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Structure of a Minimal α-Carboxysome-Derived Shell and Its Utility in Enzyme Stabilization
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Structure of a Minimal α-Carboxysome-Derived Shell and Its Utility in Enzyme Stabilization
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  • Yong Quan Tan
    Yong Quan Tan
    Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore (NUS), 8 Medical Drive, Singapore 117597
    NUS Synthetic Biology for Clinical and Technological Innovation, 28 Medical Drive, Singapore 117456
    Graduate School for Integrative Sciences and Engineering, NUS, Singapore 119077
    More by Yong Quan Tan
    Orcidhttps://orcid.org/0000-0002-6497-7756
  • Samson Ali
    Samson Ali
    Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
    Research Institute for Interdisciplinary Science (RIIS), Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan
    More by Samson Ali
  • Bo Xue
    Bo Xue
    Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore (NUS), 8 Medical Drive, Singapore 117597
    NUS Synthetic Biology for Clinical and Technological Innovation, 28 Medical Drive, Singapore 117456
    Synthetic Biology Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, Singapore 117599
    More by Bo Xue
  • Wei Zhe Teo
    Wei Zhe Teo
    Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore (NUS), 8 Medical Drive, Singapore 117597
    NUS Synthetic Biology for Clinical and Technological Innovation, 28 Medical Drive, Singapore 117456
    Synthetic Biology Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, Singapore 117599
    More by Wei Zhe Teo
  • Lay Hiang Ling
    Lay Hiang Ling
    Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore (NUS), 8 Medical Drive, Singapore 117597
    NUS Synthetic Biology for Clinical and Technological Innovation, 28 Medical Drive, Singapore 117456
    Graduate School for Integrative Sciences and Engineering, NUS, Singapore 119077
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  • Maybelle Kho Go
    Maybelle Kho Go
    Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore (NUS), 8 Medical Drive, Singapore 117597
    NUS Synthetic Biology for Clinical and Technological Innovation, 28 Medical Drive, Singapore 117456
    Synthetic Biology Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, Singapore 117599
    More by Maybelle Kho Go
  • Hong Lv
    Hong Lv
    Shanghai Engineering Research Center of Industrial Microorganisms, Shanghai 200438, People’s Republic of China
    State Key Laboratory of Genetic Engineering, School of Life Science, Fudan University, Shanghai 200438, People’s Republic of China
    More by Hong Lv
  • Robert C. Robinson
    Robert C. Robinson
    Research Institute for Interdisciplinary Science (RIIS), Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan
    School of Biomolecular Science and Engineering (BSE), Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong 21210, Thailand
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  • Akihiro Narita
    Akihiro Narita
    Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
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  • Wen Shan Yew*
    Wen Shan Yew
    Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore (NUS), 8 Medical Drive, Singapore 117597
    NUS Synthetic Biology for Clinical and Technological Innovation, 28 Medical Drive, Singapore 117456
    Graduate School for Integrative Sciences and Engineering, NUS, Singapore 119077
    Synthetic Biology Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, Singapore 117599
    *Email: [email protected]
    More by Wen Shan Yew
    Orcidhttps://orcid.org/0000-0002-3021-0469
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Biomacromolecules

Cite this: Biomacromolecules 2021, 22, 10, 4095–4109
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https://doi.org/10.1021/acs.biomac.1c00533
Published August 12, 2021

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Abstract

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Bacterial microcompartments are proteinaceous shells that encase specialized metabolic processes in bacteria. Recent advances in simplification of these intricate shells have encouraged bioengineering efforts. Here, we construct minimal shells derived from the Halothiobacillus neapolitanus α-carboxysome, which we term Cso-shell. Using cryogenic electron microscopy, the atomic-level structures of two shell forms were obtained, reinforcing notions of evolutionarily conserved features in bacterial microcompartment shell architecture. Encapsulation peptide sequences that facilitate loading of heterologous protein cargo within the shells were identified. We further provide a first demonstration in utilizing minimal bacterial microcompartment-derived shells for hosting heterologous enzymes. Cso-shells were found to stabilize enzymatic activities against heat shock, presence of methanol co-solvent, consecutive freeze–thawing, and alkaline environments. This study yields insights into α-carboxysome assembly and advances the utility of synthetic bacterial microcompartments as nanoreactors capable of stabilizing enzymes with varied properties and reaction chemistries.

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Introduction

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Bacterial microcompartments (BMCs) are protein shells found in some bacterial species that compartmentalize specialized biochemical reactions. (1) The carboxysome, found in cyanobacteria and some chemotrophic bacterial species, is the earliest known example of a BMC. (2,3) This semipermeable shell encapsulates ribulose-1,5-bisphosphate carboxylase (RuBisCO) and improves its catalytic efficiency by concentrating CO2 close to RuBisCO. (4,5) Carboxysomes are classified into two main groups depending on the class of RuBisCO that is encapsulated. α-Carboxysomes encase Form 1A RuBisCO that is found in α-cyanobacteria and chemoautotrophs, while β-carboxysomes house Form 1B RuBisCO that are observed in β-cyanobacteria. (2)
Numerous atomic-scale structures of the subunits that comprise BMC shells, along with that of three minimal BMC-derived shells (Table 1), have been reported. (6−10) Despite the diversity in the overall morphologies and functions of BMCs, the tertiary structures of the shell subunits are conserved. (11) The BMC-P (or BMV) protein is a minor but important subunit in a BMC shell that occupies the shell vertices as homopentamers, giving rise to the polyhedral morphology distinctive of BMCs. (12) The BMC-H domain protein is the stoichiometrically major subunit and assembles as a homohexamer. The BMC-P and BMC-H domains are structurally and evolutionarily distinct from each other and belong to two separate protein families, Pfam03319 and Pfam00936, respectively. (12) The BMC-T protein is formed by a tandem repeat of two BMC-H domains and assembles as a single or double stack of trimers, giving rise to a pseudo-hexameric appearance. BMC-H and BMC-T subunits constitute the shell facets and influence the diffusion of small molecules across the shell membrane. (1) Detailed understanding of the architecture of BMC components has been instrumental for BMC engineering toward synthetic biology applications. (13,14) Such endeavors include targeting heterologous cargo into the shell via the use of encapsulation peptides, which are peptides derived from cognate luminal proteins or by installation of peptide conjugation moieties into shell modules. (15−18)
Table 1. Summary of Structurally Elucidated Minimal BMC-Derived Shells Reported to Date
shellorganism of originnative shell functionshell structural models knownconstituent shell proteins identifiedreference
HO-BMCHaliangium ochraceumundeterminedT = 9BMC-P, BMC-H, BMC-TSutter et al. (7)
synthetic β-carboxysome shellsHalothece sp. PCC7428CO2 fixationT = 3, T = 4BMC-P, BMC-HSutter et al. (6)
GRM2 BMC-derived particlesKlebsiella pneumoniaecholine catabolismT = 4BMC-P, BMC-TKalnins et al. (10)
Cso-shellH. neaCO2 fixationT = 3, T = 4BMC-P, BMC-Hthis study
The α-carboxysome from Halothiobacillus neapolitanus (henceforth referred to as H. nea) has been recombinantly produced in Escherichia coli by transplantation of the α-carboxysome operon (cso) (Figure 1). (19,20) Genes found in the operon include three BMC-H paralogs (cso1ABC), one BMC-T (csoS1D) and two BMC-P paralogs (csoS4AB), along with those that encode the RuBisCO (cbbLS), carbonic anhydrase (csoSCA) and csoS2, an intrinsically disordered protein that serves as a crucial scaffold for α-carboxysome assembly. (21) A native-like α-carboxysome, expressed from six shell proteins and CsoS2, has been recently repurposed to host a hydrogenase complex for microbial hydrogen production, illustrating the bioengineering potential of the shell. (22) We reasoned that building a minimal shell derived from α-carboxysome components can yield further insights into α-carboxysome biogenesis, while improving their tractability for widespread adoption in biotechnology.

Figure 1

Figure 1. H. nea α-carboxysome operon (cso). The dotted line indicates 10 genes between cbbO and csoS1D that are unlikely to be associated with shell assembly or function. Gene lengths and intervening distances are not drawn to scale.

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We report that protein shells resembling icosahedral capsids can assemble using two subunits, representing a minimal BMC-derived shell which we term Cso-shell. Sequences derived from CsoS2 were found to mediate cargo protein encapsulation, with one variant likely displaying the highest cargo loading efficacy for a minimal BMC-derived shell to date. Atomic-scale models of the Cso-shells were obtained using cryogenic electron microscopy, revealing two shell forms. Despite the evolutionary divergence of the α-carboxysome from other BMCs, the Cso-shell shares key features with other minimal BMC-derived shells, substantiating the idea that principles of BMC shell assembly are highly conserved. (1,6,7,10,23) Hosting of cargo enzymes within the shell generally increased tolerance of the enzymes toward heat shock, methanolic buffers, consecutive freeze–thaw cycles and alkaline environments. We propose that the Cso-shell can serve as a platform for understanding α-carboxysome assembly. Additionally, the enzyme-stabilizing effect of the Cso-shell represents an advancement in harnessing BMC-based technology for catalytic applications. (24)

Materials and Methods

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Bacterial Strains and Culture

E. coli Acella(DE3) (EdgeBio) cells were used for all molecular cloning and all protein expression experiments except for expression of recombinant LacZ (either free enzyme or encapsulated within shells). BL21(DE3)-Gold Δlac (Addgene accession 99247) was used for expression of constructs containing His6-LacZ-S2-CTP to prevent co-purification of endogenous LacZ. (25) The cells were grown in Terrific Broth (BioBasic) supplemented with the appropriate antibiotics (kanamycin or streptomycin) at 50 μg/mL.

Golden Gate Assembly of Plasmids

The Golden Gate one-pot plasmid assembly largely follows previously published protocols, with slight modifications. (26) All molecular cloning reagents were purchased from NEB. For the insertion of one to three fragments, in a reaction pot was prepared 1 μL of T4 ligase buffer, 0.2 μL of 10x purified bovine serum albumin (BSA), 5 U of BsaI or Esp3I, 1 U of T4 ligase, 15 ng of destination plasmid, 1 to 3 μL of insert(s) and topped up to 10 μL with water. The reaction pot was subject to a 37 to 18 °C thermocycling process with 5 min incubation at each step for 40 cycles, followed by a 55 °C step for 15 min to digest unassembled plasmids while inhibiting ligation so as to reduce the number of colonies harboring the original destination vector.
Codon-optimized BMC genes were synthesized (BioBasic) while promoters and terminator parts were amplified from various templates as PCR products and cloned into Level 0 plasmids (termed HcKan). Modified Level 1 and 2 plasmids (Figure S1) were created using NEB HiFi assembly.

Sequence Alignment and Bioinformatics

CsoS2 sequences were aligned with Clustal Omega and output alignment file was prepared with JalView 2. (27,28) Accession numbers for the sequences used in alignment are detailed in Table S3.

Protein Purification and Densitometric Analysis

Cells were cultured in 500 mL of terrific broth (TB) supplemented with the appropriate antibiotic and shaken at 37 °C until the OD600 value of the culture reached approximately 0.6–1.0. The cultures were then cooled to 25 °C and isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to 100 μM for protein induction. The cells were shaken at 25 °C in 2 L baffled flasks at 150 rpm for 22 to 24 h before harvesting. Lysis was carried out using the M-110P microfluidizer (Microfluidics) at 15 000 psi for three passes in approximately 100 mL of lysis buffer (Tris·HCl 100 mM, NaCl 150 mM, pH 8.0). To the lysis mixture was also added 0.1 mM of phenylmethylsulfonyl fluoride (PMSF) protease inhibitor. The lysate was spun twice at 20 000g for 20 min each time. The clarified lysate was loaded at 1 mL/min linear flow rate onto either a StrepTrap HP 5 mL column (GE Life Sciences) for SII-tagged proteins or a HisPur cobalt 5 mL column (Thermo Scientific) for His6 tagged proteins. Purification was performed using an ÄKTA FPLC with 12 column volumes (CV) of washing with binding buffer (StrepTrap: Tris·HCl 100 mM, NaCl 150 mM, pH 8.0; HisPur: Tris·HCl 100 mM, NaCl 300 mM, imidazole 5 mM, pH 8.0) and 6 CVs of elution with the elution buffer (StrepTrap: binding buffer with 2.5 mM desthiobiotin; HisPur: binding buffer with 200 mM imidazole).
Shell samples were subject to anion-exchange chromatography following Strep-Tactin purification. Samples were dialyzed into ion exchange Buffer A (Tris·HCl 50 mM, pH 8.0) and loaded onto 10 mL of Q Sepharose HP resin in an XK 16/20 column (GE Life Sciences) at 1 mL/min. Buffer flow was kept at 1 mL/min and a two-step gradient protocol consisting of 0 to 50% ion exchange Buffer B (Tris·HCl 50 mM, NaCl 1.0 M, pH 8.0) over 15 CV and 60 to 100% Buffer B over 1.5 CV was used for elution. Shell-containing samples were concentrated using an Amicon centrifugal filter with 100 kDa nominal molecular weight limit (NMWL). Shells co-expressed with His6-APEX2-S2-CTP or His6-LacZ-S2-CTP were incubated with a 0.5 mL slurry of HisPur cobalt resin for 1 h at room temperature with gentle shaking to remove His6-tagged proteins not encapsulated within shells. The mixture was spun down at 20 000g for 5 min, and the supernatant was used for further analysis.
Proteins were analyzed using 13% stacking sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and stained using InstantBlue (Expedeon). Densitometric analysis was performed using the Bio-Rad image lab software in accordance with previous reports on BMC cargo quantification. (17,29) Background subtraction was performed and peak areas corresponding to bands of interest were used for quantification. Absolute protein concentrations were measured using a DeNovix spectrophotometer using calculated molar attenuation coefficients at 280 nm (ε280). The calculated ε280 of T = 3 shells, taken as the summation of the ε280 of its individual components, was 1 588 200 M–1·cm–1. The ε280 of the T = 4 shell was calculated to be 1 677 600 M–1·cm–1. The small difference in calculated ε280 values between both shell types is due to the low ε280 of CsoS1A (1490 M–1·cm–1). Most of the ε280 contribution comes from CsoS4A (23 490 M–1·cm–1), which has the same number of copies in both shell types. Concentrations of GFP were determined by fluorescence emission at 530 nm. Known concentrations of GFP were used to create calibration curves.

Immunoblotting

Proteins were transferred from SDS-PAGE gels to poly(vinylidene fluoride) (PVDF) membranes via the Bio-Rad Trans-Blot system. After transfer, membranes were blocked with phosphate-buffered saline-Tween 20 (PBS-T, Tween 20 at 0.05 v/v %) containing 3% w/v bovine serum albumin (BSA) for 1 h at room temperature with gentle shaking. Antibodies were prepared in blocking buffer at 1:1000 dilution, added to the blots, and incubated for 1 h with gentle shaking. For detection of GFP epitope, a GFP antibody conjugated with HRP (GF28R, Invitrogen) was used. For detection of His6, THE His Tag antibody conjugated with HRP (Genscript) was used. Blots were washed three times with PBS-T before chemiluminescent detection using the Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences).

Electron Microscopy

Formvar/carbon-coated copper grids (Electron Microscopy Science) were subject to glow discharge before 5 μL of purified protein samples (diluted to A280nm ∼ 0.05) were mounted for 60 s before the droplets were removed with filter paper. The grids were then negatively stained by adding a 5 μL droplet of 2.5% gadolinium(III) acetate, incubating for 90 s, and similarly blotted. Grids were imaged using a JEOL JEM-1220 TEM.

Shell Particle Size and Stability Measurements

Particle size distribution was determined by dynamic light scattering (DLS) using the UNcle instrument (Unchained labs). Samples were diluted to 1 mg/mL in TBS-50/350 pH 8.0 (Tris·HCl 50 mM, NaCl 350 mM, pH 8.0), unless otherwise specified, and spun at 20 000g for 5 min to remove aggregates prior to measurement. Care was taken to use the top-most supernatant for analysis. To the mini cuvette was added 9 μL of sample. All DLS measurements were done in triplicate and performed at 20 °C unless stated otherwise. Analysis of particle size distribution was done using the UNcle analysis software.
For shell stability measurements at various temperatures, shell samples were aliquoted into thin-walled PCR tubes and subject to temperatures ranging from 20–80 °C in 10 °C increments (indicated in Figure S8A) for 15 min in the UNcle instrument. At the end of the 15 min incubation, DLS spectra were taken.
For shell stability measurements in various buffer conditions, TBS-50/350 pH 8.0 buffers containing 10 and 20% (v/v) methanol were freshly prepared from stock solutions of Tris·HCl 1.0 M pH 8.0, NaCl 5.0 M, and 99.8% methanol (ACS reagent grade, Sigma) and used within the day of preparation. Due to the heat of mixing generated when methanol is mixed with water, methanol-containing buffers were allowed to equilibrate back to room temperature for at least 1 h after preparation. For making buffers at various pH, the following components were used at 50 mM for the pH range indicated: glycine·HCl for pH 2–4; 4-morpholineethansulfonic acid (MES) sodium salt for pH 5–7; Tris·HCl for pH 8–9; N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) sodium salt for pH 10–11; arginine·HCl for pH 12–13. All buffers contained 350 mM NaCl. Shells were incubated in the above-mentioned buffers for 15 min to allow time for possible shell dissociation/protein denaturation before particle size measurement.
For freeze–thaw stability, shell samples in TBS-50/350 pH 8.0 were aliquoted into thin-walled PCR tubes and flash-frozen in liquid N2. Samples were thawed at room temperature until no ice crystals were seen by standing for 15 min before re-freezing.

