Su, M. Design of Activated Serine-Containing Catalytic Triads with Atomic-Level Accuracy. Nat. Chem. Biol. 2014, 10 (5), 386– 391, DOI: 10.1038/nchembio.1498There is no corresponding record for this reference.
- 485Richter, F.; Blomberg, R.; Khare, S. D.; Kiss, G.; Kuzin, A. P.; Smith, A. J. T.; Gallaher, J.; Pianowski, Z.; Helgeson, R. C.; Grjasnow, A. Computational Design of Catalytic Dyads and Oxyanion Holes for Ester Hydrolysis. J. Am. Chem. Soc. 2012, 134 (39), 16197– 16206, DOI: 10.1021/ja3037367485Computational Design of Catalytic Dyads and Oxyanion Holes for Ester HydrolysisRichter, Florian; Blomberg, Rebecca; Khare, Sagar D.; Kiss, Gert; Kuzin, Alexandre P.; Smith, Adam J. T.; Gallaher, Jasmine; Pianowski, Zbigniew; Helgeson, Roger C.; Grjasnow, Alexej; Xiao, Rong; Seetharaman, Jayaraman; Su, Min; Vorobiev, Sergey; Lew, Scott; Forouhar, Farhad; Kornhaber, Gregory J.; Hunt, John F.; Montelione, Gaetano T.; Tong, Liang; Houk, K. N.; Hilvert, Donald; Baker, DavidJournal of the American Chemical Society (2012), 134 (39), 16197-16206CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Nucleophilic catalysis is a general strategy for accelerating ester and amide hydrolysis. In natural active sites, nucleophilic elements such as catalytic dyads and triads are usually paired with oxyanion holes for substrate activation, but it is difficult to parse out the independent contributions of these elements or to understand how they emerged in the course of evolution. Here we explore the minimal requirements for esterase activity by computationally designing artificial catalysts using catalytic dyads and oxyanion holes. We found much higher success rates using designed oxyanion holes formed by backbone NH groups rather than by side chains or bridging water mols. and obtained four active designs in different scaffolds by combining this motif with a Cys-His dyad. Following active site optimization, the most active of the variants exhibited a catalytic efficiency (kcat/KM) of 400 M-1 s-1 for the cleavage of a p-nitrophenyl ester. Kinetic expts. indicate that the active site cysteines are rapidly acylated as programmed by design, but the subsequent slow hydrolysis of the acyl-enzyme intermediate limits overall catalytic efficiency. Moreover, the Cys-His dyads are not properly formed in crystal structures of the designed enzymes. These results highlight the challenges that computational design must overcome to achieve high levels of activity.
- 486Burke, A. J.; Lovelock, S. L.; Frese, A.; Crawshaw, R.; Ortmayer, M.; Dunstan, M.; Levy, C.; Green, A. P. Design and Evolution of an Enzyme with a Non-Canonical Organocatalytic Mechanism. Nature 2019, 570 (7760), 219– 223, DOI: 10.1038/s41586-019-1262-8486Design and evolution of an enzyme with a non-canonical organocatalytic mechanismBurke, Ashleigh J.; Lovelock, Sarah L.; Frese, Amina; Crawshaw, Rebecca; Ortmayer, Mary; Dunstan, Mark; Levy, Colin; Green, Anthony P.Nature (London, United Kingdom) (2019), 570 (7760), 219-223CODEN: NATUAS; ISSN:0028-0836. (Nature Research)The combination of computational design and lab. evolution is a powerful and potentially versatile strategy for the development of enzymes with new functions1-4. However, the limited functionality presented by the genetic code restricts the range of catalytic mechanisms that are accessible in designed active sites. Inspired by mechanistic strategies from small-mol. organocatalysis5, here we report the generation of a hydrolytic enzyme that uses Nδ-methylhistidine as a non-canonical catalytic nucleophile. Histidine methylation is essential for catalytic function because it prevents the formation of unreactive acyl-enzyme intermediates, which has been a long-standing challenge when using canonical nucleophiles in enzyme design6-10. Enzyme performance was optimized using directed evolution protocols adapted to an expanded genetic code, affording a biocatalyst capable of accelerating ester hydrolysis with greater than 9,000-fold increased efficiency over free Nδ-methylhistidine in soln. Crystallog. snapshots along the evolutionary trajectory highlight the catalytic devices that are responsible for this increase in efficiency. Nδ-methylhistidine can be considered to be a genetically encodable surrogate of the widely employed nucleophilic catalyst dimethylaminopyridine11, and its use will create opportunities to design and engineer enzymes for a wealth of valuable chem. transformations.
- 487Bjelic, S.; Nivón, L. G.; Çelebi-Ölçüm, N.; Kiss, G.; Rosewall, C. F.; Lovick, H. M.; Ingalls, E. L.; Gallaher, J. L.; Seetharaman, J.; Lew, S. Computational Design of Enone-Binding Proteins with Catalytic Activity for the Morita-Baylis-Hillman Reaction. ACS Chem. Biol. 2013, 8 (4), 749– 757, DOI: 10.1021/cb3006227487Computational Design of Enone-Binding Proteins with Catalytic Activity for the Morita-Baylis-Hillman ReactionBjelic, Sinisa; Nivon, Lucas G.; Celebi-Olcum, Nihan; Kiss, Gert; Rosewall, Carolyn F.; Lovick, Helena M.; Ingalls, Erica L.; Gallaher, Jasmine Lynn; Seetharaman, Jayaraman; Lew, Scott; Montelione, Gaetano Thomas; Hunt, John Francis; Michael, Forrest Edwin; Houk, K. N.; Baker, DavidACS Chemical Biology (2013), 8 (4), 749-757CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)The Morita-Baylis-Hillman reaction forms a carbon-carbon bond between the α-carbon of a conjugated carbonyl compd. and a carbon electrophile. The reaction mechanism involves Michael addn. of a nucleophile catalyst at the carbonyl β-carbon, followed by bond formation with the electrophile and catalyst disassocn. to release the product. We used Rosetta to design 48 proteins contg. active sites predicted to carry out this mechanism, of which two show catalytic activity by mass spectrometry (MS). Substrate labeling measured by MS and site-directed mutagenesis expts. show that the designed active-site residues are responsible for activity, although rate acceleration over background is modest. To characterize the designed proteins, we developed a fluorescence-based screen for intermediate formation in cell lysates, carried out microsecond mol. dynamics simulations, and solved X-ray crystal structures. These data indicate a partially formed active site and suggest several clear avenues for designing more active catalysts.
- 488Hutton, A. E.; Foster, J.; Crawshaw, R.; Hardy, F. J.; Johannissen, L. O.; Lister, T. M.; Gerard, E. F.; Birch-Price, Z.; Obexer, R.; Hay, S.; Green, A. P. A Non-Canonical Nucleophile Unlocks a New Mechanistic Pathway in a Designed Enzyme. Nat Commun 2024, DOI: 10.1038/s41467-024-46123-zThere is no corresponding record for this reference.
- 489Crawshaw, R.; Crossley, A.; Johannissen, L.; Burke, A.; Hay, S.; Levy, C.; Baker, D.; Lovelock, S.; Green, A. Engineering an Efficient and Enantioselective Enzyme for the Morita-Baylis-Hillman Reaction. Nat. Chem. 2022, 14, 313, DOI: 10.1038/s41557-021-00833-9489Engineering an efficient and enantioselective enzyme for the Morita-Baylis-Hillman reactionCrawshaw, Rebecca; Crossley, Amy E.; Johannissen, Linus; Burke, Ashleigh J.; Hay, Sam; Levy, Colin; Baker, David; Lovelock, Sarah L.; Green, Anthony P.Nature Chemistry (2022), 14 (3), 313-320CODEN: NCAHBB; ISSN:1755-4330. (Nature Portfolio)Abstr.: The combination of computational design and directed evolution could offer a general strategy to create enzymes with new functions. So far, this approach has delivered enzymes for a handful of model reactions. Here we show that new catalytic mechanisms can be engineered into proteins to accelerate more challenging chem. transformations. Evolutionary optimization of a primitive design afforded an efficient and enantioselective enzyme (BH32.14) for the Morita-Baylis-Hillman (MBH) reaction. BH32.14 is suitable for preparative-scale transformations, accepts a broad range of aldehyde and enone coupling partners and is able to promote selective monofunctionalizations of dialdehydes. Crystallog., biochem. and computational studies reveal that BH32.14 operates via a sophisticated catalytic mechanism comprising a His23 nucleophile paired with a judiciously positioned Arg124. This catalytic arginine shuttles between conformational states to stabilize multiple oxyanion intermediates and serves as a genetically encoded surrogate of privileged bidentate hydrogen-bonding catalysts (for example, thioureas). This study demonstrates that elaborate catalytic devices can be built from scratch to promote demanding multi-step processes not obsd. in nature. [graphic not available: see fulltext].