Isothermal Titration Calorimetry

Isothermal titration calorimetry measurements were carried out using the MicroCal PEAQ-ITC. Receptor samples (e.g., free CsoS1A or an equivalent amount of CsoS1A within Cso-shells) were loaded in the cell while ligand samples (S2-C variants) were loaded in the syringe. Protein samples were purified via Strep-Tactin by installing the SII tag at the C-terminus of CsoS1A and at the N-termini of S2-C variants. S2-CTP was chemically synthesized (BioBasic). Protein/peptide samples were dialyzed to HEPES 20 mM pH 7.4 containing 20, 60, or 200 mM NaCl. Samples were spun at 20 000g for 5 min to remove aggregates prior to measurement. Receptor concentrations were adjusted to 10 μM while ligands were adjusted to 100 μM, with the exception of experiments involving S2-CTP. For S2-CTP, receptor concentration had to be increased to 50 μM and ligand to 500 μM before binding could be detected. The stirring speed was set to maximum and 20 injections were performed at 25.0 °C. Binding analysis was carried out using the MicroCal PEAQ-ITC Analysis software using one set of binding sites as the model.

Enzyme Steady-State Kinetics Assays

For APEX2, all reagents were made to the appropriate working concentrations using TBS-50/350 pH 8.0. Working solutions of guaiacol and H2O2, both at 10 mM, were prepared on the day of assay. The guaiacol solution was shaken vigorously at 30 °C to ensure complete dissolution before equilibration back to room temperature. The assay concentration for APEX2 was 10 nM, H2O2 1 mM, and that for guaiacol ranged from 0.20 to 2.0 mM. The total reaction volume was 200 μL. Reactions were monitored by the absorbance of tetraguaiacol formed at 470 nm using a BioTek Synergy HT microplate reader. The rate of tetraguaicol formation was found to be constant until 90 s. This time point was taken for initial rate, V0, measurement. Kinetic constants were obtained using nonlinear least-square Michaelis–Menten fitting in GraphPad Prism.
For steady-state kinetics of LacZ, all reagents were made to the appropriate working concentrations using TBS-50/350 pH 8.0 supplemented with 1 mM MgCl2. Working solutions of ONPG at 10 mM were prepared from a 50 mM stock solution in DMSO on the day of assay. The assay concentration for LacZ was 10 nM, and that for ONPG (ortho-nitrophenyl-β-galactoside) ranged from 0.050 to 1.5 mM. The total reaction volume was 100 μL. Hydrolysis of ONPG was tracked by absorbance measurements at 405 nm. Rate of production formation was found to be constant until 60 s, and this time point was taken for V0 measurement.

Enzyme/Enzyme-Shell Activity and Stability Assays

All enzyme activity measurements were performed at ambient temperature (23 °C) and working concentrations of enzymes were 10 nM. Measurements were performed in triplicate. Enzyme activity measurements were determined as initial rates of product formation using saturating substrate concentrations (i.e., near Vmax). For APEX2, this was 1.4 mM guaiacol and 1 mM H2O2. For LacZ, this was 1.5 mM ONPG.
For heat shock assays, enzyme/enzyme-shell samples were aliquoted into thin-walled PCR tubes and subject to the elevated temperatures indicated (Figure 6B) in a thermocycler for 15 min. Following incubation, samples were cooled to 20 °C and equilibrated back to ambient temperature for 15 min before assay.
For stability measurement in buffers containing methanol and under various pH conditions, enzyme/enzyme-shell samples were dialyzed to the various buffers as described in the particle size measurement section. Solutions were allowed to stand for at least 15 min before assay to allow time for possible protein denaturation.
For freeze–thaw stability, enzyme/enzyme-shell samples were aliquoted in thin-walled PCR tubes and flash-frozen in liquid N2. Samples were thawed at room temperature until no ice crystals were seen by standing for 15 min before re-freezing or assay.

Results and Discussion

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Formation of Shell-like Particles from Cso-Shell Components

We sought to derive minimal shells from the cso operon using only one member from each shell subunit class (BMC-P/-H/-T). In structurally characterized BMC-derived shells reported to date (Table 1), BMC-P and BMC-H have been essential, while BMC-T has only been unambiguously identified in HO-BMC, indicating the BMC-T subunit is sometimes present in such shells. Our strategy involved combining subunits as PTH or PH, where P represents CsoS4A or CsoS4B (BMC-P), T represents CsoS1D (BMC-T), and H represents CsoS1A, CsoS1B, or CsoS1C (BMC-H), for a total of 12 combinations. A recently reported affinity purification strategy involving the installation of the Strep-tag II (SII) to the C-terminus of the BMC-P subunit was used to expedite purification of potential shells. (17) To facilitate combinatorial synthesis of BMC pathways, we adopted an established hierarchical Golden Gate modular cloning approach (Figures 2 and S1). (26) Briefly, promoter, open reading frame (ORF) and terminator parts are maintained in Level 0 plasmids. These parts are strung together as promoter-ORF-terminator transcriptional units in Level 1 plasmids. Transcriptional units are then combined into a contiguous pathway in a Level 2 acceptor plasmid.

Figure 2

Figure 2. Identifying combinations that produce minimal α-carboxysome-derived shells. (A) Schematic of the combinatorial assembly process used to construct shell-expressing pathways. (B–E) Transmission electron microscopy (TEM) micrographs of combinations that produced capsid-like structures, (B) Cso-PATHA, (C) Cso-PAHA, (D) Cso-PATHC, and (E) Cso-PAHC. Scale bars (bottom right) represent 50 nm. (F, G) SDS-PAGE analysis of shell-containing samples following anion-exchange purification of (F) Cso-PATHA and (G) Cso-PAHA. The protein ladder is indicated by L in kDa.

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Controlling the stoichiometry of BMC components is key to their successful assembly. (6,17) As a general guideline for expression of synthetic BMCs, the target relative expression levels for BMC-P, BMC-T, and BMC-H are low, medium, and high, respectively. (16,30) Correspondingly, in our modular system, a low-expression-strength promoter (BBa_J23114 from the Anderson Collection, Table S1) was assigned to P, a medium-strength promoter (BBa_J23105) to T, and the strong T7 promoter (PT7) to H. (31,32) The BBa_J23114 and BBa_J23105 promoters are constitutively active while PT7 is regulated by the LacI repressor protein to suppress high-level expression of recombinant proteins during the initial stage of cell growth, which is deleterious to the host cell. (33) Upon reaching the mid-exponential phase of cell growth, isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to relieve the repression, enabling high-level protein expression of H.
Assembled transcriptional units were combined into pathways (Figure 2A). The 12 pathway combinations (Table S2) were expressed, affinity purified, and visualized by transmission electron microscopy (TEM). Four combinations produced shell-like particles, namely, CsoS4A-SII in combination with either CsoS1A or CsoS1C, with and without the co-expression of CsoS1D (i.e., Cso-PATHA, PAHA, PATHC, PATHC). Particles from the four samples, which can be described as capsid-like structures, were indistinguishable from each other (Figure 2B–E). These particles also closely resembled the synthetic β-carboxysome and GRM2 BMC-derived particles. (6,10) We noted that the sequence of CsoS1A is highly similar to CsoS1C, differing only by one residue near the N-terminus and another near the C-terminus (Figure S2A). Unsurprisingly, structural models of CsoS1A and CsoS1C are virtually identical (Figure S2B). As the two differing residues are in flexible regions unlikely to be important for quaternary assembly, we postulated that shells comprised CsoS4A in combination with either CsoS1A or CsoS1C should be equivalent. We thus focused on Cso-PATHA and Cso-PAHA for the remainder of this study. To determine the components necessary for shell formation, pathway combinations expressing CsoS4A-SII with CsoS1D and CsoS1D with CsoS1A-SII were created and similarly purified. No nanoscale structures could be seen. Thus, CsoS4A and CsoS1A were found to be necessary and sufficient for the formation of shell-like particles observed. We term these particles Cso-shells.
Affinity purified Cso-PATHA and Cso-PAHA shells were subject to anion-exchange chromatography to separate shells from unincorporated CsoS4A-SII. (17) Two well-separated peaks were seen in the chromatogram, corresponding to elution centered at 0.18 and 0.35 M NaCl (Figure S2C). Samples from both peaks were analyzed by TEM and shells were seen only in the 0.35 M NaCl fraction. Based on the observation that the elution peak of monomeric CsoS4A-SII was centered at 0.18 M NaCl, we reasoned that the 0.18 M NaCl peak for shell samples largely corresponded to CsoS4A-SII not incorporated into the shell. SDS-PAGE analysis of anion-exchange purified Cso-PATHA and Cso-PAHA shells showed the presence of a band that corresponds to both CsoS4A-SII and CsoS1A, which have calculated molecular masses of 10.0 kDa and could not be separated (Figure 2F,G). No band corresponding to CsoS1D (23.5 kDa) was detected in the Cso-PATHA sample, indicating CsoS1D was absent or at very low occupancy within the shell.

Identifying Encapsulation Peptide Sequences for Cso-Shell Cargo Loading

We endeavored to repurpose the Cso-shell for encapsulating protein cargo using encapsulation peptides. For the purposes of engineering BMC shells, encapsulation peptides are sequences that interact with the shell subunits, facilitating encapsulation of protein cargo fused to such peptides before the shell is fully assembled. (16,18,30) Bioinformatic analysis on the scaffolding protein, CsoS2, suggests it can be divided into three distinct regions: the N-terminal region (S2-N), middle region (S2-M), and C-terminal region (S2-C) (Figure 3A). (20) S2-C has also been postulated to initiate carboxysome assembly by recruiting CsoS1 proteins. (20,21,34) Hence, encapsulation peptide sequences may be derived from S2-C. (22) Close inspection of S2-C shows it contains three homologous repeat sequences (R1–R3) and a highly conserved 30 residue C-terminal peptide (CTP) (Figure S3). (20) To probe the ability of S2-C-derived peptides to mediate encapsulation in the Cso-shell, full-length S2-C and truncated sequences─S2-C-ΔR1, S2-C-ΔR1-2, and S2-CTP were fused to the C-terminus of a monomeric green fluorescent protein (GFP). An E. coli N-terminal degradation tag, UmuD (1−40) (first 40 residues of UmuD), which is recognized for proteolysis by the endogenous ClpXP protease, was installed on the N-terminus of GFP-S2-C variants. (35,36) Based on known structures of BMC-derived shells, the shell subunits are tightly interdigitated and impermeable toward biological macromolecules, such as proteases (Figure 3B). (7,17,37) On the other hand, UmuD1-40-tagged proteins that are not encapsulated or only interact with the shell periphery would be exposed to and degraded by ClpXP. Hence, this assay served to identify successfully encapsulated cargo proteins within intact shells.

Figure 3

Figure 3. Identifying S2-CR sequences for mediating cargo encapsulation within the Cso-shell. (A) CsoS2 can be split into three distinct regions based on sequence homology. Regions are not drawn to scale. (B) Schematic of the encapsulation-degradation assay. Encapsulation of GFP cargo within the shell prevents its degradation by the ClpXP protease. The blue and yellow blocks represent CsoS1A (BMC-H) and CsoS4A (BMC-P), respectively. (C) Pathway design for co-expressing UmuD (1−40)-GFP-S2-Cx (S2-Cx represents S2-C variants) with the Cso-PAHA shells. (D) Western blot detection of GFP in purified shell samples. The identities of the proteins in the ladder (L) lane are: His6-GFP-SpyTag (29.7 kDa), His6-GFP-SpyCatcher (38.2 kDa), and His6-GFP-SpyCatcher-SpyTag-GFP (67.9 kDa). Lanes 1–5 are Cso-PAHA co-expressed with the UmuD (1−40)-GFP variant indicated below the lanes. (E) DLS analysis of shells co-expressed with various cargo. The numbers to the right of the spectra correspond to the samples loaded in (D). The baseline is vertically displaced by 0.2 for each subsequent spectrum so that all peaks can be seen in one graph.

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The UmuD (1−40)-GFP-S2-C variants were co-expressed with Cso-PAHA shells (Figure 3C). To account for stochastic encapsulation, a strain expressing UmuD (1−40)-GFP with shells was included. Purified shells were subject to Western blot analysis for detection of GFP (Figure 3D). Coomassie blue densitometric analysis of the bands corresponding to shell proteins was within ±0.4% of the mean value (Figure S4A). Hence, the quantity of shells loaded into each lane was approximately the same and the abundance of each UmuD (1−40)-GFP-S2-C variant, measured via immunoblotting, could be compared with each other (Table 2). S2-CTP mediated the highest level of cargo encapsulation, followed by S2-C-ΔR1-2. A faint band could be seen for UmuD (1−40)-GFP-S2-C-ΔR1 while both UmuD (1−40)-GFP and UmuD (1−40)-GFP-S2-C could not be detected, signifying very low occupancy or absence of encapsulated GFP. Encapsulation of the various cargo had no significant impact on shell size distribution (Figure 3E) and average hydrodynamic diameters (Table 2), as determined by dynamic light scattering (DLS). There was also no discernable difference in morphology among shell samples (Figure S4B–F).
Table 2. Densitometric Analyses of UmuD (1−40)-GFP Tagged with S2-C Variants, along with Average Hydrodynamic Diameters of the Shells Co-expressed with These Cargo Molecules
cargo (UmuD (1−40)-GFP-)cargo molecular mass (kDa)normalized peak area (immunoblot densitometry)average hydrodynamic diameter (nm)
No tag31.5not detected24.4
S2-C59.9not detected24.6
S2-C-ΔR150.90.03524.4
S2-C-ΔR1-242.20.41923.7
S2-CTP34.41.00024.0

Atomic-Scale Models of the Cso-Shell Reinforce Notions on Plasticity and Generalizability of BMC Shell Architecture

To better understand the molecular principles behind Cso-shell assembly and cargo encapsulation, cryogenic electron microscopy was used to obtain atomic-scale models of shells expressed from Cso-PAHA and Cso-PATHA. We wished to visualize GFP-S2-CTP density within the shell without possible interference from the UmuD (1−40) tag and thus created Cso-PAHA + GFP-S2-CTP (Figure 4A). For Cso-PATHA and Cso-PAHA, shells with icosahedral triangulation numbers T = 3 were seen, and maps were obtained at resolutions of 3.35 and 3.14 Å, respectively. For Cso-PAHA + GFP-S2-CTP, T = 3 and T = 4 shell forms were observed (Figure S5A), at proportions of 14 and 86%, respectively. Maps were obtained at resolutions of 3.24 Å for T = 3 and 2.90 Å for T = 4. Known structural models of H. nea CsoS1A and CsoS4A were used for model fitting. (8,38) We also solved the structure of H. nea CsoS1D at 2.70 Å resolution using X-ray crystallography. H. nea CsoS1D assembles as a stacked dimer of trimers with pseudo-hexameric symmetry (Figure S5B), similar to a homolog from Prochlorococcus marinus MED4. (39) However, in the Cso-PATHA model, no double-stacking densities were observed. The densities corresponding to hexameric units in the Cso-PATHA map could be modeled unequivocally with CsoS1A hexamers but not with a single layer of CsoS1D trimers. This observation, along with the absence of a detectable CsoS1D band in SDS-PAGE analysis of shells purified from Cso-PATHA (Figure 2F), led us to conclude that CsoS1D was unlikely to be incorporated within these shells.

Figure 4

Figure 4. Atomic-scale models of the minimal α-carboxysome shells. (A) Operon schematic of Cso-PAHA + GFP-S2-CTP. (B, C) Surface representations of the shells, with the CsoS1A colored blue and CsoS4A in yellow. The blue and red patches over yellow indicate N- and C-termini of a CsoS4A monomer, and the cyan and red patches over blue indicate those of a CsoS1A monomer.

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Unfortunately, cargo density was not resolved within Cso-PAHA + GFP-S2-CTP shells. We surmised that the cargo was not rigidly bound within the shell, resulting in nonuniform luminal densities that prevented resolution. Nonetheless, in light of a theoretical study on the α-carboxysome that suggests encapsulated scaffold molecules may increase shell size, it is conceivable that the T = 4 shell, seen only for Cso-PAHA + GFP-S2-CTP, could result from cargo encapsulation. (40) As the densities for all T = 3 shell models were largely identical, we focused on the model produced by Cso-PAHA + GFP-S2-CTP for further refinement. The T = 3 shell contains 12 homopentamers of CsoS4A and 20 homohexamers of CsoS1A (Figure 4B) while the T = 4 shell contains 12 homopentamers and 30 homohexamers (Figure 4C). Both shell forms are similar in other aspects.
In both T = 3 and 4 shell forms, the concave sides of CsoS1A and CsoS4A, wherein the N- and C-termini reside, face away from the shell (Figure 4B,C). Compared with their structures in isolation, both CsoS1A and CsoS4A expand slightly in the shell (Figure S5C,D and Table S6). Expansion of CsoS4A brings its V11 and T13 residues closer to a neighboring CsoS1A hexamer (Figure 5A). Furthermore, the C-terminus of CsoS4A flips inward and anchors itself into a pocket formed by two adjacent CsoS4A monomers. This conformational change is stabilized by π–π interactions between W80 and W49 and cation−π interactions between W80 and K8 from an adjacent CsoS4A monomer. Conformational changes for the CsoS1A hexamer following shell incorporation include slight outward movement of the helices sandwiching the central β-sheet in the CsoS1A chain, which largely accounts for the hexamer expansion and repositioning of several solvent-exposed side chains (Figure 5B).

Figure 5

Figure 5. Close examination of the interfacial contacts in Cso-shells reinforcing notions of conserved features in BMC shell assembly. (A) CsoS4A conformations pre-shell (gray) and post-shell integration (yellow). The dashed arrow indicates movement of CsoS4A’s C-terminal loop. W80 is highlighted with dark gray (pre-integration) and orange (post-integration). (B) CsoS1A hexamers (gray) become expanded after shell incorporation (blue). Arrow indicates movement of the helices. (C) Pentamer–hexamer interface, T = 3. (D) Pentamer–hexamer interface, T = 4. (E) Hexamer–hexamer interface, T = 3. (F) Hexamer–hexamer interface, T = 4. For clarity, only half of the polar interactions left to the pseudo-2-fold axis (orange oval) at the hexamer–hexamer interface are indicated in (E) and (F). For (C)–(F), CsoS4A is colored yellow, while CsoS1A chains are colored blue and green. Dotted lines represent interatomic distances in Å.

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Examination of the shell subunit interface reveals that hydrophobic patches contribute to interface assembly through shape complementarity while hydrogen bonds and salt bridges dictate spatial orientations of subunits (Figure 5C–F). The interfaces between CsoS1A and CsoS4A are conserved in both shell forms. The most prominent interfacial residue on CsoS4A is D48, which forms a salt bridge with R83 on CsoS1A (Figure 5C,D). In BMC-P orthologs, this position is generally found to be occupied by an acidic residue, denoting the importance of this polar engagement to the pentamer–hexamer interface. (1,7) Two interfacial motifs on CsoS1A, KAA (residues 29–31) and RxH (residues 83–85), constitute a large portion of both the pentamer–hexamer and the hexamer–hexamer interfaces. Key polar interactions are provided through K29 of the KAA motif and R83 of RxH. These motifs are known for their critical roles in BMC shells. (7,41) The side chain of R83 adopts two alternative conformations in the T = 3 shell; in the T = 4 shell, it takes a single, more engaging pose (Figures 5E,F and S5E,F). The morphological plasticity frequently observed in both native and synthetic BMC shells may be partly attributed to residues with flexible side chains in the KAA and RxH motifs that permit subunits to adjoin with differing interfacial orientations. (6,7,10)
Phylogenetic analyses of BMC-H and BMC-P domain proteins indicate that the α-carboxysomes shell proteins are evolutionarily divergent from β-carboxysomes and other BMCs, such as the metabolosomes that encase catabolic reactions. (23,42) Despite carrying out essentially the same function as α-carboxysomes, β-carboxysomes are considered to be more closely related to metabolosomes in terms of shell protein sequences and assembly mechanism. (1,23) Notwithstanding the distinct phylogenetic distance between the α-carboxysome and other BMCs, there are key features shared between the Cso-shell and other BMC-derived shells. The overall structural models of both Cso-shell forms are highly similar to that of the synthetic β-carboxysome, for which T = 3 and 4 forms are known, and also to the GRM2 BMC-derived particle, for which the T = 4 structure has been resolved. (6,10) In all BMC-derived shell models reported so far, the convex faces of the BMC-H and BMC-P subunits always face the shell lumen, and the KAA and RxH motifs are vital to interfacial contacts (Figure S5G). (6,7,10) The striking similarities observed among shells derived from evolutionarily and functionally divergent BMCs substantiate the idea that principles of BMC shell architecture are generalizable. (1,7)

Sequence Length of S2-C Peptides Appears to Influence Encapsulation Efficacies of S2-C Peptides In Vivo

The average copy numbers of UmuD (1−40)-GFP-S2-C variant encapsulated per shell could be quantified using the shell structures and GFP fluorescence. (17) Based on cryogenic electron microscopy observations, for all calculations involving shell molecular masses we presumed all shells co-expressed with cargo to be a mixture of T = 3 and 4 forms. As the proportions of shell forms may vary among samples, two values, calculated by presuming all shells in a sample were either T = 3 or 4, are provided. In agreement with results from immunoblotting, S2-CTP was the most efficacious encapsulation peptide, followed by S2-C-ΔR1-2 and S2-C-ΔR1 (Table 3 and Figure S6). Virtually no signal was detected for UmuD (1−40)-GFP-S2-C and untagged UmuD (1−40)-GFP.
Table 3. Quantifying the Average Number of Encapsulated UmuD (1−40)-GFP Cargo Per Shell as Mediated by S2-C-Derived Peptidesa
 average number of UmuD (1−40)-GFP cargo per shell  
S2-C variantT = 3T = 4number of residuesestimated hydrodynamic radius (Å)
no tag0.0230.024N.A.N.A.
S2-C0.110.1126642.7
S2-C-ΔR10.480.5018035.0
S2-C-ΔR1-22.93.09725.6
S2-CTP7.78.03014.1
a

As the proportions of the shell forms are unknown, values calculated by presuming all shells are either T = 3 or 4 are given. The estimated hydrodynamic radius of the peptides may be a factor contributing to the encapsulation efficacies observed.

While we were unable to obtain atomic-scale models of how S2-CTP interacts with the shell lumen, it is still of interest to consider possible factors that contribute to the observed encapsulation efficacies of the S2-C-derived peptides. Savage et. al. recently hypothesized that S2-C interacts with CsoS1 proteins during α-carboxysome biogenesis, and formation of a protein condensate arising from high local concentrations of CsoS2 lowers the ionic strength of the immediate environment, strengthening electrostatic interactions between α-carboxysome components. (20,21,34) However, no direct evidence of S2-C interacting with CsoS1A has been reported. This prompted us to investigate putative interactions of S2-C-derived peptides with CsoS1A using isothermal titration calorimetry (ITC). In HEPES 20/200 (HEPES 20 mM pH 7.4, NaCl 200 mM), which emulates physiological salt concentration, no binding was detected between 100 μM of S2-C peptides separately titrated against 10 μM of isolated CsoS1A. (43) When solution ionic strength was lowered by decreasing NaCl concentration to 60 mM, binding was detected for all S2-C peptides (Figure 6A). The standard binding free energies, ΔG°, corresponded to a dissociation constant (KD) of 13 μM for S2-CTP and approximately 2 to 3 μM for S2-C, S2-C-ΔR1, and S2-C-ΔR1-2 (Table S7). When NaCl was further decreased to 20 mM, KD of S2-C and S2-C-ΔR1 decreased marginally to around 0.7 μM, while that of S2-C-ΔR1-2 and S2-CTP were not significantly affected.

Figure 6

Figure 6. Investigating a possible molecular mechanism contributing to encapsulation by S2-C-derived peptides in the Cso-shell. (A) Standard free energies of binding, ΔG°, of the S2-C-derived peptides titrated against CsoS1A under various NaCl concentrations. Error bars indicate uncertainties of isotherm fitting. (B) Electrostatic potential map of a cross-sectional view of the Cso-shell lumen. Negative potential is colored red, while positive potential is represented by blue over a ±4 kT/e range. Maps were calculated using APBS. (46) (C) Electrostatic potential map of the Cso-shell exterior. (D) Calculated isoelectric points and net charge at pH 7.4 of the distinct regions on S2-C.

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To probe if the shell exterior face of CsoS1A might contribute to the binding energies observed in low-ionic-strength solutions, we made use of the fact that in an intact and empty Cso-PAHA shell, only the shell exterior side of CsoS1A is able to physically interact with externally added S2-C peptides. Binding analysis at 20 and 60 mM NaCl was carried out as before, with isolated CsoS1A replaced by Cso-PAHA shells containing an equivalent concentration of CsoS1A. No binding was detected, supporting the idea that interactions of S2-C peptides with CsoS1A are confined to its luminal face. We note that the luminal face carries a distinct negative electrostatic potential (Figure 6B) due to surface exposure of multiple acidic residues. (38) Conversely, the shell exterior face of CsoS1A holds a funnel-like structure carrying a positive electrostatic potential, which has been proposed to aid in channeling of HCO3– to the central pore (Figure 6C). (44) With the exception of the R1 region, all regions of S2-C carry high isoelectric points, which is reflected in their positive net charges calculated at pH 7.4 (Figure 6D). The presence of basic regions on S2-C and the acidic character of the CsoS1A luminal face, together with the observation that moderate binding affinities (KD ∼ 1–10 μM) detected under low ionic strength were significantly weakened under a near-physiological ionic strength condition, may suggest that complementary electrostatic interactions exist between the basic regions on S2-C and the acidic CsoS1A luminal face. However, due to the unavailability of atomic-level structures of S2-C peptides in complex with CsoS1A, we are unable to provide further details on the molecular nature of binding.
The absence of detectable binding between S2-C peptides and CsoS1A under low-ionic-strength solutions in vitro appears to contradict with the observed encapsulation efficacies in vivo, in which at least two of the peptides (S2-C-ΔR1-2 and S2-CTP) could mediate cargo encapsulation into the Cso-shell at levels substantially higher than stochastic encapsulation (Table 3). We reason that while in vitro binding assessment of S2-C peptides and CsoS1A homomer may provide clues regarding the molecular characteristics of binding, it is not necessarily an accurate or sole indicator of encapsulation efficacy into the Cso-shell in vivo. We note that the length of the S2-C peptide correlates well with encapsulation efficacies (Table 3), with shorter S2-C peptides yielding higher levels of encapsulation. Full-length S2-C has been experimentally characterized to be almost entirely disordered, with only low levels of predicted local secondary structural elements. (34) Intrinsically disordered proteins tend to adopt extended conformations that occupy significantly larger volumes than globular proteins with similar masses. (45) Based on the estimated hydrodynamic radii of the S2-C peptides (Table 3), it is conceivable that the likelihood of the Cso-shell encapsulating S2-C peptides occupying smaller volumes is higher than those occupying larger volumes.
While the molecular mechanism by which S2-C peptides interact with CsoS1A is currently unascertained, it is known that in vivo, the presence of S2-CTP is sufficient for encapsulation within the Cso-shell. On the other hand, it is unknown if other repeat regions on S2-C contribute to binding under the same condition. If these regions do not contribute to binding, then their presence with S2-CTP on the same peptide sequence would only add unproductive volume onto the cargo. If there is an increase in affinity by introducing these regions, the benefit might be offset by the decreased propensity of the longer peptide to adopt a conformation that packs well into the Cso-shell before the shell is fully closed off. Hence, we propose that under intracellular conditions, sequence length of the S2-C peptide appears to be an important factor in influencing encapsulation of cargo into the Cso-shell. Given its short sequence and comparatively high GFP encapsulation efficacy, S2-CTP should be the best encapsulation peptide sequence for targeting heterologous protein cargo into the Cso-shell.

Stabilization of Enzymatic Activities Using Cso-Shells

Protein shells have been garnering attention as platforms for conferring stability on enzymes against physical insults, such as heating or freezing, or chemical insults, such as the presence of organic co-solvents or nonphysiological pH. (47,48) Enzyme confinement often reduces their conformational flexibility, which sometimes confers stability against structural changes that lead to denaturation. (49,50) Currently, homomeric protein shells are more established for hosting enzymes, attributable to their relative ease of assembly and particle size homogeneity, which improves predictability and tractability during engineering. (48,51−53) Due to their heteromeric composition, minimal BMC-derived shells represent emerging scaffolds for hosting enzymes, as these shells can provide more avenues for purposeful modifications, while their generally homogeneous particle size still confers predictability to facilitate engineering. (2,11,51,52,54) However, minimal BMC-derived shells have yet to be explored for hosting heterologous enzymes. (16,17) This encouraged us to investigate if the Cso-shell could host and stabilize enzymes. Empty Cso-shells (Cso-PAHA) were first tested for their stability against heat shock, freezing, presence of methanol co-solvent, and environments with pH from 2 to 13. The DLS spectra of shells subject to these conditions were compared to that of shells in Tris·HCl 50/350 (Tris·HCl 50 mM pH 8.0, NaCl 350 mM). Significant changes in particle size distributions and/or appearance of multiple peaks indicate protein shell disassembly. (55) Based on the conditions tested, Cso-shells were considered to be stable up to 70 °C for 15 min, 20% v/v methanol, seven consecutive freeze–thawing, and between pH 5–11 (Figure 7A–D).

Figure 7

Figure 7. Assessing the tolerance of empty Cso-shell against common denaturing factors for potential stabilization of encapsulated enzymes. (A–D) DLS spectra of empty Cso-shells tested against the conditions indicated. The baseline is vertically displaced by 0.2 for each subsequent spectrum so that all spectra can be seen in one graph. (E) Operon schematic for co-expressing Cso-shells with enzymes.

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To probe the capacity of the Cso-shell for encasing enzymes of considerably different molecular sizes, an evolved pea cytosolic ascorbate peroxidase (APEX2), a 27.0 kDa monomer, and E. coli β-galactosidase (LacZ), a 466.0 kDa homotetramer, were chosen for encapsulation. (56,57) S2-CTP was fused to the C-termini of the enzymes to mediate encapsulation. Enzymes were also N-terminally tagged with the hexahistidine (His6) tag to facilitate downstream removal of unencapsulated enzymes that might co-purify with shells. (29) Cso-PAHA shells co-expressed with enzymes were constructed (Figure 7E) and purified. Immunoblotting confirmed the presence of the target enzymes in shell samples (Figure S8A,B), and the average copy numbers of enzymes per shell were estimated by Coomassie blue densitometry (Table 4). (17,29) Encapsulation of these enzymes did not appear to significantly affect Cso-shell size and morphology (Figure S8C–E).
Table 4. Quantification of the Average Copy Number of Enzymes Encapsulated Per Shell and Kinetic Constants of Encapsulated and Free Enzymesa
 average enzyme copy number per shell   
enzyme sampleT = 3T = 4kcat (s–1)KM (mM)kcat/KM × 105 (s–1·M–1)
Free APEX2N.A.N.A.460 ± 200.58 ± 0.077.93 ± 1.02
APEX2 + shell11.014.6208 ± 140.79 ± 0.122.63 ± 0.44
LacZN.A.N.A.258 ± 170 17 ± 0.0215.2 ± 2.05
LacZ + shell3.294.37200 ± 140.14 ± 0.0214.3 ± 2.27
a

For average enzyme copy number per shell, values calculated by presuming all shells are either T = 3 or 4 are provided. Kinetic measurements were performed in triplicate, and the mean values are shown with the standard error.

Encapsulation of an enzyme into a protein shell is known to alter the enzyme’s catalytic properties in some cases. To probe how encapsulation by the Cso-shell might affect the catalytic efficiencies of APEX2 and LacZ, we performed steady-state kinetics of both free and encapsulated enzymes and fitted the data to the Michaelis–Menten model to obtain the turnover number (kcat), Michaelis–Menten constant (KM), and catalytic efficiency (kcat/KM) (Table 4 and Figure S9). For encapsulated APEX2, kcat/KM decreased to approximately 30% of the free enzyme. For encapsulated LacZ, kcat/KM was not significantly different from the free enzyme. The kinetic constants, kcat and KM,, obtained for both free enzymes were in reasonable agreement with previous work, suggesting the presence of S2-CTP did not affect the activities of the free enzymes. (56,58)
Decrease in catalytic efficiencies of enzymes upon encapsulation into protein shells has been well documented and may arise from restrictions on enzyme conformational changes during substrate binding, catalysis, and product release, with the effects on kinetic parameters appearing to be enzyme-specific. (52,53,59) As an illustration, when encapsulated within the P22 capsid, kcat of an alcohol dehydrogenase decreased by approximately 85%, that of a cytochrome P450 by 30%, while that for a lipase was largely unchanged. (51−53) This is not unexpected, as different enzymes carry out catalysis via different mechanisms and conformational changes. In our study, KM of both APEX2 and LacZ were not significantly changed upon encapsulation, suggesting that substrate affinities of these enzymes were apparently not affected. On the other hand, kcat of APEX2 decreased by approximately 50% and that of LacZ by 20%. This may be attributed to loss in conformational flexibility during substrate catalysis. Impeded diffusion of substrate through the protein shell membrane into the enzyme active site has also been proposed to contribute to decrease in catalytic efficiencies. (51−53,60) A molecular dynamics study by Mahinthichaichan et al. on transport across the CsoS1A pore suggests that the pore selects for polar and anionic molecules, such as HCO3–, and acts as a diffusional barrier against nonpolar molecules, such as O2 and CO2. (44) This is supported by the electrostatic potential map of the CsoS1A (Figure 6C), which depicts the pore as having a positive electrostatic potential. However, the study focused on small molecules cognate to the native α-carboxysome and the generalizability of these findings toward noncognate small molecules, as were used in this study (guaiacol and ortho-nitrophenyl-β-galactoside, ONPG), cannot be ascertained. Another molecular dynamics study on CcmK2, a β-carboxysome BMC-H protein that is structurally similar to CsoS1A, suggests that the central pores may accommodate the transit of relatively large metabolites, such as ribulose-1,5-bisphosphate, though it is likely to be less favored compared to transit of smaller metabolites. (61,62) The molecular permeability of CsoS4A, the other structural component of the Cso-shell, has not been as well investigated. Nevertheless, given that the CsoS4A pore is similar to that of CsoS1A in terms of the positive electrostatic character (Figure 6C) and size (Table S6), it would not be surprising if the permeabilities of CsoS4A and CsoS1A pores are akin to each other. Unlike in the bona fide α-carboxysome where CsoS4A is a minor component, in the Cso-shell the stoichiometry of CsoS4A is comparable to that of CsoS1A. (12) Hence, the pores of CsoS4A should be expected to significantly influence the overall permeability of the Cso-shell. In-depth investigation of the dynamics behind the permeability of the Cso-shell may provide further insights on how diffusion of small molecules across the shell barrier affects the catalytic activities of the encapsulated enzyme.
To determine possible stabilizing effects of the Cso-shell on enzymes, free enzyme and shell-encapsulated enzyme samples were challenged with the above-mentioned conditions under which empty shells were found to be stable. Enzyme activities were normalized to that of the pristine sample to determine the residual activity (Figure 8A–D). The Cso-shell conferred a moderate level of thermostability on both enzymes. Encapsulated enzymes retained approximately 90% of their activities following incubation at 40 °C for 15 min, in contrast to 40% for free enzymes. At 50 °C, encapsulated APEX2 retained about half its activity while the free enzyme was essentially inactive. However, the activity of encapsulated LacZ was only marginally higher than free enzyme at 50 °C. At 60 °C and above, all enzyme samples were inactive. The Cso-shell had a protective effect on APEX2 up to 20% v/v methanol. On the other hand, for both free and encapsulated LacZ in methanol, increases in activities were seen. It has been reported that the presence of up to 40% v/v methanol does not denature LacZ but rather enhances its activity. (63) Hence, Cso-shell was unlikely to have stabilized LacZ against methanol. For freeze–thaw stability, the Cso-shell stabilized both enzyme species up to seven consecutive cycles.