- 490Agten, S. M.; Dawson, P. E.; Hackeng, T. M. Oxime Conjugation in Protein Chemistry: From Carbonyl Incorporation to Nucleophilic Catalysis. J. Pept. Sci. 2016, 22 (5), 271– 279, DOI: 10.1002/psc.2874490Oxime conjugation in protein chemistry: from carbonyl incorporation to nucleophilic catalysisAgten, Stijn M.; Dawson, Philip E.; Hackeng, Tilman M.Journal of Peptide Science (2016), 22 (5), 271-279CODEN: JPSIEI; ISSN:1075-2617. (John Wiley & Sons Ltd.)A review. Use of oxime forming reactions has become a widely applied strategy for peptide and protein bioconjugation. The efficiency of the reaction and robust stability of the oxime product led to the development of a growing list of methods to introduce the required ketone or aldehyde functionality site specifically into proteins. Early methods focused on site-specific oxidn. of an N-terminal serine or threonine and more recently transamination methods have been developed to convert a broader set of N-terminal amino acids into a ketone or aldehyde. More recently, site-specific modification of protein has been attained through engineering enzymes involved in posttranslational modifications to accommodate aldehyde-contg. substrates. Similarly, a growing list of unnatural amino acids can be introduced through development of selective amino-acyl tRNA synthetase/tRNA pairs combined with codon reassignment. In the case of glycoproteins, glycans can be selectively modified chem. or enzymically to introduce aldehyde functional groups. Finally, the total chem. synthesis of proteins complements these biol. and chemoenzymic approaches. Once introduced, the oxime ligation of these aldehyde and ketone groups can be catalyzed by aniline or a variety of aniline derivs. to tune the activity, pH preference, stability and soly. of the catalyst.
- 491Kölmel, D. K.; Kool, E. T. Oximes and Hydrazones in Bioconjugation: Mechanism and Catalysis. Chem. Rev. 2017, 117 (15), 10358– 10376, DOI: 10.1021/acs.chemrev.7b00090491Oximes and Hydrazones in Bioconjugation: Mechanism and CatalysisKolmel, Dominik K.; Kool, Eric T.Chemical Reviews (Washington, DC, United States) (2017), 117 (15), 10358-10376CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. The formation of oximes and hydrazones is employed in numerous scientific fields as a simple and versatile conjugation strategy. This imine-forming reaction is applied in fields as diverse as polymer chem., biomaterials and hydrogels, dynamic combinatorial chem., org. synthesis, and chem. biol. Here we outline chem. developments in this field, with special focus on the past ∼10 years of developments. Recent strategies for installing reactive carbonyl groups and α-nucleophiles into biomols. are described. The basic chem. properties of reactants and products in this reaction are then reviewed, with an eye to understanding the reaction's mechanism and how reactant structure controls rates and equil. in the process. Recent work that has uncovered structural features and new mechanisms for speeding the reaction, sometimes by orders of magnitude, is discussed. We describe recent studies that have identified esp. fast reacting aldehyde/ketone substrates and structural effects that lead to rapid-reacting α-nucleophiles as well. Among the most effective new strategies has been the development of substituents near the reactive aldehyde group that either transfer protons at the transition state or trap the initially formed tetrahedral intermediates. In addn., the recent development of efficient nucleophilic catalysts for the reaction is outlined, improving greatly upon aniline, the classical catalyst for imine formation. A no. of uses of such second- and third-generation catalysts in bioconjugation and in cellular applications are highlighted. While formation of hydrazone and oxime has been traditionally regarded as being limited by slow rates, developments in the past 5 years have resulted in completely overturning this limitation; indeed, the reaction is now one of the fastest and most versatile reactions available for conjugations of biomols. and biomaterials.
- 492Drienovská, I.; Mayer, C.; Dulson, C.; Roelfes, G. A Designer Enzyme for Hydrazone and Oxime Formation Featuring an Unnatural Catalytic Aniline Residue. Nat. Chem. 2018, 10 (9), 946– 952, DOI: 10.1038/s41557-018-0082-z492A designer enzyme for hydrazone and oxime formation featuring an unnatural catalytic aniline residueDrienovska, Ivana; Mayer, Clemens; Dulson, Christopher; Roelfes, GerardNature Chemistry (2018), 10 (9), 946-952CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Creating designer enzymes with the ability to catalyze abiol. transformations is a formidable challenge. Efforts toward this goal typically consider only canonical amino acids in the initial design process. However, incorporating unnatural amino acids that feature uniquely reactive side chains could significantly expand the catalytic repertoire of designer enzymes. To explore the potential of such artificial building blocks for enzyme design, here we selected p-aminophenylalanine (pAF) as a potentially novel catalytic residue. We demonstrate that the catalytic activity of the aniline side chain for hydrazone and oxime formation reactions is increased by embedding p-aminophenylalanine into the hydrophobic pore of the multidrug transcriptional regulator LmrR from Lactococcus lactis. Both the recruitment of reactants by the promiscuous binding pocket and a judiciously placed aniline that functions as a catalytic residue contribute to the success of the identified artificial enzyme. We anticipate that our design strategy will prove rewarding to significantly expand the catalytic repertoire of designer enzymes in the future.
- 493Ofori Atta, L.; Zhou, Z.; Roelfes, G. In vivo Biocatalytic Cascades Featuring an Artificial-Enzyme-Catalysed New-to-Nature Reaction. Angew. Chem. Int. Ed. 2023, 62 (1), e202214191 DOI: 10.1002/anie.202214191There is no corresponding record for this reference.
- 494Leveson-Gower, R. B.; de Boer, R. M.; Roelfes, G. Tandem Friedel-Crafts-Alkylation-Enantioselective-Protonation by Artificial Enzyme Iminium Catalysis. ChemCatChem 2022, 14 (8), e202101875 DOI: 10.1002/cctc.202101875494Tandem Friedel-Crafts-Alkylation-Enantioselective-Protonation by Artificial Enzyme Iminium CatalysisLeveson-Gower, Reuben B.; de Boer, Ruben M.; Roelfes, GerardChemCatChem (2022), 14 (8), e202101875CODEN: CHEMK3; ISSN:1867-3880. (Wiley-VCH Verlag GmbH & Co. KGaA)The incorporation of organocatalysts into protein scaffolds holds the promise of overcoming some of the limitations of this powerful catalytic approach. Previously, we showed that incorporation of the non-canonical amino acid para-aminophenylalanine into the non-enzymic protein scaffold LmrR forms a proficient and enantioselective artificial enzyme (LmrR_pAF) for the Friedel-Crafts alkylation of indoles with enals. The unnatural aniline side-chain is directly involved in catalysis, operating via a well-known organocatalytic iminium-based mechanism. In this study, we show that LmrR_pAF can enantioselectively form tertiary carbon centers not only during C-C bond formation, but also by enantioselective protonation, delivering a proton to one face of a prochiral enamine intermediate. The importance of various side-chains in the pocket of LmrR is distinct from the Friedel-Crafts reaction without enantioselective protonation, and two particularly important residues were probed by exhaustive mutagenesis.