Figure 8

Figure 8. Investigating the stability of the Cso-shell and scaffolded enzymes in potentially denaturing conditions. Residual enzymes activities of free and encapsulated (+ shell) enzymes were obtained by normalizing the activity to that of the pristine sample, shown as (B) 23 °C, (C) 0% v/v methanol, (D) no freeze–thawing, and (E) pH 8. Error bars represent one standard deviation of the mean.

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Encapsulated enzymes displayed higher activities within the Cso-shell at pH 10–11, but lower activities at pH 5–6. We reasoned the acidic microenvironment within the Cso-shell likely shifted the pH-activity profile of encapsulated enzymes to more alkaline conditions compared to free enzyme. The influence of anionic scaffolds on the pH-dependent activities of enzymes has been observed for synthetic maleic acid polymer scaffolds on trypsin and chymotrypsin, and more recently for the DNA polyphosphate backbone on the glucose oxidase-horseradish peroxidase (GOx-HRP) cascade. (64−66) The maleic acid polymer scaffolds increased the activities of immobilized trypsin and chymotrypsin under alkaline conditions compared to free enzymes. Similarly, the DNA scaffold emulated the acidic environment preferred by GOx and HRP, increasing the activity of the cascade relative to free enzymes in alkaline buffer. (66)
To date, the Cso-shell likely demonstrates the highest heterologous cargo loading via encapsulation peptides among minimal BMC-derived shells. The use of encapsulation peptides for such shells has been largely inefficacious and cargo often could not be detected via Coomassie blue staining, requiring more sensitive techniques such as immunoblotting or fluorescence. (16,17,30) In contrast, for the Cso-shell and S2-CTP system all three heterologous protein cargo tested (GFP, APEX2, LacZ) could be clearly identified in Coomassie-blue-stained gels (Figures S4 and S8). While orthogonal methods of cargo loading, such as structural permutation of the BMC-H subunit and installation of peptide conjugation domains into shell subunits and cargo, were developed to circumvent the hitherto low efficacy of encapsulation peptides, these peptides remain the most straightforward and widely used method for targeting protein cargo into synthetic BMCs. (17,18,24,29,37) Based on structural generalizability of BMC shell subunits, it is conceivable that for the Cso-shell, subunit engineering can be employed with encapsulation peptides for hosting of multiple cargo species in tandem, enabling more elaborate reactions to be supported by the shell. (24)

Conclusions

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Understanding α-carboxysome assembly is of interest as its biotechnological potential is increasingly recognized. (1,2,22) While the Cso-shell cannot fully capture the intricacies of the bona fide α-carboxysome, the establishment of a minimal shell scaffold and associated encapsulation peptides provides a platform for gaining deeper insights into the native shell. In tandem with emerging mechanistic elucidation of α-carboxysome biogenesis, the Cso-shell can be leveraged toward fully modular reconstitution and modification of the α-carboxysome, enabling applications in synthetic biology and soft matter. (34,40)
Enzyme stability often limits their utility. (47) We have shown that the Cso-shell is able to host and stabilize enzymes with markedly different molecular sizes and reaction chemistries against common denaturing factors. (47) Stabilization of APEX2 and β-galactosidase may expand their usefulness in cellular imaging and food processing applications, respectively. (56,67) Among known protein shells, a distinctive feature of the Cso-shell is its ability to stabilize encapsulated enzymes under basic environments. (48) The utility of the Cso-shell can be further explored in remediation of alkaline industrial effluent. (68) Enzymes that are currently used for wastewater treatment, such as laccases, peroxidases, and nitrilases, often suffer from diminished activities in high-pH environments. It may thus be worth exploring if these enzymes are able to benefit from encapsulation within the Cso-shell for wastewater treatment applications. (69−71) We have also identified an encapsulation peptide for the Cso-shell that mediates high levels of protein cargo encapsulation. These demonstrations advance the utility of minimal BMC-derived shells beyond its current proof-of-concept stage of encapsulating fluorescent proteins. (6,17) Characterization of the Cso-shell presented herein should inform its future employment as a bespoke nanoreactor.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biomac.1c00533.

  • Detailed procedures on data collection and analysis of structural models obtained from cryo-electron microscopy and X-ray crystallography; adopting and adapting an established Golden Gate-based genetic toolkit for combinatorial assembly of synthetic BMC pathways; list of constitutively active promoters from the Anderson collection; summary of recombinant combinatorial expression of Cso-shell components; multiple sequence alignment of 100 CsoS2 orthologs, focusing on the C-terminal region; and comparison of pore sizes of minimal BMC-derived shells with known atomic-scale structures and sequences of genetic constructs (PDF)

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Author Information

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  • Corresponding Author
    • Wen Shan Yew - Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore (NUS), 8 Medical Drive, Singapore 117597;  NUS Synthetic Biology for Clinical and Technological Innovation, 28 Medical Drive, Singapore 117456;  Graduate School for Integrative Sciences and Engineering, NUS, Singapore 119077;  Synthetic Biology Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, Singapore 117599;  Orcidhttps://orcid.org/0000-0002-3021-0469;  Email: [email protected]
  • Authors
    • Yong Quan Tan - Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore (NUS), 8 Medical Drive, Singapore 117597;  NUS Synthetic Biology for Clinical and Technological Innovation, 28 Medical Drive, Singapore 117456;  Graduate School for Integrative Sciences and Engineering, NUS, Singapore 119077;  Orcidhttps://orcid.org/0000-0002-6497-7756
    • Samson Ali - Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan;  Research Institute for Interdisciplinary Science (RIIS), Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan
    • Bo Xue - Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore (NUS), 8 Medical Drive, Singapore 117597;  NUS Synthetic Biology for Clinical and Technological Innovation, 28 Medical Drive, Singapore 117456;  Synthetic Biology Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, Singapore 117599
    • Wei Zhe Teo - Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore (NUS), 8 Medical Drive, Singapore 117597;  NUS Synthetic Biology for Clinical and Technological Innovation, 28 Medical Drive, Singapore 117456;  Synthetic Biology Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, Singapore 117599
    • Lay Hiang Ling - Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore (NUS), 8 Medical Drive, Singapore 117597;  NUS Synthetic Biology for Clinical and Technological Innovation, 28 Medical Drive, Singapore 117456;  Graduate School for Integrative Sciences and Engineering, NUS, Singapore 119077
    • Maybelle Kho Go - Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore (NUS), 8 Medical Drive, Singapore 117597;  NUS Synthetic Biology for Clinical and Technological Innovation, 28 Medical Drive, Singapore 117456;  Synthetic Biology Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, Singapore 117599
    • Hong Lv - Shanghai Engineering Research Center of Industrial Microorganisms, Shanghai 200438, People’s Republic of China;  State Key Laboratory of Genetic Engineering, School of Life Science, Fudan University, Shanghai 200438, People’s Republic of China
    • Robert C. Robinson - Research Institute for Interdisciplinary Science (RIIS), Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan;  School of Biomolecular Science and Engineering (BSE), Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong 21210, Thailand
    • Akihiro Narita - Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
  • Author Contributions

    Y.Q.T., S.A., and B.X. contributed equally to this work. Y.Q.T. and W.S.Y. conceptualized the experiments. Y.Q.T., S.A., B.X., W.Z.T., and L.H.L. performed investigations. Y.Q.T., S.A., B.X., R.C.R., and A.N. conducted formal analysis on data. Y.Q.T., S.A., B.X., and W.S.Y. prepared the manuscript. M.K.G. and H.L. reviewed the manuscript. W.S.Y. supervised the project. All authors have given approval to the final version of the manuscript.

  • Funding

    This work was supported by grants from the National Research Foundation Synthetic Biology Research and Development Program (SBP-P3). A part of this work was supported by JSPS KAKENHI (18H02410) to A.N. Y.Q.T. and L.H.L. are supported by the NUS Graduate School for Integrative Sciences and Engineering scholarship.

  • Notes
    The authors declare no competing financial interest.

    A patent application (SG Non-Provisional Application No. 10202010547W) has been filed on the development and use of Cso-shells.


    The coordinates of the shells have been deposited in the Protein Data Bank (PDB) under the accession numbers 7CKB (T = 3) and 7CKC (T = 4), and Electron Microscopy Data Bank (EMDB) under the accession numbers EMD-30384 (T = 3) and EMD-30385 (T = 4). The coordinates of H. nea CsoS1D have been deposited in PDB with accession number 7DHQ.

Acknowledgments

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BL21(DE3)-Gold Δlac strain was a kind gift from Jeff Hasty. The authors acknowledge Dr. Lu Ting Liow for providing advice on bacterial promoters. They also thank Professors Kaoru Mitsuoka, Research Center for Ultra-High Voltage Electron Microscopy (supported by Nanotechnology Platform Program, MEXT, Japan, A-19-OS-0052), and Kenji Iwasaki, Institute for Protein Research (supported by the Cooperative Research Program of Institute for Protein Research, Osaka University), both in Osaka University for access to the cryo-electron microscopy microscopes for data optimization and collection. This research was undertaken on the MX1 beamline at the Australian Synchrotron, part of ANSTO.

Abbreviations

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BMC

bacterial microcompartments

BMC–P/ −T/ −H

bacterial microcompartment–pentamer/–trimer/–hexamer

DLS

dynamic light scattering

GFP

green fluorescent protein

ITC

isothermal titration calorimetry

IPTG

isopropyl β-d-1-thiogalactopyranoside

ONPG

ortho-nitrophenyl galactoside

ORF

open reading frame

PT7

T7 promoter

RuBisCO

ribulose-1,5-bisphosphate carboxylase

S2-CTP

CsoS2 C-terminal peptide

S2–N/ −M/ −C

CsoS2 N-terminal/–middle/ −C-terminal region

SII

strep-tag II

TEM

transmission electron microscopy

References

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    Hagen, A. R.; Plegaria, J. S.; Sloan, N.; Ferlez, B.; Aussignargues, C.; Burton, R.; Kerfeld, C. A. In Vitro Assembly of Diverse Bacterial Microcompartment Shell Architectures. Nano Lett. 2018, 18, 7030– 7037,  DOI: 10.1021/acs.nanolett.8b02991
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    13
    In Vitro Assembly of Diverse Bacterial Microcompartment Shell Architectures
    Hagen, Andrew R.; Plegaria, Jefferson S.; Sloan, Nancy; Ferlez, Bryan; Aussignargues, Clement; Burton, Rodney; Kerfeld, Cheryl A.
    Nano Letters (2018), 18 (11), 7030-7037CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
    Bacterial microcompartments (BMCs) are organelles composed of a selectively permeable protein shell that encapsulates enzymes involved in CO2 fixation (carboxysomes) or carbon catabolism (metabolosomes). Confinement of sequential reactions by the BMC shell presumably increases the efficiency of the pathway by reducing the crosstalk of metabolites, release of toxic intermediates, and accumulation of inhibitory products. Because BMCs are composed entirely of protein and self-assemble, they are an emerging platform for engineering nanoreactors and mol. scaffolds. However, testing designs for assembly and function through in vivo expression is labor-intensive and has limited the potential of BMCs in bioengineering. Here, we developed a new method for in vitro assembly of defined nanoscale BMC architectures: shells and nanotubes. By inserting a "protecting group", a short ubiquitin-like modifier (SUMO) domain, self-assembly of shell proteins in vivo was thwarted, enabling prepn. of concs. of shell building blocks. Addn. of the cognate protease removes the SUMO domain and subsequent mixing of the constituent shell proteins in vitro results in the self-assembly of three types of supramol. architectures: a metabolosome shell, a carboxysome shell, and a BMC protein-based nanotube. We next applied our method to generate a metabolosome shell engineered with a hyper-basic luminal surface, allowing for the encapsulation of biotic or abiotic cargos functionalized with an acidic accessory group. This is the first demonstration of using charge complementarity to encapsulate diverse cargos in BMC shells. Collectively, our work provides a generally applicable method for in vitro assembly of natural and engineered BMC-based architectures.
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  14. 14
    Aussignargues, C.; Pandelia, M.-E.; Sutter, M.; Plegaria, J. S.; Zarzycki, J.; Turmo, A.; Huang, J.; Ducat, D. C.; Hegg, E. L.; Gibney, B. R.; Kerfeld, C. A. Structure and Function of a Bacterial Microcompartment Shell Protein Engineered to Bind a [4Fe-4S] Cluster. J. Am. Chem. Soc. 2016, 138, 5262– 5270,  DOI: 10.1021/jacs.5b11734
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    14
    Structure and Function of a Bacterial Microcompartment Shell Protein Engineered to Bind a [4Fe-4S] Cluster
    Aussignargues, Clement; Pandelia, Maria-Eirini; Sutter, Markus; Plegaria, Jefferson S.; Zarzycki, Jan; Turmo, Aiko; Huang, Jingcheng; Ducat, Daniel C.; Hegg, Eric L.; Gibney, Brian R.; Kerfeld, Cheryl A.
    Journal of the American Chemical Society (2016), 138 (16), 5262-5270CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Bacterial microcompartments (BMCs) are self-assembling organelles composed of a selectively permeable protein shell and encapsulated enzymes. They are considered promising templates for the engineering of designed bionanoreactors for biotechnol. In particular, encapsulation of oxidoreductive reactions requiring electron transfer between the lumen of the BMC and the cytosol relies on the ability to conduct electrons across the shell. We detd. the crystal structure of a component protein of a synthetic BMC shell, which informed the rational design of a [4Fe-4S] cluster-binding site in its pore. We also solved the structure of the [4Fe-4S] cluster-bound, engineered protein to 1.8 Å resoln., providing the first structure of a BMC shell protein contg. a metal center. The [4Fe-4S] cluster was characterized by optical and EPR spectroscopies; it has a redn. potential of -370 mV vs the std. hydrogen electrode (SHE) and is stable through redox cycling. This remarkable stability may be attributable to the hydrogen-bonding network provided by the main chain of the protein scaffold. The properties of the [4Fe-4S] cluster resemble those in low-potential bacterial ferredoxins, while its ligation to three cysteine residues is reminiscent of enzymes such as aconitase and radical S-adenosymethionine (SAM) enzymes. This engineered shell protein provides the foundation for conferring electron-transfer functionality to BMC shells.
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  15. 15
    Lawrence, A. D.; Frank, S.; Newnham, S.; Lee, M. J.; Brown, I. R.; Xue, W.-F.; Rowe, M. L.; Mulvihill, D. P.; Prentice, M. B.; Howard, M. J.; Warren, M. J. Solution Structure of a Bacterial Microcompartment Targeting Peptide and Its Application in the Construction of an Ethanol Bioreactor. ACS Synth. Biol. 2014, 3, 454– 465,  DOI: 10.1021/sb4001118
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    15
    Solution Structure of a Bacterial Microcompartment Targeting Peptide and Its Application in the Construction of an Ethanol Bioreactor
    Lawrence, Andrew D.; Frank, Stefanie; Newnham, Sarah; Lee, Matthew J.; Brown, Ian R.; Xue, Wei-Feng; Rowe, Michelle L.; Mulvihill, Daniel P.; Prentice, Michael B.; Howard, Mark J.; Warren, Martin J.
    ACS Synthetic Biology (2014), 3 (7), 454-465CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    Targeting of proteins to bacterial microcompartments (BMCs) is mediated by an 18-amino-acid peptide sequence. Herein, we report the soln. structure of the N-terminal targeting peptide (P18) of PduP, the aldehyde dehydrogenase assocd. with the 1,2-propanediol utilization metabolosome from Citrobacter freundii. The soln. structure reveals the peptide to have a well-defined helical conformation along its whole length. Satn. transfer difference and transferred NOE NMR has highlighted the obsd. interaction surface on the peptide with its main interacting shell protein, PduK. By tagging both a pyruvate decarboxylase and an alc. dehydrogenase with targeting peptides, it has been possible to direct these enzymes to empty BMCs in vivo and to generate an ethanol bioreactor. Not only are the purified, redesigned BMCs able to transform pyruvate into ethanol efficiently, but the strains contg. the modified BMCs produce elevated levels of alc.
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  16. 16
    Cai, F.; Bernstein, S. L.; Wilson, S. C.; Kerfeld, C. A. Production and Characterization of Synthetic Carboxysome Shells with Incorporated Luminal Proteins. Plant Physiol. 2016, 170, 1868– 1877,  DOI: 10.1104/pp.15.01822
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    16
    Production and characterization of synthetic carboxysome shells with incorporated luminal proteins
    Cai, Fei; Bernstein, Susan L.; Wilson, Steven C.; Kerfeld, Cheryl A.
    Plant Physiology (2016), 170 (3), 1868-1877CODEN: PLPHAY; ISSN:1532-2548. (American Society of Plant Biologists)
    Spatial segregation of metab., such as cellular-localized CO2 fixation in C4 plants or in the cyanobacterial carboxysome, enhances the activity of inefficient enzymes by selectively concg. them with their substrates. The carboxysome and other bacterial microcompartments (BMCs) have drawn particular attention for bioengineering of nanoreactors because they are selfassembling proteinaceous organelles. All BMCs share an architecturally similar, selectively permeable shell that encapsulates enzymes. Fundamental to engineering carboxysomes and other BMCs for applications in plant synthetic biol. and metabolic engineering is understanding the structural determinants of cargo packaging and shell permeability. Here we describe the expression of a synthetic operon in Escherichia coli that produces carboxysome shells. Protein domains native to the carboxysome core were used to encapsulate foreign cargo into the synthetic shells. These synthetic shells can be purified to homogeneity with or without luminal proteins. Our results not only further the understanding of protein-protein interactions governing carboxysome assembly, but also establish a platform to study shell permeability and the structural basis of the function of intact BMC shells both in vivo and in vitro. This system will be esp. useful for developing synthetic carboxysomes for plant engineering.
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  17. 17
    Hagen, A.; Sutter, M.; Sloan, N.; Kerfeld, C. A. Programmed loading and rapid purification of engineered bacterial microcompartment shells. Nat. Commun. 2018, 9, 2881  DOI: 10.1038/s41467-018-05162-z
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    17
    Programmed loading and rapid purification of engineered bacterial microcompartment shells
    Hagen Andrew; Sutter Markus; Sloan Nancy; Kerfeld Cheryl A; Sutter Markus; Kerfeld Cheryl A; Kerfeld Cheryl A
    Nature communications (2018), 9 (1), 2881 ISSN:.
    Bacterial microcompartments (BMCs) are selectively permeable proteinaceous organelles which encapsulate segments of metabolic pathways across bacterial phyla. They consist of an enzymatic core surrounded by a protein shell composed of multiple distinct proteins. Despite great potential in varied biotechnological applications, engineering efforts have been stymied by difficulties in their isolation and characterization and a dearth of robust methods for programming cores and shell permeability. We address these challenges by functionalizing shell proteins with affinity handles, enabling facile complementation-based affinity purification (CAP) and specific cargo docking sites for efficient encapsulation via covalent-linkage (EnCo). These shell functionalizations extend our knowledge of BMC architectural principles and enable the development of minimal shell systems of precisely defined structure and composition. The generalizability of CAP and EnCo will enable their application to functionally diverse microcompartment systems to facilitate both characterization of natural functions and the development of bespoke shells for selectively compartmentalizing proteins.
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  18. 18
    Fan, C.; Cheng, S.; Liu, Y.; Escobar, C. M.; Crowley, C. S.; Jefferson, R. E.; Yeates, T. O.; Bobik, T. A. Short N-terminal sequences package proteins into bacterial microcompartments. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 7509– 7514,  DOI: 10.1073/pnas.0913199107
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    18
    Short N-terminal sequences package proteins into bacterial microcompartments
    Fan, Chenguang; Cheng, Shouqiang; Liu, Yu; Escobar, Cristina M.; Crowley, Christopher S.; Jefferson, Robert E.; Yeates, Todd O.; Bobik, Thomas A.
    Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (16), 7509-7514, S7509/1-S7509/327CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Hundreds of bacterial species produce proteinaceous microcompartments (MCPs) that act as simple organelles by confining the enzymes of metabolic pathways that have toxic or volatile intermediates. A fundamental unanswered question about bacterial MCPs is how enzymes are packaged within the protein shell that forms their outer surface. Here, we report that a short N-terminal peptide is necessary and sufficient for packaging enzymes into the lumen of an MCP involved in B12-dependent 1,2-propanediol utilization (Pdu MCP). Deletion of 10 or 14 amino acids from the N terminus of the propionaldehyde dehydrogenase (PduP) enzyme, which is normally found within the Pdu MCP, substantially impaired packaging, with minimal effects on its enzymic activity. Fusion of the 18 N-terminal amino acids from PduP to GFP, GST, or maltose-binding protein resulted in their encapsulation within MCPs. Bioinformatic analyses revealed N-terminal extensions in two addnl. Pdu proteins and three proteins from two unrelated MCPs, suggesting that N-terminal peptides may be used to package proteins into diverse MCPs. The potential uses of MCP assembly principles in nature and in biotechnol. are discussed.
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  19. 19
    Bonacci, W.; Teng, P. K.; Afonso, B.; Niederholtmeyer, H.; Grob, P.; Silver, P. A.; Savage, D. F. Modularity of a carbon-fixing protein organelle. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 478– 483,  DOI: 10.1073/pnas.1108557109
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    19
    Modularity of a carbon-fixing protein organelle
    Bonacci, Walter; Teng, Poh K.; Afonso, Bruno; Niederholtmeyer, Henrike; Grob, Patricia; Silver, Pamela A.; Savage, David F.
    Proceedings of the National Academy of Sciences of the United States of America (2012), 109 (2), 478-483, S478/1-S478/4CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Bacterial microcompartments are proteinaceous complexes that catalyze metabolic pathways in a manner reminiscent of organelles. Although microcompartment structure is well understood, much less is known about their assembly and function in vivo. We show here that carboxysomes, CO2-fixing microcompartments encoded by 10 genes, can be heterologously produced in Escherichia coli. Expression of carboxysomes in E. coli resulted in the prodn. of icosahedral complexes similar to those from the native host. In vivo, the complexes were capable of both assembling with carboxysomal proteins and fixing CO2. Characterization of purified synthetic carboxysomes indicated that they were well formed in structure, contained the expected mol. components, and were capable of fixing CO2 in vitro. In addn., we verify assocn. of the postulated pore-forming protein CsoS1D with the carboxysome and show how it may modulate function. We have developed a genetic system capable of producing modular carbon-fixing microcompartments in a heterologous host. In doing so, we lay the groundwork for understanding these elaborate protein complexes and for the synthetic biol. engineering of self-assembling mol. structures.
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  20. 20
    Chaijarasphong, T.; Nichols, R. J.; Kortright, K. E.; Nixon, C. F.; Teng, P. K.; Oltrogge, L. M.; Savage, D. F. Programmed Ribosomal Frameshifting Mediates Expression of the Alpha-Carboxysome. J. Mol. Biol. 2016, 428, 153– 164,  DOI: 10.1016/j.jmb.2015.11.017
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    20
    Programmed ribosomal frameshifting mediates expression of the α-carboxysome
    Chaijarasphong, Thawatchai; Nichols, Robert J.; Kortright, Kaitlyn E.; Nixon, Charlotte F.; Teng, Poh K.; Oltrogge, Luke M.; Savage, David F.
    Journal of Molecular Biology (2016), 428 (1), 153-164CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    Many bacteria employ a protein organelle, the carboxysome, to catalyze carbon dioxide fixation in the Calvin Cycle. Only 10 genes from Halothiobacillus neapolitanus are sufficient for heterologous expression of carboxysomes in Escherichia coli, opening the door to detailed mechanistic anal. of the assembly process of this complex (more than 200 MDa). One of these genes, csoS2, has been implicated in assembly but ascribing a mol. function is confounded by the observation that the single csoS2 gene yields expression of two gene products and both display an apparent mol. wt. incongruent with the predicted amino acid sequence. Here, we elucidate the co-translational mechanism responsible for the expression of the two protein isoforms. Specifically, csoS2 was found to possess - 1 frameshifting elements that lead to the prodn. of the full-length protein, CsoS2B, and a truncated protein, CsoS2A, which possesses a C-terminus translated from the alternate frame. The frameshifting elements comprise both a ribosomal slippery sequence and a 3' secondary structure, and ablation of either sequence is sufficient to eliminate the slip. Using these mutants, we investigated the individual roles of CsoS2B and CsoS2A on carboxysome formation. In this in vivo formation assay, cells expressing only the CsoS2B isoform were capable of producing intact carboxysomes, while those with only CsoS2A were not. Thus, we have answered a long-standing question about the nature of CsoS2 in this model microcompartment and demonstrate that CsoS2B is functionally distinct from CsoS2A in the assembly of α-carboxysomes.
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  21. 21
    Cai, F.; Dou, Z.; Bernstein, S. L.; Leverenz, R.; Williams, E. B.; Heinhorst, S.; Shively, J.; Cannon, G. C.; Kerfeld, C. A. Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component. Life 2015, 5, 1141– 1171,  DOI: 10.3390/life5021141
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    21
    Advances in understanding carboxysome assembly in Prochlorococcus and Synechococcus implicate CsoS2 as a critical component
    Cai, Fei; Dou, Zhicheng; Bernstein, Susan L.; Leverenz, Ryan; Williams, Eric B.; Heinhorst, Sabine; Shively, Jessup; Cannon, Gordon C.; Kerfeld, Cheryl A.
    Life (Basel, Switzerland) (2015), 5 (2), 1141-1171CODEN: LBSIB7; ISSN:2075-1729. (MDPI AG)
    The marine Synechococcus and Prochlorococcus are the numerically dominant cyanobacteria in the ocean and important in global carbon fixation. They have evolved a CO2-concg.-mechanism, of which the central component is the carboxysome, a self-assembling proteinaceous organelle. Two types of carboxysome, α and β, encapsulating form IA and form IB D-ribulose-1,5-bisphosphate carboxylase/oxygenase, resp.,differ in gene organization and assocd. proteins. In contrast to the β-carboxysome, the assembly process of the α-carboxysome is enigmatic. Moreover, an absolutely conserved α-carboxysome protein, CsoS2, is of unknown function and has proven recalcitrant to crystn. Here, we present studies on the CsoS2 protein in three model organisms and show that CsoS2 is vital for α-carboxysome biogenesis. The primary structure of CsoS2 appears tripartite, composed of an N-terminal, middle (M)-, and C-terminal region. Repetitive motifs can be identified in the N- and M-regions. Multiple lines of evidence suggest CsoS2 is highly flexible, possibly an intrinsically disordered protein. Based on our results from bioinformatic, biophys., genetic and biochem. approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-carboxysome. Analogies between the pathway for β-carboxysome biogenesis and our model for α-carboxysome assembly are discussed.
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  22. 22
    Li, T.; Jiang, Q.; Huang, J.; Aitchison, C. M.; Huang, F.; Yang, M.; Dykes, G. F.; He, H.-L.; Wang, Q.; Sprick, R. S.; Cooper, A. I.; Liu, L.-N. Reprogramming bacterial protein organelles as a nanoreactor for hydrogen production. Nat. Commun. 2020, 11, 5448  DOI: 10.1038/s41467-020-19280-0
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    22
    Reprogramming bacterial protein organelles as a nanoreactor for hydrogen production
    Li, Tianpei; Jiang, Qiuyao; Huang, Jiafeng; Aitchison, Catherine M.; Huang, Fang; Yang, Mengru; Dykes, Gregory F.; He, Hai-Lun; Wang, Qiang; Sprick, Reiner Sebastian; Cooper, Andrew I.; Liu, Lu-Ning
    Nature Communications (2020), 11 (1), 5448CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Abstr.: Compartmentalization is a ubiquitous building principle in cells, which permits segregation of biol. elements and reactions. The carboxysome is a specialized bacterial organelle that encapsulates enzymes into a virus-like protein shell and plays essential roles in photosynthetic carbon fixation. The naturally designed architecture, semi-permeability, and catalytic improvement of carboxysomes have inspired rational design and engineering of new nanomaterials to incorporate desired enzymes into the protein shell for enhanced catalytic performance. Here, we build large, intact carboxysome shells (over 90 nm in diam.) in the industrial microorganism Escherichia coli by expressing a set of carboxysome protein-encoding genes. We develop strategies for enzyme activation, shell self-assembly, and cargo encapsulation to construct a robust nanoreactor that incorporates catalytically active [FeFe]-hydrogenases and functional partners within the empty shell for the prodn. of hydrogen. We show that shell encapsulation and the internal microenvironment of the new catalyst facilitate hydrogen prodn. of the encapsulated oxygen-sensitive hydrogenases. The study provides insights into the assembly and formation of carboxysomes and paves the way for engineering carboxysome shell-based nanoreactors to recruit specific enzymes for diverse catalytic reactions.
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    Kerfeld, C. A.; Melnicki, M. R. Assembly, function and evolution of cyanobacterial carboxysomes. Curr. Opin. Plant Biol. 2016, 31, 66– 75,  DOI: 10.1016/j.pbi.2016.03.009
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    23
    Assembly, function and evolution of cyanobacterial carboxysomes
    Kerfeld, Cheryl A.; Melnicki, Matthew R.
    Current Opinion in Plant Biology (2016), 31 (), 66-75CODEN: COPBFZ; ISSN:1369-5266. (Elsevier Ltd.)