- 495Zhou, Z.; Roelfes, G. Synergistic Catalysis in an Artificial Enzyme by Simultaneous Action of Two Abiological Catalytic Sites. Nat. Catal. 2020, 3 (3), 289– 294, DOI: 10.1038/s41929-019-0420-6495Synergistic catalysis in an artificial enzyme by simultaneous action of two abiological catalytic sitesZhou, Zhi; Roelfes, GerardNature Catalysis (2020), 3 (3), 289-294CODEN: NCAACP; ISSN:2520-1158. (Nature Research)Abstr.: Artificial enzymes, which are hybrids of proteins with abiol. catalytic groups, have emerged as a powerful approach towards the creation of enzymes for new-to-nature reactions. Typically, only a single abiol. catalytic moiety is incorporated. Here we introduce a design of an artificial enzyme that comprises two different abiol. catalytic moieties and show that these can act synergistically to achieve high activity and enantioselectivity (up to >99% e.e.) in the catalyzed Michael addn. reaction. The design is based on the lactococcal multidrug resistance regulator as the protein scaffold and combines a genetically encoded unnatural p-aminophenylalanine residue (which activates an enal through iminium ion formation) and a supramolecularly bound Lewis acidic Cu(II) complex (which activates the Michael donor by enolization and delivers it to one preferred prochiral face of the activated enal). This study demonstrates that synergistic combination of abiol. catalytic groups is a robust way to achieve catalysis that is normally outside of the realm of artificial enzymes.
- 496Zhou, Z.; Roelfes, G. Synergistic Catalysis of Tandem Michael Addition/Enantioselective Protonation Reactions by an Artificial Enzyme. ACS Catal. 2021, 11 (15), 9366– 9369, DOI: 10.1021/acscatal.1c02298There is no corresponding record for this reference.
- 497Gran-Scheuch, A.; Bonandi, E.; Drienovská, I. Expanding the Genetic Code: Incorporation of Functional Secondary Amines via Stop Codon Suppression. ChemCatChem 2024, 16, e202301004 DOI: 10.1002/cctc.202301004There is no corresponding record for this reference.
- 498Longwitz, L.; Leveson-Gower, R. B.; Rozeboom, H. J.; Thunnissen, A.-M. W. H.; Roelfes, G. Boron Catalysis in a Designer Enzyme. Nature 2024, 629, 824, DOI: 10.1038/s41586-024-07391-3There is no corresponding record for this reference.
- 499Garrido-Castro, A. F.; Maestro, M. C.; Alemán, J. Asymmetric Induction in Photocatalysis - Discovering a New Side to Light-Driven Chemistry. Tetrahedron Lett. 2018, 59 (14), 1286– 1294, DOI: 10.1016/j.tetlet.2018.02.040There is no corresponding record for this reference.
- 500Yao, W.; Bazan-Bergamino, E. A.; Ngai, M.-Y. Asymmetric Photocatalysis Enabled by Chiral Organocatalysts. ChemCatChem 2022, 14 (1), e202101292 DOI: 10.1002/cctc.202101292500Asymmetric Photocatalysis Enabled by Chiral OrganocatalystsYao, Wang; Bazan-Bergamin, Emmanuel A.; Ngai, Ming-YuChemCatChem (2022), 14 (1), e202101292CODEN: CHEMK3; ISSN:1867-3880. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Visible-light photocatalysis has advanced as a versatile tool in org. synthesis. However, attaining precise stereocontrol in photocatalytic reactions has been a longstanding challenge due to undesired photochem. background reactions and the involvement of highly reactive radicals or radical ion intermediates generated under photocatalytic conditions. To address this problem and expand the synthetic utility of photocatalytic reactions, a no. of innovative strategies, including mono- and dual-catalytic approaches, have recently emerged. Of these, exploiting chiral organocatalysis, such as enamine catalysis, iminium-ion catalysis, Broensted acid/base catalysis, and N-heterocyclic carbene catalysis, to induce chirality transfer of photocatalytic reactions has been widely explored. This Review aims to provide a current, comprehensive overview of asym. photocatalytic reactions enabled by chiral organocatalysts published through June 2021. The substrate scope, advantages, limitations, and proposed reaction mechanisms of each reaction are discussed. This review should serve as a ref. for the development of visible-light-induced asym. photocatalysis and promote the improvement of the chem. reactivity and stereoselectivity of these reactions.
- 501Heyes, D. J.; Lakavath, B.; Hardman, S. J. O.; Sakuma, M.; Hedison, T. M.; Scrutton, N. S. Photochemical Mechanism of Light-Driven Fatty Acid Photodecarboxylase. ACS Catal. 2020, 10 (12), 6691– 6696, DOI: 10.1021/acscatal.0c01684501Photochemical Mechanism of Light-Driven Fatty Acid PhotodecarboxylaseHeyes, Derren J.; Lakavath, Balaji; Hardman, Samantha J. O.; Sakuma, Michiyo; Hedison, Tobias M.; Scrutton, Nigel S.ACS Catalysis (2020), 10 (12), 6691-6696CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)Fatty acid photodecarboxylase (FAP) is a promising target for the prodn. of biofuels and fine chems. It contains a FAD cofactor and catalyzes the blue-light-dependent decarboxylation of fatty acids to generate the corresponding alkane. However, little is known about the catalytic mechanism of FAP, or how light is used to drive enzymic decarboxylation. Here, we have used a combination of time-resolved and cryogenic trapping UV-visible absorption spectroscopy to characterize a red-shifted flavin intermediate obsd. in the catalytic cycle of FAP. We show that this intermediate can form below the "glass transition" temp. of proteins, whereas the subsequent decay of the species proceeds only at higher temps., implying a role for protein motions in the decay of the intermediate. Solvent isotope effect measurements, combined with analyses of selected site-directed variants of FAP, suggest that the formation of the red-shifted flavin species is directly coupled with hydrogen atom transfer from a nearby active site cysteine residue, yielding the final alkane product. Our study suggests that this cysteine residue forms a thiolate-flavin charge-transfer species, which is assigned as the red-shifted flavin intermediate. Taken together, our data provide insights into light-dependent decarboxylase mechanisms catalyzed by FAP and highlight important considerations in the (re)design of flavin-based photoenzymes.
- 502Heyes, D. J.; Zhang, S.; Taylor, A.; Johannissen, L. O.; Hardman, S. J. O.; Hay, S.; Scrutton, N. S. Photocatalysis as the ‘Master Switch’ of Photomorphogenesis in Early Plant Development. Nature Plants 2021, 7 (3), 268– 276, DOI: 10.1038/s41477-021-00866-5There is no corresponding record for this reference.
- 503Tan, C.; Liu, Z.; Li, J.; Guo, X.; Wang, L.; Sancar, A.; Zhong, D. The Molecular Origin of High DNA-Repair Efficiency by Photolyase. Nature Commun. 2015, 6 (1), 7302, DOI: 10.1038/ncomms8302There is no corresponding record for this reference.
- 504Black, M. J.; Biegasiewicz, K. F.; Meichan, A. J.; Oblinsky, D. G.; Kudisch, B.; Scholes, G. D.; Hyster, T. K. Asymmetric Redox-Neutral Radical Cyclization Catalysed by Flavin-Dependent ‘Ene’-Reductases. Nat. Chem. 2020, 12 (1), 71– 75, DOI: 10.1038/s41557-019-0370-2504Asymmetric redox-neutral radical cyclization catalysed by flavin-dependent 'ene'-reductasesBlack Michael J; Biegasiewicz Kyle F; Meichan Andrew J; Oblinsky Daniel G; Kudisch Bryan; Scholes Gregory D; Hyster Todd KNature chemistry (2020), 12 (1), 71-75 ISSN:.Flavin-dependent 'ene'-reductases (EREDs) are exquisite catalysts for effecting stereoselective reductions. Although these reactions typically proceed through a hydride transfer mechanism, we recently found that EREDs can also catalyse reductive dehalogenations and cyclizations via single electron transfer mechanisms. Here, we demonstrate that these enzymes can catalyse redox-neutral radical cyclizations to produce enantioenriched oxindoles from α-haloamides. This transformation is a C-C bond-forming reaction currently unknown in nature and one for which there are no catalytic asymmetric examples. Mechanistic studies indicate the reaction proceeds via the flavin semiquinone/quinone redox couple, where ground-state flavin semiquinone provides the electron for substrate reduction and flavin quinone oxidizes the vinylogous α-amido radical formed after cyclization. This mechanistic manifold was previously unknown for this enzyme family, highlighting the versatility of EREDs in asymmetric synthesis.