    All cyanobacteria contain carboxysomes, RuBisCO-encapsulating bacterial microcompartments that function as prokaryotic organelles. The two carboxysome types, alpha and beta, differ fundamentally in components, assembly, and species distribution. Alpha carboxysomes share a highly-conserved gene organization, with evidence of horizontal gene transfer from chemoautotrophic proteobacteria to the picocyanobacteria, and seem to co-assemble shells concomitantly with aggregation of cargo enzymes. In contrast, beta carboxysomes assemble an enzymic core first, with an encapsulation peptide playing a crit. role in formation of the surrounding shell. Based on similarities in assembly, and phylogenetic anal. of the pentameric shell protein conserved across all bacterial microcompartments, beta carboxysomes appear to be more closely related to the microcompartments of heterotrophic bacteria (metabolosomes) than to alpha carboxysomes, which appear deeply divergent. Beta carboxysomes can be found in the basal cyanobacterial clades that diverged before the ancestor of the chloroplast and have recently been shown to be able to encapsulate functional RuBisCO enzymes resurrected from ancestrally-reconstructed sequences, consistent with an ancient origin. Alpha and beta carboxysomes are not only distinct units of evolution, but are now emerging as genetic/metabolic modules for synthetic biol.; heterologous expression and redesign of both the shell and the enzymic core have recently been achieved.
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    Plegaria, J. S.; Kerfeld, C. A. Engineering nanoreactors using bacterial microcompartment architectures. Curr. Opin. Biotechnol. 2018, 51, 1– 7,  DOI: 10.1016/j.copbio.2017.09.005
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    24
    Engineering nanoreactors using bacterial microcompartment architectures
    Plegaria, Jefferson S.; Kerfeld, Cheryl A.
    Current Opinion in Biotechnology (2018), 51 (), 1-7CODEN: CUOBE3; ISSN:0958-1669. (Elsevier B.V.)
    Bacterial microcompartments (BMCs) are organelles that encapsulate enzymes involved in CO2 fixation or carbon catabolism in a selectively permeable protein shell. Here, we highlight recent advances in the bioengineering of these protein-based nanoreactors in heterologous systems, including transfer and expression of BMC gene clusters, the prodn. of template empty shells, and the encapsulation of non-native enzymes.
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    Didovyk, A.; Tonooka, T.; Tsimring, L.; Hasty, J. Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression. ACS Synth. Biol. 2017, 6, 2198– 2208,  DOI: 10.1021/acssynbio.7b00253
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    25
    Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression
    Didovyk, Andriy; Tonooka, Taishi; Tsimring, Lev; Hasty, Jeff
    ACS Synthetic Biology (2017), 6 (12), 2198-2208CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    Cell-free gene expression systems are emerging as an important platform for a diverse range of synthetic biol. and biotechnol. applications, including prodn. of robust field-ready biosensors. Here, the authors combine programmed cellular autolysis with a freeze-thaw or freeze-dry cycle to create a practical, reproducible, and a labor- and cost-effective approach for rapid prodn. of bacterial lysates for cell-free gene expression. Using this method, robust and highly active bacterial cell lysates can be produced without specialized equipment at a wide range of scales, making cell-free gene expression easily and broadly accessible. Moreover, live autolysis strain can be freeze-dried directly and subsequently lysed upon rehydration to produce active lysate. The authors demonstrate the utility of autolyzates for synthetic biol. by regulating protein prodn. and degrdn., implementing quorum sensing, and showing quant. protection of linear DNA templates by GamS protein. To allow versatile and sensitive β-galactosidase (LacZ) based readout the authors produce autolyzates with no detectable background LacZ activity and use them to produce sensitive mercury(II) biosensors with LacZ-mediated colorimetric and fluorescent outputs. The autolysis approach can facilitate wider adoption of cell-free technol. for cell-free gene expression as well as other synthetic biol. and biotechnol. applications, such as metabolic engineering, natural product biosynthesis, or proteomics.
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  26. 26
    Guo, Y.; Dong, J.; Zhou, T.; Auxillos, J.; Li, T.; Zhang, W.; Wang, L.; Shen, Y.; Luo, Y.; Zheng, Y.; Lin, J.; Chen, G. Q.; Wu, Q.; Cai, Y.; Dai, J. YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae. Nucleic Acids Res. 2015, 43, e88  DOI: 10.1093/nar/gkv464
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    26
    YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae
    Guo, Yakun; Dong, Junkai; Zhou, Tong; Auxillos, Jamie; Li, Tianyi; Zhang, Weimin; Wang, Lihui; Shen, Yue; Luo, Yisha; Zheng, Yijing; Lin, Jiwei; Chen, Guo-Qiang; Wu, Qingyu; Cai, Yizhi; Dai, Junbiao
    Nucleic Acids Research (2015), 43 (13), e88/1-e88/14CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    It is a routine task in metabolic engineering to introduce multicomponent pathways into a heterologous host for prodn. of metabolites. However, this process sometimes may take weeks to months due to the lack of standardized genetic tools. Here, we present a method for the design and construction of biol. parts based on the native genes and regulatory elements in Saccharomyces cerevisiae. We have developed highly efficient protocols (termed YeastFab Assembly) to synthesize these genetic elements as standardized biol. parts, which can be used to assemble transcriptional units in a single-tube reaction. In addn., standardized characterization assays are developed using reporter constructs to calibrate the function of promoters. Furthermore, the assembled transcription units can be either assayed individually or applied to construct multi-gene metabolic pathways, which targets a genomic locus or a receiving plasmid effectively, through a simple in vitro reaction. Finally, using β-carotene biosynthesis pathway as an example, we demonstrate that our method allows us not only to construct and test a metabolic pathway in several days, but also to optimize the prodn. through combinatorial assembly of a pathway using hundreds of regulatory biol. parts.
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  27. 27
    Waterhouse, A. M.; Procter, J. B.; Martin, D. M. A.; Clamp, M.; Barton, G. J. Jalview Version 2─a multiple sequence alignment editor and analysis workbench. Bioinformatics 2009, 25, 1189– 1191,  DOI: 10.1093/bioinformatics/btp033
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    Jalview Version 2-a multiple sequence alignment editor and analysis workbench
    Waterhouse, Andrew M.; Procter, James B.; Martin, David M. A.; Clamp, Michele; Barton, Geoffrey J.
    Bioinformatics (2009), 25 (9), 1189-1191CODEN: BOINFP; ISSN:1367-4803. (Oxford University Press)
    Summary: Jalview Version 2 is a system for interactive WYSIWYG editing, anal. and annotation of multiple sequence alignments. Core features include keyboard and mouse-based editing, multiple views and alignment overviews, and linked structure display with Jmol. Jalview 2 is available in two forms: a lightwt. Java applet for use in web applications, and a powerful desktop application that employs web services for sequence alignment, secondary structure prediction and the retrieval of alignments, sequences, annotation and structures from public databases and any DAS 1.53 compliant sequence or annotation server. Availability: The Jalview 2 Desktop application and JalviewLite applet are made freely available under the GPL, and can be downloaded from www.jalview.org.
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  28. 28
    Sievers, F.; Higgins, D. G. Clustal Omega, accurate alignment of very large numbers of sequences. Methods Mol. Biol. 2014, 1079, 105– 116,  DOI: 10.1007/978-1-62703-646-7_6
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    Clustal Omega, Accurate Alignment of Very Large Numbers of Sequences
    Sievers, Fabian; Higgins, Desmond G.
    Methods in Molecular Biology (New York, NY, United States) (2014), 1079 (Multiple Sequence Alignment Methods), 105-116CODEN: MMBIED; ISSN:1940-6029. (Springer)
    Clustal Omega is a completely rewritten and revised version of the widely used Clustal series of programs for multiple sequence alignment. It can deal with very large nos. (many tens of thousands) of DNA/RNA or protein sequences due to its use of the mBED algorithm for calcg. guide trees. This algorithm allows very large alignment problems to be tackled very quickly, even on personal computers. The accuracy of the program has been considerably improved over earlier Clustal programs, through the use of the HHalign method for aligning profile hidden Markov models. The program currently is used from the command line or can be run on line.
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    Nichols, T. M.; Kennedy, N. W.; Tullman-Ercek, D. Cargo encapsulation in bacterial microcompartments: Methods and analysis. Methods Enzymol. 2019, 617, 155– 186,  DOI: 10.1016/bs.mie.2018.12.009
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    Cargo encapsulation in bacterial microcompartments: methods and analysis
    Nichols, Taylor M.; Kennedy, Nolan W.; Tullman-Ercek, Danielle
    Methods in Enzymology (2019), 617 (Metabolons and Supramolecular Enzyme Assemblies), 155-186CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)
    A review. Metabolic engineers seek to produce high-value products from inexpensive starting materials in a sustainable and cost-effective manner by using microbes as cellular factories. However, pathway development and optimization can be arduous tasks, complicated by pathway bottlenecks and toxicity. Pathway organization has emerged as a potential soln. to these issues, and the use of protein- or DNA-based scaffolds has successfully increased the prodn. of several industrially relevant compds. These efforts demonstrate the usefulness of pathway colocalization and spatial organization for metabolic engineering applications. In particular, scaffolding within an enclosed, subcellular compartment shows great promise for pathway optimization, offering benefits such as increased local enzyme and substrate concns., sequestration of toxic or volatile intermediates, and alleviation of cofactor and resource competition with the host. Here, we describe the 1,2-propanediol utilization (Pdu) bacterial microcompartment (MCP) as an enclosed scaffold for pathway sequestration and organization. We first describe methods for controlling Pdu MCP formation, expressing and encapsulating heterologous cargo, and tuning cargo loading levels. We further describe assays for analyzing Pdu MCPs and assessing encapsulation levels. These methods will enable the repurposing of MCPs as tunable nanobioreactors for heterologous pathway encapsulation.
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    Lassila, J. K.; Bernstein, S. L.; Kinney, J. N.; Axen, S. D.; Kerfeld, C. A. Assembly of robust bacterial microcompartment shells using building blocks from an organelle of unknown function. J. Mol. Biol. 2014, 426, 2217– 2228,  DOI: 10.1016/j.jmb.2014.02.025
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    Assembly of Robust Bacterial Microcompartment Shells Using Building Blocks from an Organelle of Unknown Function
    Lassila, Jonathan K.; Bernstein, Susan L.; Kinney, James N.; Axen, Seth D.; Kerfeld, Cheryl A.
    Journal of Molecular Biology (2014), 426 (11), 2217-2228CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    Bacterial microcompartments (BMCs) sequester enzymes from the cytoplasmic environment by encapsulation inside a selectively permeable protein shell. Bioinformatic analyses indicate that many bacteria encode BMC clusters of unknown function and with diverse combinations of shell proteins. The genome of the halophilic myxobacterium Haliangium ochraceum encodes one of the most atypical sets of shell proteins in terms of compn. and primary structure. We found that microcompartment shells could be purified in high yield when all seven H. ochraceum BMC shell genes were expressed from a synthetic operon in Escherichia coli. These shells differ substantially from previously isolated shell systems in that they are considerably smaller and more homogeneous, with measured diams. of 39±2 nm. The size and nearly uniform geometry allowed the development of a structural model for the shells composed of 260 hexagonal units and 13 hexagons per icosahedral face. We found that new proteins could be recruited to the shells by fusion to a predicted targeting peptide sequence, setting the stage for the use of these remarkably homogeneous shells for applications such as three-dimensional scaffolding and the construction of synthetic BMCs. Our results demonstrate the value of selecting from the diversity of BMC shell building blocks found in genomic sequence data for the construction of novel compartments.
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    Anderson, J. C. Anderson Promoter Library Registry of Standard Biological Parts. http://parts.igem.org/Promoters/Catalog/Anderson (accessed 1 May, 2019).
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    Yadav, V. G.; De Mey, M.; Lim, C. G.; Ajikumar, P. K.; Stephanopoulos, G. The future of metabolic engineering and synthetic biology: towards a systematic practice. Metab. Eng. 2012, 14, 233– 241,  DOI: 10.1016/j.ymben.2012.02.001
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    The future of metabolic engineering and synthetic biology: Towards a systematic practice
    Yadav, Vikramaditya G.; De Mey, Marjan; Giaw Lim, Chin; Kumaran Ajikumar, Parayil; Stephanopoulos, Gregory
    Metabolic Engineering (2012), 14 (3), 233-241CODEN: MEENFM; ISSN:1096-7176. (Elsevier B. V.)
    Industrial biotechnol. promises to revolutionize conventional chem. manufg. in the years ahead, largely owing to the excellent progress in our ability to re-engineer cellular metab. However, most successes of metabolic engineering have been confined to over-producing natively synthesized metabolites in E. coli and S. cerevisiae. A major reason for this development has been the descent of metabolic engineering, particularly secondary metabolic engineering, to a collection of demonstrations rather than a systematic practice with generalizable tools. Synthetic biol., a more recent development, faces similar criticisms. Herein, we attempt to lay down a framework around which bioreaction engineering can systematize itself just like chem. reaction engineering. Central to this undertaking is a new approach to engineering secondary metab. known as 'multivariate modular metabolic engineering' (MMME), whose novelty lies in its assessment and elimination of regulatory and pathway bottlenecks by re-defining the metabolic network as a collection of distinct modules. After introducing the core principles of MMME, we shall then present a no. of recent developments in secondary metabolic engineering that could potentially serve as its facilitators. It is hoped that the ever-declining costs of de novo gene synthesis; the improved use of bioinformatic tools to mine, sort and analyze biol. data; and the increasing sensitivity and sophistication of investigational tools will make the maturation of microbial metabolic engineering an autocatalytic process. Encouraged by these advances, research groups across the world would take up the challenge of secondary metabolite prodn. in simple hosts with renewed vigor, thereby adding to the range of products synthesized using metabolic engineering.
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    Baneyx, F. Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 1999, 10, 411– 421,  DOI: 10.1016/S0958-1669(99)00003-8
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    Recombinant protein expression in Escherichia coli
    Baneyx, Francois
    Current Opinion in Biotechnology (1999), 10 (5), 411-421CODEN: CUOBE3; ISSN:0958-1669. (Current Biology Publications)
    A review with 69 refs. Escherichia coli is one of the most widely used hosts for the prodn. of heterologous proteins and its genetics are far better characterized than those of any other microorganism. Recent progress in the fundamental understanding of transcription, translation, and protein folding in E. coli, together with serendipitous discoveries and the availability of improved genetic tools are making this bacterium more valuable than ever for the expression of complex eukaryotic proteins.
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    Oltrogge, L. M.; Chaijarasphong, T.; Chen, A. W.; Bolin, E. R.; Marqusee, S.; Savage, D. F. Multivalent interactions between CsoS2 and Rubisco mediate α-carboxysome formation. Nat. Struct. Mol. Biol. 2020, 27, 281– 287,  DOI: 10.1038/s41594-020-0387-7
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    Multivalent interactions between CsoS2 and Rubisco mediate α-carboxysome formation
    Oltrogge, Luke M.; Chaijarasphong, Thawatchai; Chen, Allen W.; Bolin, Eric R.; Marqusee, Susan; Savage, David F.
    Nature Structural & Molecular Biology (2020), 27 (3), 281-287CODEN: NSMBCU; ISSN:1545-9993. (Nature Research)
    Abstr.: Carboxysomes are bacterial microcompartments that function as the centerpiece of the bacterial CO2-concg. mechanism by facilitating high CO2 concns. near the carboxylase Rubisco. The carboxysome self-assembles from thousands of individual proteins into icosahedral-like particles with a dense enzyme cargo encapsulated within a proteinaceous shell. In the case of the α-carboxysome, there is little mol. insight into protein-protein interactions that drive the assembly process. Here, studies on the α-carboxysome from Halothiobacillus neapolitanus demonstrate that Rubisco interacts with the N terminus of CsoS2, a multivalent, intrinsically disordered protein. X-ray structural anal. of the CsoS2 interaction motif bound to Rubisco reveals a series of conserved electrostatic interactions that are only made with properly assembled hexadecameric Rubisco. Although biophys. measurements indicate that this single interaction is weak, its implicit multivalency induces high-affinity binding through avidity. Taken together, our results indicate that CsoS2 acts as an interaction hub to condense Rubisco and enable efficient α-carboxysome formation.
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    Gonzalez, M.; Frank, E. G.; Levine, A. S.; Woodgate, R. Lon-mediated proteolysis of the Escherichia coli UmuD mutagenesis protein: in vitro degradation and identification of residues required for proteolysis. Genes Dev. 1998, 12, 3889– 3899,  DOI: 10.1101/gad.12.24.3889
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    Lon-mediated proteolysis of the Escherichia coli UmuD mutagenesis protein: in vitro degradation and identification of residues required for proteolysis
    Gonzalez, Martin; Frank, Ekaterina G.; Levine, Arthur S.; Woodgate, Roger
    Genes & Development (1998), 12 (24), 3889-3899CODEN: GEDEEP; ISSN:0890-9369. (Cold Spring Harbor Laboratory Press)
    Most SOS mutagenesis in Escherichia coli is dependent on the UmuD and UmuC proteins. Perhaps as a consequence, the activity of these proteins is exquisitely regulated. The intracellular level of UmuD and UmuC is normally quite low but increases dramatically in Ion- strains, suggesting that both proteins are substrates of the Lon protease. We report here that the highly purified UmuD protein is specifically degraded in vitro by Lon in an ATP-dependent manner. To identify the regions of UmuD necessary for Lon-mediated proteolysis, we performed 'alanine-stretch' mutagenesis on umuD and followed the stability of the mutant protein in vivo. Such an approach allowed us to localize the site(s) within UmuD responsible for Lon-mediated proteolysis. The primary signal is located between residues 15 and 18 (FPLF), with an auxiliary site between residues 26 and 29 (FPSP), of the amino terminus of UmuD. Transfer of the amino terminus of UmuD (residues 1-40) to an otherwise stable protein imparts Lon-mediated proteolysis, thereby indicating that the amino terminus of UmuD is sufficient for Lon recognition and the ensuing degrdn. of the protein.
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    Neher, S. B.; Sauer, R. T.; Baker, T. A. Distinct peptide signals in the UmuD and UmuD′ subunits of UmuD/D′ mediate tethering and substrate processing by the ClpXP protease. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13219– 13224,  DOI: 10.1073/pnas.2235804100
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    Distinct peptide signals in the UmuD and UmuD' subunits of UmuD/D' mediate tethering and substrate processing by the ClpXP protease
    Neher, Saskia B.; Sauer, Robert T.; Baker, Tania A.
    Proceedings of the National Academy of Sciences of the United States of America (2003), 100 (23), 13219-13224CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The Escherichia coli UmuD' protein is a component of DNA polymerase V, an error-prone polymerase that carries out translesion synthesis on damaged DNA templates. The intracellular concn. of UmuD' is strictly controlled by regulated transcription, by posttranslational processing of UmuD to UmuD', and by ClpXP degrdn. UmuD' is a substrate for the ClpXP protease but must form a heterodimer with its unabbreviated precursor, UmuD, for efficient degrdn. to occur. Here, we show that UmuD functions as a UmuD' delivery protein for ClpXP. UmuD can also deliver a UmuD partner for degrdn. UmuD resembles SspB, a well-characterized substrate-delivery protein for ClpX, in that both proteins use related peptide motifs to bind to the N-terminal domain of ClpX, thereby tethering substrate complexes to ClpXP. The combined use of a weak substrate recognition signal and a delivery factor that tethers the substrate to the protease allows regulated proteolysis of UmuD/D' in the cell. Dual recognition strategies of this type may be a relatively common feature of intracellular protein turnover.
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    Ferlez, B.; Sutter, M.; Kerfeld, C. A. A designed bacterial microcompartment shell with tunable composition and precision cargo loading. Metab. Eng. 2019, 54, 286– 291,  DOI: 10.1016/j.ymben.2019.04.011
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    A designed bacterial microcompartment shell with tunable composition and precision cargo loading
    Ferlez, Bryan; Sutter, Markus; Kerfeld, Cheryl A.
    Metabolic Engineering (2019), 54 (), 286-291CODEN: MEENFM; ISSN:1096-7176. (Elsevier B.V.)
    Microbes often augment their metab. by conditionally constructing proteinaceous organelles, known as bacterial microcompartments (BMCs), that encapsulate enzymes to degrade org. compds. or assimilate CO2. BMCs self-assemble and are spatially delimited by a semi-permeable shell made up of hexameric, trimeric, and pentameric shell proteins. Bioengineers aim to recapitulate the organization and efficiency of these complex biol. architectures by redesigning the shell to incorporate non-native enzymes from biotechnol. relevant pathways. To meet this challenge, a diverse set of synthetic biol. tools are required, including methods to manipulate the properties of the shell as well as target and organize cargo encapsulation. We designed and detd. the crystal structure of a synthetic shell protein building block with an inverted sidedness of its N- and C-terminal residues relative to its natural counterpart; the inversion targets genetically fused protein cargo to the lumen of the shell. Moreover, the titer of fluorescent protein cargo encapsulated using this strategy is controllable using an inducible tetracycline promoter. These results expand the available set of building blocks for precision engineering of BMC-based nanoreactors and are compatible with orthogonal methods which will facilitate the installation and organization of multi-enzyme pathways.
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    Tsai, Y.; Sawaya, M. R.; Cannon, G. C.; Cai, F.; Williams, E. B.; Heinhorst, S.; Kerfeld, C. A.; Yeates, T. O. Structural Analysis of CsoS1A and the Protein Shell of the Halothiobacillus neapolitanus Carboxysome. PLoS Biol. 2007, 5, e144  DOI: 10.1371/journal.pbio.0050144
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    Klein, M. G.; Zwart, P.; Bagby, S. C.; Cai, F.; Chisholm, S. W.; Heinhorst, S.; Cannon, G. C.; Kerfeld, C. A. Identification and structural analysis of a novel carboxysome shell protein with implications for metabolite transport. J. Mol. Biol. 2009, 392, 319– 333,  DOI: 10.1016/j.jmb.2009.03.056
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    Identification and Structural Analysis of a Novel Carboxysome Shell Protein with Implications for Metabolite Transport
    Klein, Michael G.; Zwart, Peter; Bagby, Sarah C.; Cai, Fei; Chisholm, Sallie W.; Heinhorst, Sabine; Cannon, Gordon C.; Kerfeld, Cheryl A.
    Journal of Molecular Biology (2009), 392 (2), 319-333CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    Bacterial microcompartments (BMCs) are polyhedral bodies, composed entirely of proteins, that function as organelles in bacteria; they promote subcellular processes by encapsulating and co-localizing targeted enzymes with their substrates. The best-characterized BMC is the carboxysome, a central part of the carbon-concg. mechanism that greatly enhances carbon fixation in cyanobacteria and some chemoautotrophs. Here we report the first structural insights into the carboxysome of Prochlorococcus, the numerically dominant cyanobacterium in the world's oligotrophic oceans. Bioinformatic methods, substantiated by anal. of gene expression data, were used to identify a new carboxysome shell component, CsoS1D, in the genome of Prochlorococcus strain MED4; orthologs were subsequently found in all cyanobacteria. Two independent crystal structures of Prochlorococcus MED4 CsoS1D reveal three features not seen in any BMC-domain protein structure solved to date. First, CsoS1D is composed of a fused pair of BMC domains. Second, this double-domain protein trimerizes to form a novel pseudohexameric building block for incorporation into the carboxysome shell, and the trimers further dimerize, forming a two-tiered shell building block. Third, and most strikingly, the large pore formed at the 3-fold axis of symmetry appears to be gated. Each dimer of trimers contains one trimer with an open pore and one whose pore is obstructed due to side-chain conformations of two residues that are invariant among all CsoS1D orthologs. This is the first evidence of the potential for gated transport across the carboxysome shell and reveals a new type of building block for BMC shells.
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    Mohajerani, F.; Sayer, E.; Neil, C.; Inlow, K.; Hagan, M. F. Mechanisms of Scaffold-Mediated Microcompartment Assembly and Size Control. ACS Nano 2021, 15, 4197– 4212,  DOI: 10.1021/acsnano.0c05715
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    Mechanisms of Scaffold-Mediated Microcompartment Assembly and Size Control
    Mohajerani, Farzaneh; Sayer, Evan; Neil, Christopher; Inlow, Koe; Hagan, Michael F.
    ACS Nano (2021), 15 (3), 4197-4212CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    This article describes a theor. and computational study of the dynamical assembly of a protein shell around a complex consisting of many cargo mols. and long, flexible scaffold mols. Our study is motivated by bacterial microcompartments, which are proteinaceous organelles that assemble around a condensed droplet of enzymes and reactants. As in many examples of cytoplasmic liq.-liq. phase sepn., condensation of the microcompartment interior cargo is driven by flexible scaffold proteins that have weak multivalent interactions with the cargo. Our results predict that the shell size, amt. of encapsulated cargo, and assembly pathways depend sensitively on properties of the scaffold, including its length and valency of scaffold-cargo interactions. Moreover, the ability of self-assembling protein shells to change their size to accommodate scaffold mols. of different lengths depends crucially on whether the spontaneous curvature radius of the protein shell is smaller or larger than a characteristic elastic length scale of the shell. Beyond natural microcompartments, these results have important implications for synthetic biol. efforts to target alternative mols. for encapsulation by microcompartments or viral shells. More broadly, the results elucidate how cells exploit coupling between self-assembly and liq.-liq. phase sepn. to organize their interiors.
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    Sinha, S.; Cheng, S.; Sung, Y. W.; McNamara, D. E.; Sawaya, M. R.; Yeates, T. O.; Bobik, T. A. Alanine scanning mutagenesis identifies an asparagine-arginine-lysine triad essential to assembly of the shell of the Pdu microcompartment. J. Mol. Biol. 2014, 426, 2328– 2345,  DOI: 10.1016/j.jmb.2014.04.012
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    Alanine Scanning Mutagenesis Identifies an Asparagine-Arginine-Lysine Triad Essential to Assembly of the Shell of the Pdu Microcompartment
    Sinha, Sharmistha; Cheng, Shouqiang; Sung, Yea Won; McNamara, Dan E.; Sawaya, Michael R.; Yeates, Todd O.; Bobik, Thomas A.
    Journal of Molecular Biology (2014), 426 (12), 2328-2345CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    Bacterial microcompartments (MCPs) are the simplest organelles known. They function to enhance metabolic pathways by confining several related enzymes inside an all-protein envelope called the shell. In this study, we investigated the factors that govern MCP assembly by performing scanning mutagenesis on the surface residues of PduA, a major shell protein of the MCP used for 1,2-propanediol degrdn. Biochem., genetic, and structural anal. of 20 mutants allowed us to det. that PduA K26, N29, and R79 are crucial residues that stabilize the shell of the 1,2-propanediol MCP. In addn., we identify two PduA mutants (K37A and K55A) that impair MCP function most likely by altering the permeability of its protein shell. These are the first studies to examine the phenotypic effects of shell protein structural mutations in an MCP system. The findings reported here may be applicable to engineering protein containers with improved stability for biotechnol. applications.
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    Cai, F.; Sutter, M.; Cameron, J. C.; Stanley, D. N.; Kinney, J. N.; Kerfeld, C. A. The structure of CcmP, a tandem bacterial microcompartment domain protein from the β-carboxysome, forms a subcompartment within a microcompartment. J. Biol. Chem. 2013, 288, 16055– 16063,  DOI: 10.1074/jbc.M113.456897
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    The Structure of CcmP, a Tandem Bacterial Microcompartment Domain Protein from the β-Carboxysome, Forms a Subcompartment Within a Microcompartment
    Cai, Fei; Sutter, Markus; Cameron, Jeffrey C.; Stanley, Desiree N.; Kinney, James N.; Kerfeld, Cheryl A.
    Journal of Biological Chemistry (2013), 288 (22), 16055-16063CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The carboxysome is a bacterial organelle found in all cyanobacteria; it encapsulates CO2 fixation enzymes within a protein shell. The most abundant carboxysome shell protein contains a single bacterial microcompartment (BMC) domain. We present in vivo evidence that a hypothetical protein (dubbed CcmP) encoded in all β-cyanobacterial genomes is part of the carboxysome. We show that CcmP is a tandem BMC domain protein, the first to be structurally characterized from a β-carboxysome. CcmP forms a dimer of tightly stacked trimers, resulting in a nanocompartment-contg. shell protein that may weakly bind 3-phosphoglycerate, the product of CO2 fixation. The trimers have a large central pore through which metabolites presumably pass into the carboxysome. Conserved residues surrounding the pore have alternate side-chain conformations suggesting that it can be open or closed. Furthermore, CcmP and its orthologs in α-cyanobacterial genomes form a distinct clade of shell proteins. Members of this subgroup are also found in numerous heterotrophic BMC-assocd. gene clusters encoding functionally diverse bacterial organelles, suggesting that the potential to form a nanocompartment within a microcompartment shell is widespread. Given that carboxysomes and architecturally related bacterial organelles are the subject of intense interest for applications in synthetic biol./metabolic engineering, our results describe a new type of building block with which to functionalize BMC shells.
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    Szatmári, D.; Sárkány, P.; Kocsis, B.; Nagy, T.; Miseta, A.; Barkó, S.; Longauer, B.; Robinson, R. C.; Nyitrai, M. Intracellular ion concentrations and cation-dependent remodelling of bacterial MreB assemblies. Sci. Rep. 2020, 10, 12002  DOI: 10.1038/s41598-020-68960-w
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    Intracellular ion concentrations and cation-dependent remodelling of bacterial MreB assemblies
    Szatmari, David; Sarkany, Peter; Kocsis, Bela; Nagy, Tamas; Miseta, Attila; Barko, Szilvia; Longauer, Beata; Robinson, Robert C.; Nyitrai, Miklos
    Scientific Reports (2020), 10 (1), 12002CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
    Abstr.: Here, we measured the concns. of several ions in cultivated Gram-neg. and Gram-pos. bacteria, and analyzed their effects on polymer formation by the actin homolog MreB. We measured potassium, sodium, chloride, calcium and magnesium ion concns. in Leptospira interrogans, Bacillus subtilis and Escherichia coli. Intracellular ionic strength contributed from these ions varied within the 130-273 mM range. The intracellular sodium ion concn. range was between 122 and 296 mM and the potassium ion concn. range was 5 and 38 mM. However, the levels were significantly influenced by extracellular ion levels. L. interrogans, Rickettsia rickettsii and E. coli MreBs were heterologously expressed and purified from E. coli using a novel filtration method to prep. MreB polymers. The structures and stability of Alexa-488 labeled MreB polymers, under varying ionic strength conditions, were investigated by confocal microscopy and MreB polymn. rates were assessed by measuring light scattering. MreB polymn. was fastest in the presence of monovalent cations in the 200-300 mM range. MreB filaments showed high stability in this concn. range and formed large assemblies of tape-like bundles that transformed to extensive sheets at higher ionic strengths. Changing the calcium concn. from 0.2 to 0 mM and then to 2 mM initialized rapid remodelling of MreB polymers.
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    Mahinthichaichan, P.; Morris, D. M.; Wang, Y.; Jensen, G. J.; Tajkhorshid, E. Selective Permeability of Carboxysome Shell Pores to Anionic Molecules. J. Phys. Chem. B 2018, 122, 9110– 9118,  DOI: 10.1021/acs.jpcb.8b06822
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    Selective Permeability of Carboxysome Shell Pores to Anionic Molecules
    Mahinthichaichan, Paween; Morris, Dylan M.; Wang, Yi; Jensen, Grant J.; Tajkhorshid, Emad
    Journal of Physical Chemistry B (2018), 122 (39), 9110-9118CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
    Carboxysomes are closed polyhedral cellular microcompartments that increase the efficiency of carbon fixation in autotrophic bacteria. Carboxysome shells consist of small proteins that form hexameric units with semi-permeable central pores contg. binding sites for anions. This feature is thought to selectively allow access to RuBisCO enzymes inside the carboxysome by HCO3- (the dominant form of CO2 in the aq. soln. at pH 7.4) but not O2, which leads to a non-productive reaction. To test this hypothesis, here we use mol. dynamics simulations to characterize the energetics and permeability of CO2, O2, and HCO3- through the central pores of two different shell proteins, namely, CsoS1A of α-carboxysome and CcmK4 of β-carboxysome shells. We find that the central pores are in fact selectively permeable to anions such as HCO3-, as predicted by the model.
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    Marsh, J. A.; Forman-Kay, J. D. Sequence determinants of compaction in intrinsically disordered proteins. Biophys. J. 2010, 98, 2383– 2390,  DOI: 10.1016/j.bpj.2010.02.006
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    Sequence determinants of compaction in intrinsically disordered proteins
    Marsh, Joseph A.; Forman-Kay, Julie D.
    Biophysical Journal (2010), 98 (10), 2383-2390CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    Intrinsically disordered proteins (IDPs), which lack folded structure and are disordered under nondenaturing conditions, have been shown to perform important functions in a large no. of cellular processes. These proteins have interesting structural properties that deviate from the random-coil-like behavior exhibited by chem. denatured proteins. In particular, IDPs are often obsd. to exhibit significant compaction. In this study, we have analyzed the hydrodynamic radii of a no. of IDPs to investigate the sequence determinants of this compaction. Net charge and proline content are obsd. to be strongly correlated with increased hydrodynamic radii, suggesting that these are the dominant contributors to compaction. Hydrophobicity and secondary structure, on the other hand, appear to have negligible effects on compaction, which implies that the determinants of structure in folded and intrinsically disordered proteins are profoundly different. Finally, we observe that polyhistidine tags seem to increase IDP compaction, which suggests that these tags have significant perturbing effects and thus should be removed before any structural characterizations of IDPs. Using the relationships obsd. in this anal., we have developed a sequence-based predictor of hydrodynamic radius for IDPs that shows substantial improvement over a simple model based upon chain length alone.
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    Baker, N. A.; Sept, D.; Joseph, S.; Holst, M. J.; McCammon, J. A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 10037– 10041,  DOI: 10.1073/pnas.181342398
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    Electrostatics of nanosystems: application to microtubules and the ribosome
    Baker, Nathan A.; Sept, David; Joseph, Simpson; Holst, Michael J.; McCammon, J. Andrew
    Proceedings of the National Academy of Sciences of the United States of America (2001), 98 (18), 10037-10041CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Evaluation of the electrostatic properties of biomols. has become a std. practice in mol. biophysics. Foremost among the models used to elucidate the electrostatic potential is the Poisson-Boltzmann equation; however, existing methods for solving this equation have limited the scope of accurate electrostatic calcns. to relatively small biomol. systems. Here we present the application of numerical methods to enable the trivially parallel soln. of the Poisson-Boltzmann equation for supramol. structures that are orders of magnitude larger in size. As a demonstration of this methodol., electrostatic potentials have been calcd. for large microtubule and ribosome structures. The results point to the likely role of electrostatics in a variety of activities of these structures.
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    Silva, C.; Martins, M.; Jing, S.; Fu, J.; Cavaco-Paulo, A. Practical insights on enzyme stabilization. Crit. Rev. Biotechnol. 2018, 38, 335– 350,  DOI: 10.1080/07388551.2017.1355294
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    Practical insights on enzyme stabilization
    Silva, Carla; Martins, Madalena; Jing, Su; Fu, Jiajia; Cavaco-Paulo, Artur
    Critical Reviews in Biotechnology (2018), 38 (3), 335-350CODEN: CRBTE5; ISSN:0738-8551. (Taylor & Francis Ltd.)
    Enzymes are efficient catalysts designed by nature to work in physiol. environments of living systems. The best operational conditions to access and convert substrates at the industrial level are different from nature and normally extreme. Strategies to isolate enzymes from extremophiles can redefine new operational conditions, however not always solving all industrial requirements. The stability of enzymes is therefore a key issue on the implementation of the catalysts in industrial processes which require the use of extreme environments that can undergo enzyme instability. Strategies for enzyme stabilization have been exhaustively reviewed, however they lack a practical approach. This review intends to compile and describe the most used approaches for enzyme stabilization highlighting case studies in a practical point of view.
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    Tan, Y. Q.; Xue, B.; Yew, W. S. Genetically Encodable Scaffolds for Optimizing Enzyme Function. Molecules 2021, 26, 1389  DOI: 10.3390/molecules26051389
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    Genetically encodable scaffolds for optimizing enzyme function
    Tan, Yong Quan; Xue, Bo; Yew, Wen Shan
    Molecules (2021), 26 (5), 1389CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
    Enzyme engineering is an indispensable tool in the field of synthetic biol., where enzymes are challenged to carry out novel or improved functions. Achieving these goals sometimes goes beyond modifying the primary sequence of the enzyme itself. The use of protein or nucleic acid scaffolds to enhance enzyme properties has been reported for applications such as microbial prodn. of chems., biosensor development and bioremediation. Key advantages of using these assemblies include optimizing reaction conditions, improving metabolic flux and increasing enzyme stability. This review summarizes recent trends in utilizing genetically encodable scaffolds, developed in line with synthetic biol. methodologies, to complement the purposeful deployment of enzymes. Current mol. tools for constructing these synthetic enzyme-scaffold systems are also highlighted.
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    Küchler, A.; Yoshimoto, M.; Luginbühl, S.; Mavelli, F.; Walde, P. Enzymatic reactions in confined environments. Nat. Nanotechnol. 2016, 11, 409– 420,  DOI: 10.1038/nnano.2016.54
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    Enzymatic reactions in confined environments
    Kuchler, Andreas; Yoshimoto, Makoto; Luginbuhl, Sandra; Mavelli, Fabio; Walde, Peter
    Nature Nanotechnology (2016), 11 (5), 409-420CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
    A review. Within each biol. cell, surface- and vol.-confined enzymes control a highly complex network of chem. reactions. These reactions are efficient, timely, and spatially defined. Efforts to transfer such appealing features to in vitro systems have led to several successful examples of chem. reactions catalyzed by isolated and immobilized enzymes. In most cases, these enzymes are either bound or adsorbed to an insol. support, phys. trapped in a macromol. network, or encapsulated within compartments. Advanced applications of enzymic cascade reactions with immobilized enzymes include enzymic fuel cells and enzymic nanoreactors, both for in vitro and possible in vivo applications. Here, the authors discuss some of the general principles of enzymic reactions confined on surfaces, at interfaces, and inside small vols. The authors also highlight the similarities and differences between the in vivo and in vitro cases and attempt to critically evaluate some of the necessary future steps to improve the fundamental understanding of these systems.