- 505Emmanuel, M. A.; Greenberg, N. R.; Oblinsky, D. G.; Hyster, T. K. Accessing Non-Natural Reactivity by Irradiating Nicotinamide-Dependent Enzymes with Light. Nature 2016, 540 (7633), 414– 417, DOI: 10.1038/nature20569505Accessing non-natural reactivity by irradiating nicotinamide-dependent enzymes with lightEmmanuel, Megan A.; Greenberg, Norman R.; Oblinsky, Daniel G.; Hyster, Todd K.Nature (London, United Kingdom) (2016), 540 (7633), 414-417CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Enzymes are ideal for use in asym. catalysis by the chem. industry, because their chem. compns. can be tailored to a specific substrate and selectivity pattern while providing efficiencies and selectivities that surpass those of classical synthetic methods. However, enzymes are limited to reactions that are found in nature and, as such, facilitate fewer types of transformation than do other forms of catalysis. Thus, a longstanding challenge in the field of biol. mediated catalysis has been to develop enzymes with new catalytic functions. Here we describe a method for achieving catalytic promiscuity that uses the photoexcited state of nicotinamide cofactors (mols. that assist enzyme-mediated catalysis). Under irradn. with visible light, the nicotinamide-dependent enzyme known as ketoreductase (KRED) can be transformed from a carbonyl reductase into an initiator of radical species and a chiral source of hydrogen atoms. We demonstrate this new reactivity through a highly enantioselective radical dehalogenation of lactones - a challenging transformation for small-mol. catalysts. Mechanistic expts. support the theory that a radical species acts as an intermediate in this reaction, with NADH and NADPH (the reduced forms of nicotinamide adenine nucleotide and NADP, resp.) serving as both a photoreductant and the source of hydrogen atoms. To our knowledge, this method represents the first example of photoinduced enzyme promiscuity, and highlights the potential for accessing new reactivity from existing enzymes simply by using the excited states of common biol. cofactors. This represents a departure from existing light-driven biocatalytic techniques, which are typically explored in the context of cofactor regeneration.
- 506Liu, X.; Kang, F.; Hu, C.; Wang, L.; Xu, Z.; Zheng, D.; Gong, W.; Lu, Y.; Ma, Y.; Wang, J. A Genetically Encoded Photosensitizer Protein Facilitates the Rational Design of a Miniature Photocatalytic CO2-Reducing Enzyme. Nat. Chem. 2018, 10 (12), 1201– 1206, DOI: 10.1038/s41557-018-0150-4506A genetically encoded photosensitizer protein facilitates the rational design of a miniature photocatalytic CO2-reducing enzymeLiu, Xiaohong; Kang, Fuying; Hu, Cheng; Wang, Li; Xu, Zhen; Zheng, Dandan; Gong, Weimin; Lu, Yi; Ma, Yanhe; Wang, JiangyunNature Chemistry (2018), 10 (12), 1201-1206CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Photosensitizers, which harness light energy to upgrade weak reductants to strong reductants, are pivotal components of the natural and artificial photosynthesis machineries. However, it has proved difficult to enhance and expand their functions through genetic engineering. Here the authors report a genetically encoded, 27 kDa photosensitizer protein (PSP), which facilitates the rational design of miniature photocatalytic CO2-reducing enzymes. Visible light drives PSP efficiently into a long-lived triplet excited state (PSP*), which reacts rapidly with reduced NAD to generate a super-reducing radical (PSP•), which is strong enough to reduce many CO2-reducing catalysts. The authors detd. the three-dimensional structure of PSP• at 1.8 Å resoln. by x-ray crystallog. Genetic engineering enabled the site-specific attachment of a nickel-terpyridine complex and the modular optimization of the photochem. properties of PSP, the chromophore/catalytic center distance and the catalytic center microenvironment, which culminated in a miniature photocatalytic CO2-reducing enzyme that has a CO2/CO conversion quantum efficiency of 2.6%.
- 507Siegel, J. B.; Zanghellini, A.; Lovick, H. M.; Kiss, G.; Lambert, A. R.; St. Clair, J. L.; Gallaher, J. L.; Hilvert, D.; Gelb, M. H.; Stoddard, B. L.; Houk, K. N.; Michael, F. E.; Baker, D. Computational Design of an Enzyme Catalyst for a Stereoselective Bimolecular Diels-Alder Reaction. Science 2010, 329, 309– 313, DOI: 10.1126/science.1190239507Computational Design of an Enzyme Catalyst for a Stereoselective Bimolecular Diels-Alder ReactionSiegel, Justin B.; Zanghellini, Alexandre; Lovick, Helena M.; Kiss, Gert; Lambert, Abigail R.; St. Clair, Jennifer L.; Gallaher, Jasmine L.; Hilvert, Donald; Gelb, Michael H.; Stoddard, Barry L.; Houk, Kendall N.; Michael, Forrest E.; Baker, DavidScience (Washington, DC, United States) (2010), 329 (5989), 309-313CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The Diels-Alder reaction is a cornerstone in org. synthesis, forming two carbon-carbon bonds and up to four new stereogenic centers in one step. No naturally occurring enzymes have been shown to catalyze bimol. Diels-Alder reactions. We describe the de novo computational design and exptl. characterization of enzymes catalyzing a bimol. Diels-Alder reaction with high stereoselectivity and substrate specificity. X-ray crystallog. confirms that the structure matches the design for the most active of the enzymes, and binding site substitutions reprogram the substrate specificity. Designed stereoselective catalysts for carbon-carbon bond-forming reactions should be broadly useful in synthetic chem.
- 508Sun, N.; Huang, J.; Qian, J.; Zhou, T.-P.; Guo, J.; Tang, L.; Zhang, W.; Deng, Y.; Zhao, W.; Wu, G. Enantioselective [2 + 2]-Cycloadditions with Triplet Photoenzymes. Nature 2022, 611 (7937), 715– 720, DOI: 10.1038/s41586-022-05342-4508Enantioselective [2+2]-cycloadditions with triplet photoenzymesSun, Ningning; Huang, Jianjian; Qian, Junyi; Zhou, Tai-Ping; Guo, Juan; Tang, Langyu; Zhang, Wentao; Deng, Yaming; Zhao, Weining; Wu, Guojiao; Liao, Rong-Zhen; Chen, Xi; Zhong, Fangrui; Wu, YuzhouNature (London, United Kingdom) (2022), 611 (7937), 715-720CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)Naturally evolved enzymes, despite their astonishingly large variety and functional diversity, operate predominantly through thermochem. activation. Integrating prominent photocatalysis modes into proteins, such as triplet energy transfer, could create artificial photoenzymes that expand the scope of natural biocatalysis1-3. Here, we exploit genetically reprogrammed, chem. evolved photoenzymes embedded with a synthetic triplet photosensitizer that are capable of excited-state enantio-induction4-6. Structural optimization through four rounds of directed evolution afforded proficient variants for the enantioselective intramol. [2+2]-photocycloaddn. of indole derivs. with good substrate generality and excellent enantioselectivities (up to 99% enantiomeric excess). A crystal structure of the photoenzyme-substrate complex elucidated the non-covalent interactions that mediate the reaction stereochem. This study expands the energy transfer reactivity7-10 of artificial triplet photoenzymes in a supramol. protein cavity and unlocks an integrated approach to valuable enantioselective photochem. synthesis that is not accessible with either the synthetic or the biol. world alone.
- 509Allen, A. R.; Noten, E. A.; Stephenson, C. R. J. Aryl Transfer Strategies Mediated by Photoinduced Electron Transfer. Chem. Rev. 2022, 122 (2), 2695– 2751, DOI: 10.1021/acs.chemrev.1c00388509Aryl Transfer Strategies Mediated by Photoinduced Electron TransferAllen, Anthony R.; Noten, Efrey A.; Stephenson, Corey R. J.Chemical Reviews (Washington, DC, United States) (2022), 122 (2), 2695-2751CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. This review encapsulates progress in radical aryl migration enabled by photochem. methods-particularly photoredox catalysis-since 2015. Special attention is paid to descriptions of scope, mechanism, and synthetic applications of each method.
- 510Dadashi-Silab, S.; Doran, S.; Yagci, Y. Photoinduced Electron Transfer Reactions for Macromolecular Syntheses. Chem. Rev. 2016, 116 (17), 10212– 10275, DOI: 10.1021/acs.chemrev.5b00586510Photoinduced Electron Transfer Reactions for Macromolecular SynthesesDadashi-Silab, Sajjad; Doran, Sean; Yagci, YusufChemical Reviews (Washington, DC, United States) (2016), 116 (17), 10212-10275CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)Photochem. reactions, particularly those involving photoinduced electron transfer processes, establish a substantial contribution to the modern synthetic chem., and the polymer community has been increasingly interested in exploiting and developing novel photochem. strategies. These reactions are efficiently utilized in almost every aspect of macromol. architecture synthesis, involving initiation, control of the reaction kinetics and mol. structures, functionalization, and decoration, etc. Merging with polymn. techniques, photochem. has opened up new intriguing and powerful avenues for macromol. synthesis. Construction of various polymers with incredibly complex structures and specific control over the chain topol., as well as providing the opportunity to manipulate the reaction course through spatiotemporal control, are one of the unique abilities of such photochem. reactions. This review paper provides a comprehensive account of the fundamentals and applications of photoinduced electron transfer reactions in polymer synthesis. Besides traditional photopolymn. methods, namely free radical and cationic polymns., step-growth polymns. involving electron transfer processes are included. In addn., controlled radical polymn. and "Click Chem." methods have significantly evolved over the last few decades allowing access to narrow mol. wt. distributions, efficient regulation of the mol. wt. and the monomer sequence and incredibly complex architectures, and polymer modifications and surface patterning are covered. Potential applications including synthesis of block and graft copolymers, polymer-metal nanocomposites, various hybrid materials and bioconjugates, and sequence defined polymers through photoinduced electron transfer reactions are also investigated in detail.
- 511Reid, B. G.; Flynn, G. C. Chromophore Formation in Green Fluorescent Protein. Biochemistry 1997, 36 (22), 6786– 6791, DOI: 10.1021/bi970281w511Chromophore formation in green fluorescent proteinReid, Brian G.; Flynn, Gregory C.Biochemistry (1997), 36 (22), 6786-6791CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)The green fluorescent protein (GFP) from the jellyfish Aequorea victoria forms an intrinsic chromophore through cyclization and oxidn. of an internal tripeptide motif. Here, the authors monitored the formation of the chromophore in vitro using the S65T-GFP chromophore mutant. S65T-GFP recovered from inclusion bodies in Escherichia coli lacked the mature chromophore, suggesting that protein destined for inclusion bodies aggregated prior to productive folding. This material was used to follow the steps leading to chromophore formation. The process of chromophore formation in S65T-GFP was detd. to be an ordered reaction consisting of 3 distinct kinetic steps. Protein folding occurred fairly slowly (kf = 2.44 × 10-3 s-1) and prior to any chromophore modification. Next, an intermediate step occurred that included, but was not necessarily limited to, cyclization of the tripeptide chromophore motif (kc = 3.8 × 10-3 s-1). The final and slow step (kox = 1.51 × 10-4 s-1) in chromophore formation involved oxidn. of the cyclized chromophore. Since the chromophore formed de novo from purified denatured protein which was a 1st-order process, it was conclude that GFP chromophore formation is an autocatalytic process.
- 512Fu, Y.; Huang, J.; Wu, Y.; Liu, X.; Zhong, F.; Wang, J. Biocatalytic Cross-Coupling of Aryl Halides with a Genetically Engineered Photosensitizer Artificial Dehalogenase. J. Am. Chem. Soc. 2021, 143 (2), 617– 622, DOI: 10.1021/jacs.0c10882There is no corresponding record for this reference.
- 513Gu, Y.; Ellis-Guardiola, K.; Srivastava, P.; Lewis, J. C. Preparation, Characterization, and Oxygenase Activity of a Photocatalytic Artificial Enzyme. ChemBioChem 2015, 16 (13), 1880– 1883, DOI: 10.1002/cbic.201500165There is no corresponding record for this reference.
- 514Zubi, Y. S.; Liu, B.; Gu, Y.; Sahoo, D.; Lewis, J. C. Controlling the Optical and Catalytic Properties of Artificial Metalloenzyme Photocatalysts Using Chemogenetic Engineering. Chem. Sci. 2022, 13 (5), 1459– 1468, DOI: 10.1039/D1SC05792HThere is no corresponding record for this reference.
- 515Liu, B.; Zubi, Y. S.; Lewis, J. C. Iridium(iii) Polypyridine Artificial Metalloenzymes with Tunable Photophysical Properties: A New Platform for Visible Light Photocatalysis in Aqueous Solution. Dalton Trans. 2023, 52 (16), 5034– 5038, DOI: 10.1039/D3DT00932GThere is no corresponding record for this reference.
- 516Lee, J.; Song, W. J. Photocatalytic C-O Coupling Enzymes That Operate via Intramolecular Electron Transfer. J. Am. Chem. Soc. 2023, 145 (9), 5211– 5221, DOI: 10.1021/jacs.2c12226There is no corresponding record for this reference.
- 517Mills, J. H.; Sheffler, W.; Ener, M. E.; Almhjell, P. J.; Oberdorfer, G.; Pereira, J. H.; Parmeggiani, F.; Sankaran, B.; Zwart, P. H.; Baker, D. Computational Design of a Homotrimeric Metalloprotein with a Trisbipyridyl Core. Proc. Natl. Acad. Sci. U.S.A. 2016, 113 (52), 15012– 15017, DOI: 10.1073/pnas.1600188113517Computational design of a homotrimeric metalloprotein with a trisbipyridyl coreMills, Jeremy H.; Sheffler, William; Ener, Maraia E.; Almhjell, Patrick J.; Oberdorfer, Gustav; Pereira, Jose Henrique; Parmeggiani, Fabio; Sankaran, Banumathi; Zwart, Peter H.; Baker, DavidProceedings of the National Academy of Sciences of the United States of America (2016), 113 (52), 15012-15017CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Metal-chelating heteroaryl small mols. have found widespread use as building blocks for coordination-driven, self-assembling nanostructures. The metal-chelating noncanonical amino acid (2,2'-bipyridin-5yl)alanine (Bpy-ala) could, in principle, be used to nucleate specific metalloprotein assemblies if introduced into proteins such that one assembly had much lower free energy than all alternatives. Here we describe the use of the Rosetta computational methodol. to design a self-assembling homotrimeric protein with [Fe(Bpy-ala)3]2+ complexes at the interface between monomers. X-ray crystallog. anal. of the homotrimer showed that the design process had near-at.-level accuracy: The all-atom rmsd between the design model and crystal structure for the residues at the protein interface is ∼1.4 Å. These results demonstrate that computational protein design together with genetically encoded noncanonical amino acids can be used to drive formation of precisely specified metal-mediated protein assemblies that could find use in a wide range of photophys. applications.
- 518Duan, H.-Z.; Hu, C.; Li, Y.-L.; Wang, S.-H.; Xia, Y.; Liu, X.; Wang, J.; Chen, Y.-X. Genetically Encoded Phosphine Ligand for Metalloprotein Design. J. Am. Chem. Soc. 2022, 144 (50), 22831– 22837, DOI: 10.1021/jacs.2c09683There is no corresponding record for this reference.
- 519Beattie, A. T.; Dunkelmann, D. L.; Chin, J. W. Quintuply Orthogonal Pyrrolysyl-tRNA Synthetase/tRNAPyl Pairs. Nat. Chem. 2023, 15 (7), 948– 959, DOI: 10.1038/s41557-023-01232-yThere is no corresponding record for this reference.
- 520Dunkelmann, D. L.; Willis, J. C. W.; Beattie, A. T.; Chin, J. W. Engineered Triply Orthogonal Pyrrolysyl-tRNA Synthetase/tRNA Pairs Enable the Genetic Encoding of Three Distinct Non-Canonical Amino Acids. Nat. Chem. 2020, 12 (6), 535– 544, DOI: 10.1038/s41557-020-0472-x520Engineered triply orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs enable the genetic encoding of three distinct non-canonical amino acidsDunkelmann, Daniel L.; Willis, Julian C. W.; Beattie, Adam T.; Chin, Jason W.Nature Chemistry (2020), 12 (6), 535-544CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)Expanding and reprogramming the genetic code of cells for the incorporation of multiple distinct non-canonical amino acids (ncAAs), and the encoded biosynthesis of non-canonical biopolymers, requires the discovery of multiple orthogonal aminoacyl-tRNA synthetase/tRNA pairs. These pairs must be orthogonal to both the host synthetases and tRNAs and to each other. Pyrrolysyl-tRNA synthetase (PylRS)/PyltRNA pairs are the most widely used system for genetic code expansion. Here, we reveal that the sequences of ΔNPylRS/ΔNPyltRNA pairs (which lack N-terminal domains) form two distinct classes. We show that the measured specificities of the ΔNPylRSs and ΔNPyltRNAs correlate with sequence-based clustering, and most ΔNPylRSs preferentially function with ΔNPyltRNAs from their class. We then identify 18 mutually orthogonal pairs from the 88 ΔNPylRS/ΔNPyltRNA combinations tested. Moreover, we generate a set of 12 triply orthogonal pairs, each composed of three new PylRS/PyltRNA pairs. Finally, we diverge the ncAA specificity and decoding properties of each pair, within a triply orthogonal set, and direct the incorporation of three distinct non-canonical amino acids into a single polypeptide.
- 521Italia, J. S.; Addy, P. S.; Erickson, S. B.; Peeler, J. C.; Weerapana, E.; Chatterjee, A. Mutually Orthogonal Nonsense-Suppression Systems and Conjugation Chemistries for Precise Protein Labeling at up to Three Distinct Sites. J. Am. Chem. Soc. 2019, 141 (15), 6204– 6212, DOI: 10.1021/jacs.8b12954521Mutually Orthogonal Nonsense-Suppression Systems and Conjugation Chemistries for Precise Protein Labeling at up to Three Distinct SitesItalia, James S.; Addy, Partha Sarathi; Erickson, Sarah B.; Peeler, Jennifer C.; Weerapana, Eranthie; Chatterjee, AbhishekJournal of the American Chemical Society (2019), 141 (15), 6204-6212CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Site-specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into a protein is an emerging technol. with tremendous potential. It relies on mutually orthogonal engineered aminoacyl-tRNA synthetase/tRNA pairs that suppress different nonsense/frameshift codons. So far, up to two distinct ncAAs have been incorporated into proteins expressed in E. coli, using archaea-derived tyrosyl and pyrrolysyl pairs. Here we report that the E. coli derived tryptophanyl pair can be combined with the archaeal tyrosyl or the pyrrolysyl pair in ATMW1 E. coli to incorporate two different ncAAs into one protein with high fidelity and efficiency. By combining all three orthogonal pairs, we further demonstrate simultaneous site-specific incorporation of three different ncAAs into one protein. To use this technol. for chemoselectively labeling proteins with multiple distinct entities at predefined sites, we also sought to identify different bioconjugation handles that can be coincorporated into proteins as ncAA-side chains and subsequently functionalized through mutually compatible labeling chemistries. To this end, we show that the recently developed chemoselective rapid azo-coupling reaction (CRACR) directed to 5-hydroxytryptophan (5HTP) is compatible with strain-promoted azide-alkyne cycloaddn. (SPAAC) targeted to p-azidophenylalanine (pAzF) and strain-promoted inverse electron-demand Diels-Alder cycloaddn. (SPIEDAC) targeted to cyclopropene-lysine (CpK) for rapid, catalyst-free protein labeling at multiple sites. Combining these mutually orthogonal nonsense suppression systems and the mutually compatible bioconjugation handles they incorporate, we demonstrate site-specific labeling of recombinantly expressed proteins at up to three distinct sites.
- 522Hashimoto, K.; Fischer, E. C.; Romesberg, F. E. Efforts toward Further Integration of an Unnatural Base Pair into the Biology of a Semisynthetic Organism. J. Am. Chem. Soc. 2021, 143 (23), 8603– 8607, DOI: 10.1021/jacs.1c03860There is no corresponding record for this reference.
- 523Ledbetter, M. P.; Karadeema, R. J.; Romesberg, F. E. Reprograming the Replisome of a Semisynthetic Organism for the Expansion of the Genetic Alphabet. J. Am. Chem. Soc. 2018, 140 (2), 758– 765, DOI: 10.1021/jacs.7b11488523Reprograming the Replisome of a Semisynthetic Organism for the Expansion of the Genetic AlphabetLedbetter, Michael P.; Karadeema, Rebekah J.; Romesberg, Floyd E.Journal of the American Chemical Society (2018), 140 (2), 758-765CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Semi-synthetic organisms (SSOs) created from Escherichia coli can replicate a plasmid contg. an unnatural base pair (UBP) formed between the synthetic nucleosides dNaM and dTPT3 (dNaM-dTPT3) when the corresponding unnatural triphosphates are imported via expression of a nucleoside triphosphate transporter. The UBP can also be transcribed and used to translate proteins contg. unnatural amino acids. However, UBPs are not well retained in all sequences, limiting the information that can be encoded, and are invariably lost upon extended growth. Here we explore the contributions of the E. coli DNA replication and repair machinery to the propagation of DNA contg. dNaM-dTPT3 and show that replication by DNA polymerase III, supplemented with the activity of polymerase II and methyl-directed mismatch repair contribute to retention of the UBP and that recombinational repair of stalled forks is responsible for the majority of its loss. This work elucidates fundamental aspects of how bacteria replicate DNA and we use this information to reprogram the replisome of the SSO for increased UBP retention, which then allowed for the first time the construction of SSOs harboring a UBP in their chromosome.
- 524Stucki, A.; Vallapurackal, J.; Ward, T. R.; Dittrich, P. S. Droplet Microfluidics and Directed Evolution of Enzymes: An Intertwined Journey. Angew. Chem. Int. Ed. 2021, 60 (46), 24368– 24387, DOI: 10.1002/anie.202016154524Droplet Microfluidics and Directed Evolution of Enzymes: An Intertwined JourneyStucki, Ariane; Vallapurackal, Jaicy; Ward, Thomas R.; Dittrich, Petra S.Angewandte Chemie, International Edition (2021), 60 (46), 24368-24387CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Evolution is essential to the generation of complexity and ultimately life. It relies on the propagation of the properties, traits, and characteristics that allow an organism to survive in a challenging environment. It is evolution that shaped our world over about four billion years by slow and iterative adaptation. While natural evolution based on selection is slow and gradual, directed evolution allows the fast and streamlined optimization of a phenotype under selective conditions. The potential of directed evolution for the discovery and optimization of enzymes is mostly limited by the throughput of the tools and methods available for screening. Over the past twenty years, versatile tools based on droplet microfluidics have been developed to address the need for higher throughput. In this Review, we provide a chronol. overview of the intertwined development of microfluidics droplet-based compartmentalization methods and in vivo directed evolution of enzymes.
- 525Wicky, B. I. M.; Milles, L. F.; Courbet, A.; Ragotte, R. J.; Dauparas, J.; Kinfu, E.; Tipps, S.; Kibler, R. D.; Baek, M.; DiMaio, F. Hallucinating Symmetric Protein Assemblies. Science 2022, 378 (6615), 56– 61, DOI: 10.1126/science.add1964525Hallucinating symmetric protein assembliesWicky, B. I. M.; Milles, L. F.; Courbet, A.; Ragotte, R. J.; Dauparas, J.; Kinfu, E.; Tipps, S.; Kibler, R. D.; Baek, M.; DiMaio, F.; Li, X.; Carter, L.; Kang, A.; Nguyen, H.; Bera, A. K.; Baker, D.Science (Washington, DC, United States) (2022), 378 (6615), 56-61CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Deep learning generative approaches provide an opportunity to broadly explore protein structure space beyond the sequences and structures of natural proteins. Here, we use deep network hallucination to generate a wide range of sym. protein homo-oligomers given only a specification of the no. of protomers and the protomer length. Crystal structures of seven designs are very similar to the computational models (median root mean square deviation: 0.6 angstroms), as are three cryo-electron microscopy structures of giant 10-nm rings with up to 1550 residues and C33 symmetry; all differ considerably from previously solved structures. Our results highlight the rich diversity of new protein structures that can be generated using deep learning and pave the way for the design of increasingly complex components for nanomachines and biomaterials.
- 526Wang, J.; Lisanza, S.; Juergens, D.; Tischer, D.; Watson, J. L.; Castro, K. M.; Ragotte, R.; Saragovi, A.; Milles, L. F.; Baek, M. Scaffolding Protein Functional Sites Using Deep Learning. Science 2022, 377 (6604), 387– 394, DOI: 10.1126/science.abn2100526Scaffolding protein functional sites using deep learningWang, Jue; Lisanza, Sidney; Juergens, David; Tischer, Doug; Watson, Joseph L.; Castro, Karla M.; Ragotte, Robert; Saragovi, Amijai; Milles, Lukas F.; Baek, Minkyung; Anishchenko, Ivan; Yang, Wei; Hicks, Derrick R.; Exposit, Marc; Schlichthaerle, Thomas; Chun, Jung-Ho; Dauparas, Justas; Bennett, Nathaniel; Wicky, Basile I. M.; Muenks, Andrew; DiMaio, Frank; Correia, Bruno; Ovchinnikov, Sergey; Baker, DavidScience (Washington, DC, United States) (2022), 377 (6604), 387-394CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)A review. The binding and catalytic functions of proteins are generally mediated by a small no. of functional residues held in place by the overall protein structure. Here, we describe deep learning approaches for scaffolding such functional sites without needing to prespecify the fold or secondary structure of the scaffold. The first approach, "constrained hallucination," optimizes sequences such that their predicted structures contain the desired functional site. The second approach, "inpainting," starts from the functional site and fills in addnl. sequence and structure to create a viable protein scaffold in a single forward pass through a specifically trained RoseTTAFold network. We use these two methods to design candidate immunogens, receptor traps, metalloproteins, enzymes, and protein-binding proteins and validate the designs using a combination of in silico and exptl. tests.
- 527Krishna, R.; Wang, J.; Ahern, W.; Sturmfels, P.; Venkatesh, P.; Kalvet, I.; Lee, G. R.; Morey-Burros, F. S.; Anishchenko, I.; Humphreys, I. R. Generalized Biomolecular Modeling and Design with RoseTTAFold All-Atom. Science 2024, DOI: 10.1126/science.adl2528There is no corresponding record for this reference.
- 528Jumper, J.; Evans, R.; Pritzel, A.; Green, T.; Figurnov, M.; Ronneberger, O.; Tunyasuvunakool, K.; Bates, R.; Žídek, A.; Potapenko, A. Highly Accurate Protein Structure Prediction with AlphaFold. Nature 2021, 596 (7873), 583– 589, DOI: 10.1038/s41586-021-03819-2528Highly accurate protein structure prediction with AlphaFoldJumper, John; Evans, Richard; Pritzel, Alexander; Green, Tim; Figurnov, Michael; Ronneberger, Olaf; Tunyasuvunakool, Kathryn; Bates, Russ; Zidek, Augustin; Potapenko, Anna; Bridgland, Alex; Meyer, Clemens; Kohl, Simon A. A.; Ballard, Andrew J.; Cowie, Andrew; Romera-Paredes, Bernardino; Nikolov, Stanislav; Jain, Rishub; Adler, Jonas; Back, Trevor; Petersen, Stig; Reiman, David; Clancy, Ellen; Zielinski, Michal; Steinegger, Martin; Pacholska, Michalina; Berghammer, Tamas; Bodenstein, Sebastian; Silver, David; Vinyals, Oriol; Senior, Andrew W.; Kavukcuoglu, Koray; Kohli, Pushmeet; Hassabis, DemisNature (London, United Kingdom) (2021), 596 (7873), 583-589CODEN: NATUAS; ISSN:0028-0836. (Nature Portfolio)Proteins are essential to life, and understanding their structure can facilitate a mechanistic understanding of their function. Through an enormous exptl. effort, the structures of around 100,000 unique proteins have been detd., but this represents a small fraction of the billions of known protein sequences. Structural coverage is bottlenecked by the months to years of painstaking effort required to det. a single protein structure. Accurate computational approaches are needed to address this gap and to enable large-scale structural bioinformatics. Predicting the three-dimensional structure that a protein will adopt based solely on its amino acid sequence-the structure prediction component of the 'protein folding problem'-has been an important open research problem for more than 50 years. Despite recent progress, existing methods fall far short of at. accuracy, esp. when no homologous structure is available. Here we provide the first computational method that can regularly predict protein structures with at. accuracy even in cases in which no similar structure is known. We validated an entirely redesigned version of our neural network-based model, AlphaFold, in the challenging 14th Crit. Assessment of protein Structure Prediction (CASP14), demonstrating accuracy competitive with exptl. structures in a majority of cases and greatly outperforming other methods. Underpinning the latest version of AlphaFold is a novel machine learning approach that incorporates phys. and biol. knowledge about protein structure, leveraging multi-sequence alignments, into the design of the deep learning algorithm.
- 529Baumann, T.; Hauf, M.; Richter, F.; Albers, S.; Möglich, A.; Ignatova, Z.; Budisa, N. Computational Aminoacyl-tRNA Synthetase Library Design for Photocaged Tyrosine. Int. J. Mol. Sci. 2019, 20 (9), 2343, DOI: 10.3390/ijms20092343There is no corresponding record for this reference.
- 530Cervettini, D.; Tang, S.; Fried, S. D.; Willis, J. C. W.; Funke, L. F. H.; Colwell, L. J.; Chin, J. W. Rapid Discovery and Evolution of Orthogonal Aminoacyl-tRNA Synthetase-tRNA Pairs. Nat. Biotechnol. 2020, 38 (8), 989– 999, DOI: 10.1038/s41587-020-0479-2530Rapid discovery and evolution of orthogonal aminoacyl-tRNA synthetase-tRNA pairsCervettini, Daniele; Tang, Shan; Fried, Stephen D.; Willis, Julian C. W.; Funke, Louise F. H.; Colwell, Lucy J.; Chin, Jason W.Nature Biotechnology (2020), 38 (8), 989-999CODEN: NABIF9; ISSN:1087-0156. (Nature Research)Abstr.: A central challenge in expanding the genetic code of cells to incorporate noncanonical amino acids into proteins is the scalable discovery of aminoacyl-tRNA synthetase (aaRS)-tRNA pairs that are orthogonal in their aminoacylation specificity. Here we computationally identify candidate orthogonal tRNAs from millions of sequences and develop a rapid, scalable approach-named tRNA Extension (tREX)-to det. the in vivo aminoacylation status of tRNAs. Using tREX, we test 243 candidate tRNAs in Escherichia coli and identify 71 orthogonal tRNAs, covering 16 isoacceptor classes, and 23 functional orthogonal tRNA-cognate aaRS pairs. We discover five orthogonal pairs, including three highly active amber suppressors, and evolve new amino acid substrate specificities for two pairs. Finally, we use tREX to characterize a matrix of 64 orthogonal synthetase-orthogonal tRNA specificities. This work expands the no. of orthogonal pairs available for genetic code expansion and provides a pipeline for the discovery of addnl. orthogonal pairs and a foundation for encoding the cellular synthesis of noncanonical biopolymers.
- 531Taylor, C. J.; Hardy, F. J.; Burke, A. J.; Bednar, R. M.; Mehl, R. A.; Green, A. P.; Lovelock, S. L. Engineering Mutually Orthogonal PylRS/tRNA Pairs for Dual Encoding of Functional Histidine Analogues. Protein Sci. 2023, 32 (5), e4640 DOI: 10.1002/pro.4640There is no corresponding record for this reference.
- 532Fredens, J.; Wang, K.; de la Torre, D.; Funke, L. F. H.; Robertson, W. E.; Christova, Y.; Chia, T.; Schmied, W. H.; Dunkelmann, D. L.; Beránek, V. Total Synthesis of Escherichia coli with a Recoded Genome. Nature 2019, 569 (7757), 514– 518, DOI: 10.1038/s41586-019-1192-5532Total synthesis of Escherichia coli with a recoded genomeFredens, Julius; Wang, Kaihang; de la Torre, Daniel; Funke, Louise F. H.; Robertson, Wesley E.; Christova, Yonka; Chia, Tiongsun; Schmied, Wolfgang H.; Dunkelmann, Daniel L.; Beranek, Vaclav; Uttamapinant, Chayasith; Llamazares, Andres Gonzalez; Elliott, Thomas S.; Chin, Jason W.Nature (London, United Kingdom) (2019), 569 (7757), 514-518CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon-out of up to 6 synonyms-to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the no. of codons used to encode the canonical amino acids can be reduced, through the genome-wide substitution of target codons by defined synonyms. We create a variant of Escherichia coli with a four-megabase synthetic genome through a high-fidelity convergent total synthesis. Our synthetic genome implements a defined recoding and refactoring scheme-with simple corrections at just seven positions-to replace every known occurrence of two sense codons and a stop codon in the genome. Thus, we recode 18,214 codons to create an organism with a 61-codon genome; this organism uses 59 codons to encode the 20 amino acids, and enables the deletion of a previously essential tRNA.
- 533Lajoie, M. J.; Rovner, A. J.; Goodman, D. B.; Aerni, H.-R.; Haimovich, A. D.; Kuznetsov, G.; Mercer, J. A.; Wang, H. H.; Carr, P. A.; Mosberg, J. A. Genomically Recoded Organisms Expand Biological Functions. Science 2013, 342 (6156), 357– 360, DOI: 10.1126/science.1241459533Genomically recoded organisms expand biological functionsLajoie, Marc J.; Rovner, Alexis J.; Goodman, Daniel B.; Aerni, Hans-Rudolf; Haimovich, Adrian D.; Kuznetsov, Gleb; Mercer, Jaron A.; Wang, Harris H.; Carr, Peter A.; Mosberg, Joshua A.; Rohland, Nadin; Schultz, Peter G.; Jacobson, Joseph M.; Rinehart, Jesse; Church, George M.; Isaacs, Farren J.Science (Washington, DC, United States) (2013), 342 (6156), 357-360CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)We describe the construction and characterization of a genomically recoded organism (GRO). We replaced all known UAG stop codons in Escherichia coli MG1655 with synonymous UAA codons, which permitted the deletion of release factor 1 and reassignment of UAG translation function. This GRO exhibited improved properties for incorporation of nonstandard amino acids that expand the chem. diversity of proteins in vivo. The GRO also exhibited increased resistance to T7 bacteriophage, demonstrating that new genetic codes could enable increased viral resistance.
- 534Mukai, T.; Hoshi, H.; Ohtake, K.; Takahashi, M.; Yamaguchi, A.; Hayashi, A.; Yokoyama, S.; Sakamoto, K. Highly Reproductive Escherichia coli Cells with No Specific Assignment to the UAG Codon. Sci. Rep. 2015, 5 (1), 9699, DOI: 10.1038/srep09699534Highly reproductive Escherichia coli cells with no specific assignment to the UAG codonMukai, Takahito; Hoshi, Hiroko; Ohtake, Kazumasa; Takahashi, Mihoko; Yamaguchi, Atsushi; Hayashi, Akiko; Yokoyama, Shigeyuki; Sakamoto, KensakuScientific Reports (2015), 5 (), 9699CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)Escherichia coli is a widely used host organism for recombinant technol., and the bacterial incorporation of non-natural amino acids promises the efficient synthesis of proteins with novel structures and properties. In the present study, we developed E. coli strains in which the UAG codon was reserved for non-natural amino acids, without compromising the reproductive strength of the host cells. Ninety-five of the 273 UAG stop codons were replaced synonymously in the genome of E. coli BL21(DE3), by exploiting the oligonucleotide-mediated base-mismatch-repair mechanism. This genomic modification allowed the safe elimination of the UAG-recognizing cellular component (RF-1), thus leaving the remaining 178 UAG codons with no specific mol. recognizing them. The resulting strain B-95.ΔA grew as vigorously as BL21(DE3) in rich medium at 25-42°C, and its deriv. B-95.ΔAΔfabR was better adapted to low temps. and minimal media than B-95.ΔA. UAG was reassigned to synthetic amino acids by expressing the specific pairs of UAG-reading tRNA and aminoacyl-tRNA synthetase. Due to the preserved growth vigor, the B-95.ΔA strains showed superior productivities for hirudin mols. sulfonated on a particular tyrosine residue, and the Fab fragments of Herceptin contg. multiple azido groups.
- 535Wang, T.; Liang, C.; An, Y.; Xiao, S.; Xu, H.; Zheng, M.; Liu, L.; Wang, G.; Nie, L. Engineering the Translational Machinery for Biotechnology Applications. Mol. Biotechnol. 2020, 62 (4), 219– 227, DOI: 10.1007/s12033-020-00246-yThere is no corresponding record for this reference.
- 536Zackin, M. T.; Stieglitz, J. T.; Van Deventer, J. A. Genome-Wide Screen for Enhanced Noncanonical Amino Acid Incorporation in Yeast. ACS Synth. Biol. 2022, 11 (11), 3669– 3680, DOI: 10.1021/acssynbio.2c00267There is no corresponding record for this reference.
- 537Exner, M. P.; Kuenzl, T.; To, T. M. T.; Ouyang, Z.; Schwagerus, S.; Hoesl, M. G.; Hackenberger, C. P. R.; Lensen, M. C.; Panke, S.; Budisa, N. Design of S-Allylcysteine in Situ Production and Incorporation Based on a Novel Pyrrolysyl-tRNA Synthetase Variant. ChemBioChem 2017, 18 (1), 85– 90, DOI: 10.1002/cbic.201600537There is no corresponding record for this reference.
- 538Kim, S.; Sung, B. H.; Kim, S. C.; Lee, H. S. Genetic Incorporation of L-Dihydroxyphenylalanine (DOPA) Biosynthesized by a Tyrosine Phenol-Lyase. Chem. Commun. 2018, 54 (24), 3002– 3005, DOI: 10.1039/C8CC00281AThere is no corresponding record for this reference.
- 539Nojoumi, S.; Ma, Y.; Schwagerus, S.; Hackenberger, C. P. R.; Budisa, N. In-Cell Synthesis of Bioorthogonal Alkene Tag S-Allyl-Homocysteine and Its Coupling with Reprogrammed Translation. Int. J. Mol. Sci. 2019, 20 (9), 2299, DOI: 10.3390/ijms20092299There is no corresponding record for this reference.