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    Das, S.; Zhao, L.; Elofson, K.; Finn, M. G. Enzyme Stabilization by Virus-Like Particles. Biochemistry 2020, 59, 2870– 2881,  DOI: 10.1021/acs.biochem.0c00435
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    Enzyme Stabilization by Virus-Like Particles
    Das, Soumen; Zhao, Liangjun; Elofson, Kristen; Finn, M. G.
    Biochemistry (2020), 59 (31), 2870-2881CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The properties of enzymes packaged within the coat protein shell of virus-like particles (VLPs) were studied to provide a comprehensive assessment of such factors. Such entrainment did not seem to perturb enzyme function, but it did significantly enhance enzyme stability against several denaturing stimuli such as heat, org. solvents, and chaotropic agents. This improvement in performance is general and independent of the no. of independent subunits required and of the no. of catalytically active enzymes packaged. Packaged enzymes were found by measurements of intrinsic tryptophan fluorescence to retain some of their native folded structure even longer than their catalytic activity, suggesting that protein folding is a significant component of the obsd. catalytic benefits. While the authors are unable to distinguish between kinetic and thermodn. effects - including inhibition of enzyme unfolding, acceleration of refolding, and biasing of folding equil. - VLP packaging appears to represent a useful general strategy for the stabilization of enzymes that operate on diffusible substrates and products.
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    Patterson, D. P.; Schwarz, B.; El-Boubbou, K.; van der Oost, J.; Prevelige, P. E.; Douglas, T. Virus-like particle nanoreactors: programmed encapsulation of the thermostable CelB glycosidase inside the P22 capsid. Soft Matter 2012, 8, 10158– 10166,  DOI: 10.1039/c2sm26485d
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    Virus-like particle nanoreactors: programmed encapsulation of the thermostable CelB glycosidase inside the P22 capsid
    Patterson, Dustin P.; Schwarz, Benjamin; El-Boubbou, Kheireddine; van der Oost, John; Prevelige, Peter E.; Douglas, Trevor
    Soft Matter (2012), 8 (39), 10158-10166CODEN: SMOABF; ISSN:1744-683X. (Royal Society of Chemistry)
    Self-assembling biol. systems hold great potential for the synthetic construction of new active soft nanomaterials. Here we demonstrate the hierarchical bottom-up assembly of bacteriophage P22 virus-like particles (VLPs) that encapsulate the thermostable CelB glycosidase creating catalytically active nanoreactors. The in vivo assembly and encapsulation produces P22 VLPs with a high packaging d. of the tetrameric CelB, but without loss of enzyme activity or the ability of the P22 VLP to undergo unique morphol. transitions that modify the VLPs internal vol. and shell porosity. The P22 VLPs encapsulating CelB are also shown to retain a high percentage of the enzyme activity upon being embedded and immobilized in a polymeric matrix.
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    Patterson, D. P.; Prevelige, P. E.; Douglas, T. Nanoreactors by Programmed Enzyme Encapsulation Inside the Capsid of the Bacteriophage P22. ACS Nano 2012, 6, 5000– 5009,  DOI: 10.1021/nn300545z
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    Nanoreactors by Programmed Enzyme Encapsulation Inside the Capsid of the Bacteriophage P22
    Patterson, Dustin P.; Prevelige, Peter E.; Douglas, Trevor
    ACS Nano (2012), 6 (6), 5000-5009CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    The virus like particle (VLP) derived from bacteriophage P22 presents a unique platform for constructing catalytically functional nanomaterials by encapsulation of enzymes into its interior. Encapsulation has been engineered to be genetically programmed allowing "one pot" synthesis and incorporation of desired enzymes. The unique characteristic that separates P22 from other VLP systems is the ability to modulate the overall vol. and porosity of the VLP structure, thus controlling substrate access to the encapsulated enzyme. The present study demonstrates incorporation of an enzyme, alc. dehydrogenase D, with the highest internal loading for an active enzyme by any VLP described thus far. In addn., we show that not only does encapsulating AdhD inside P22 affect its kinetic parameters in comparison with the "free" enzyme, but transformation of P22 to different morphol. states, which changes the internal vol. of the VLP, yields changes in the overall activity of the encapsulated enzyme as well. The findings reported here clearly illustrate that P22 holds potential for synthetic approaches to create nanoreactors, by design, using the power of highly evolved enzymes for chem. transformations.
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    Sánchez-Sánchez, L.; Tapia-Moreno, A.; Juarez-Moreno, K.; Patterson, D. P.; Cadena-Nava, R. D.; Douglas, T.; Vazquez-Duhalt, R. Design of a VLP-nanovehicle for CYP450 enzymatic activity delivery. J. Nanobiotechnol. 2015, 13, 66  DOI: 10.1186/s12951-015-0127-z
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    Design of a VLP-nanovehicle for CYP450 enzymatic activity delivery
    Sanchez-Sanchez, Lorena; Tapia-Moreno, Alejandro; Juarez-Moreno, Karla; Patterson, Dustin P.; Cadena-Nava, Ruben D.; Douglas, Trevor; Vazquez-Duhalt, Rafael
    Journal of Nanobiotechnology (2015), 13 (), 66/1-66/10CODEN: JNOAAO; ISSN:1477-3155. (BioMed Central Ltd.)
    Background: The intracellular delivery of enzymes for therapeutic use has a promising future for the treatment of several diseases such as genetic disorders and cancer. Virus-like particles offer an interesting platform for enzymic delivery to targeted cells because of their great cargo capacity and the enhancement of the biocatalyst stability towards several factors important in the practical application of these nanoparticles. Results: We have designed a nano-bioreactor based on the encapsulation of a cytochrome P 450 (CYP) inside the capsid derived from the bacteriophage P22. An enhanced peroxigenase, CYPBM3, was selected as a model enzyme because of its potential in enzyme prodrug therapy. A total of 109 enzymes per capsid were encapsulated with a 70 % retention of activity for cytochromes with the correct incorporation of the heme cofactor. Upon encapsulation, the stability of the enzyme towards protease degrdn. and acidic pH was increased. Cytochrome P 450 activity was delivered into Human cervix carcinoma cells via transfecting P22-CYP nanoparticles with lipofectamine. Conclusion: This work provides a clear demonstration of the potential of biocatalytic virus-like particles as medical relevant enzymic delivery vehicles for clin. applications.
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    Azuma, Y.; Zschoche, R.; Tinzl, M.; Hilvert, D. Quantitative Packaging of Active Enzymes into a Protein Cage. Angew. Chem., Int. Ed 2016, 55, 1531– 1534,  DOI: 10.1002/anie.201508414
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    Quantitative Packaging of Active Enzymes into a Protein Cage
    Azuma, Yusuke; Zschoche, Reinhard; Tinzl, Matthias; Hilvert, Donald
    Angewandte Chemie, International Edition (2016), 55 (4), 1531-1534CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Genetic fusion of cargo proteins to a pos. supercharged variant of green fluorescent protein enables their quant. encapsulation by engineered lumazine synthase capsids possessing a neg. charged lumenal surface. This simple tagging system provides a robust and versatile means of creating hierarchically ordered protein assemblies for use as nanoreactors. The generality of the encapsulation strategy and its effect on enzyme function were investigated with eight structurally and mechanistically distinct catalysts.
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    Yu, Z.; Reid, J. C.; Yang, Y.-P. Utilizing Dynamic Light Scattering as a Process Analytical Technology for Protein Folding and Aggregation Monitoring in Vaccine Manufacturing. J. Pharm. Sci. 2013, 102, 4284– 4290,  DOI: 10.1002/jps.23746
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    Utilizing Dynamic Light Scattering as a Process Analytical Technology for Protein Folding and Aggregation Monitoring in Vaccine Manufacturing
    Yu, Zhou; Reid, Jennifer C.; Yang, Yan-Ping
    Journal of Pharmaceutical Sciences (2013), 102 (12), 4284-4290CODEN: JPMSAE; ISSN:0022-3549. (John Wiley & Sons, Inc.)
    Protein aggregation is a common challenge in the manufg. of biol. products. It is possible to minimize the extent of aggregation through timely measurement and in-depth characterization of aggregation. In this study, we demonstrated the use of dynamic light scattering (DLS) to monitor inclusion body (IB) solubilization, protein refolding, and aggregation near the prodn. line of a recombinant protein-based vaccine candidate. Our results were in good agreement with those measured by size-exclusion chromatog. DLS was also used to characterize the mechanism of aggregation. As DLS is a quick, nonperturbing technol., it can potentially be used as an at-line process anal. technol. to ensure complete IB solubilization and aggregate-free refolding. pr 2013 Wiley Periodicals, Inc. and the American Pharmacists Assocn. J Pharm Sci.
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    Lam, S. S.; Martell, J. D.; Kamer, K. J.; Deerinck, T. J.; Ellisman, M. H.; Mootha, V. K.; Ting, A. Y. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat. Methods 2015, 12, 51– 54,  DOI: 10.1038/nmeth.3179
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    Directed evolution of APEX2 for electron microscopy and proximity labeling
    Lam, Stephanie S.; Martell, Jeffrey D.; Kamer, Kimberli J.; Deerinck, Thomas J.; Ellisman, Mark H.; Mootha, Vamsi K.; Ting, Alice Y.
    Nature Methods (2015), 12 (1), 51-54CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    APEX is an engineered peroxidase that functions as an electron microscopy tag and a promiscuous labeling enzyme for live-cell proteomics. Because limited sensitivity precludes applications requiring low APEX expression, the authors used yeast-display evolution to improve its catalytic efficiency. APEX2 is far more active in cells, enabling the use of electron microscopy to resolve the submitochondrial localization of calcium uptake regulatory protein MICU1. APEX2 also permits superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins.
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    Golan, R.; Zehavi, U.; Naim, M.; Patchornik, A.; Smirnoff, P. Inhibition of Escherichia coli beta-galactosidase by 2-nitro-1-(4,5-dimethoxy-2-nitrophenyl) ethyl, a photoreversible thiol label. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1996, 1293, 238– 242,  DOI: 10.1016/0167-4838(95)00254-5
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    Inhibition of Escherichia coli β-galactosidase by 2-nitro-1-(4,5-dimethoxy-2-nitrophenyl)ethyl, a photoreversible thiol label
    Golan, Rachel; Zehavi, Uri; Naim, Michael; Patchornik, Abraham; Smirnoff, Patricia
    Biochimica et Biophysica Acta, Protein Structure and Molecular Enzymology (1996), 1293 (2), 238-42CODEN: BBAEDZ; ISSN:0167-4838. (Elsevier B.V.)
    1-Nitro-2-phenylethene (β-nitrostyrene, 1), which is a thiol-protecting reagent (Jung, G., Fouad, H. and Heusel, G. (1975) Angew. Chem. Int. Ed. Engl. 14, 817-818) was demonstrated in this work to be an irreversible inhibitor of β-galactosidase (EC 3.2.1.23), an enzyme known to be inhibited by some thiol reagents or through modifying a methionine residue at the active site. No reversal of the inhibition was obsd. upon subsequent incubation with mercaptoethanol or irradn. (350 nm). 1-(4,5-Dimethoxy-2-nitrophenyl)-2-Nitroethene (2) was also shown to be an irreversible inhibitor (94% inhibition, pH 8.3) of the enzyme. Kcat values of β-galactosidase at pH 8.3 with o-nitrophenyl β-D-galactopyranoside (ONPG) as the substrate and at the highest inhibitor concns. employed for compd. 1 (4.06·10-4 M) ranged from 1.67·104 s-1 after 30 min of preincubation to <0.07·104 s-1 after 180 min preincubation. For compd. 2 (9.5·10-5 M) Kcat values ranged from 2.70·104 s-1 following 30 min preincubation to 1.15·104 s-1 after 180 min of preincubation; the changes in Kmapp, however, were small. The activity was not recovered following incubation with mercaptoethanol. Since compd. 2 and the inhibited enzyme are 2-nitrobenzyl derivs., they are expected to be photosensitive and indeed, irradn. of the inhibited enzyme in the presence of mercaptoethanol resulted in recovery (89%, pH 8.3) of the enzyme activity.
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    Juers, D. H.; Hakda, S.; Matthews, B. W.; Huber, R. E. Structural Basis for the Altered Activity of Gly794 Variants of Escherichia coli β-Galactosidase. Biochemistry 2003, 42, 13505– 13511,  DOI: 10.1021/bi035506j
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    Structural Basis for the Altered Activity of Gly794 Variants of Escherichia coli β-Galactosidase
    Juers, Douglas H.; Hakda, Shamina; Matthews, Brian W.; Huber, Reuben E.
    Biochemistry (2003), 42 (46), 13505-13511CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The open-closed conformational switch in the active site of Escherichia coli β-galactosidase was studied by X-ray crystallog. and enzyme kinetics. Replacement of Gly794 by alanine causes the apoenzyme to adopt the closed rather than the open conformation. Binding of the competitive inhibitor iso-Pr thio-β-D-galactoside (IPTG) requires the mutant enzyme to adopt its less favored open conformation, weakening affinity relative to wild type. In contrast, transition-state inhibitors bind to the enzyme in the closed conformation, which is favored for the mutant, and display increased affinity relative to wild type. Changes in affinity suggest that the free energy difference between the closed and open forms is 1-2 kcal/mol. By favoring the closed conformation, the substitution moves the resting state of the enzyme along the reaction coordinate relative to the native enzyme and destabilizes the ground state relative to the first transition state. The result is that the rate const. for galactosylation is increased but degalactosylation is slower. The covalent intermediate may be better stabilized than the second transition state. The substitution also results in better binding of glucose to both the free and the galactosylated enzyme. However, transgalactosylation with glucose to produce allolactose (the inducer of the lac operon) is slower with the mutant than with the native enzyme. This suggests either that the glucose is misaligned for the reaction or that the galactosylated enzyme with glucose bound is stabilized relative to the transition state for transgalactosylation.
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    Zhou, H.-X.; Rivas, G.; Minton, A. P. Macromolecular Crowding and Confinement: Biochemical, Biophysical, and Potential Physiological Consequences. Annu. Rev. Biophys. 2008, 37, 375– 397,  DOI: 10.1146/annurev.biophys.37.032807.125817
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    Macromolecular crowding and confinement: Biochemical, biophysical, and potential physiological consequences
    Zhou, Huan-Xiang; Rivas, German; Minton, Allen P.
    Annual Review of Biophysics (2008), 37 (), 375-397CODEN: ARBNCV ISSN:. (Annual Reviews Inc.)
    A review. Expected and obsd. effects of vol. exclusion on the free energy of rigid and flexible macromols. in crowded and confined systems, and consequent effects of crowding and confinement on macromol. reaction rates and equil. are summarized. Findings from relevant theor./simulation and exptl. literature published from 2004 onward are reviewed. Addnl. complexity arising from the heterogeneity of local environments in biol. media, and the presence of nonspecific interactions between macromols. over and above steric repulsion, are discussed. Theor. and exptl. approaches to the characterization of crowding- and confinement-induced effects in systems approaching the complexity of living organisms are suggested.
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    Comellas-Aragonès, M.; Engelkamp, H.; Claessen, V. I.; Sommerdijk, N. A. J. M.; Rowan, A. E.; Christianen, P. C. M.; Maan, J. C.; Verduin, B. J. M.; Cornelissen, J. J. L. M.; Nolte, R. J. M. A virus-based single-enzyme nanoreactor. Nat. Nanotechnol. 2007, 2, 635– 639,  DOI: 10.1038/nnano.2007.299
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    A virus-based single-enzyme nanoreactor
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    Nature Nanotechnology (2007), 2 (10), 635-639CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
    Most enzyme studies are carried out in bulk aq. soln., at the so-called ensemble level, but more recently studies have appeared in which enzyme activity is measured at the level of a single mol., revealing previously unseen properties. To this end, enzymes have been chem. or phys. anchored to a surface, which is often disadvantageous because it may lead to denaturation. In a natural environment, enzymes are present in a confined reaction space, which inspired us to develop a generic method to carry out single-enzyme expts. in the restricted spatial environment of a virus capsid. We report here the incorporation of individual horseradish peroxidase enzymes in the inner cavity of a virus, and describe single-mol. studies on their enzymic behavior. These show that the virus capsid is permeable for substrate and product and that this permeability can be altered by changing pH.
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    Faulkner, M.; Szabó, I.; Weetman, S. L.; Sicard, F.; Huber, R. G.; Bond, P. J.; Rosta, E.; Liu, L.-N. Molecular simulations unravel the molecular principles that mediate selective permeability of carboxysome shell protein. Sci. Rep. 2020, 10, 17501  DOI: 10.1038/s41598-020-74536-5
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    Molecular simulations unravel molecular principles that mediate selective permeability of carboxysome shell protein
    Faulkner, Matthew; Szabo, Istvan; Weetman, Samantha L.; Sicard, Francois; Huber, Roland G.; Bond, Peter J.; Rosta, Edina; Liu, Lu-Ning
    Scientific Reports (2020), 10 (1), 17501CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
    Bacterial microcompartments (BMCs) are nanoscale proteinaceous organelles that encapsulate enzymes from the cytoplasm using an icosahedral protein shell that resembles viral capsids. Of particular interest are the carboxysomes (CBs), which sequester the CO2-fixing enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to enhance carbon assimilation. The carboxysome shell serves as a semi-permeable barrier for passage of metabolites in and out of the carboxysome to enhance CO2 fixation. How the protein shell directs influx and efflux of mols. in an effective manner has remained elusive. Here we use mol. dynamics and umbrella sampling calcns. to det. the free-energy profiles of the metabolic substrates, bicarbonate, CO2 and ribulose bisphosphate and the product 3-phosphoglycerate assocd. with their transition through the major carboxysome shell protein CcmK2. We elucidate the electrostatic charge-based permeability and key amino acid residues of CcmK2 functioning in mediating mol. transit through the central pore. Conformational changes of the loops forming the central pore may also be required for transit of specific metabolites. The importance of these in-silico findings is validated exptl. by site-directed mutagenesis of the key CcmK2 residue Serine 39. This study provides insight into the mechanism that mediates mol. transport through the shells of carboxysomes, applicable to other BMCs. It also offers a predictive approach to investigate and manipulate the shell permeability, with the intent of engineering BMC-based metabolic modules for new functions in synthetic biol.
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    Park, J.; Chun, S.; Bobik, T. A.; Houk, K. N.; Yeates, T. O. Molecular Dynamics Simulations of Selective Metabolite Transport across the Propanediol Bacterial Microcompartment Shell. J. Phys. Chem. B 2017, 121, 8149– 8154,  DOI: 10.1021/acs.jpcb.7b07232
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    Molecular Dynamics Simulations of Selective Metabolite Transport across the Propanediol Bacterial Microcompartment Shell
    Park, Jiyong; Chun, Sunny; Bobik, Thomas A.; Houk, Kendall N.; Yeates, Todd O.
    Journal of Physical Chemistry B (2017), 121 (34), 8149-8154CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
    Bacterial microcompartments are giant protein-based organelles that encapsulate special metabolic pathways in diverse bacteria. Structural and genetic studies indicate that metabolic substrates enter these microcompartments by passing through the central pores in hexameric assemblies of shell proteins. Limiting the escape of toxic metabolic intermediates created inside the microcompartments would confer a selective advantage for the host organism. Here, we report the first mol. dynamics (MD) simulation studies to analyze small-mol. transport across a microcompartment shell. PduA is a major shell protein in a bacterial microcompartment that metabolizes 1,2-propanediol via a toxic aldehyde intermediate, propionaldehyde. Using both metadynamics and replica-exchange umbrella sampling, we find that the pore of the PduA hexamer has a lower energy barrier for passage of the propanediol substrate compared to the toxic propionaldehyde generated within the microcompartment. The energetic effect is consistent with a lower capacity of a serine side chain, which protrudes into the pore at a point of constriction, to form hydrogen bonds with propionaldehyde relative to the more freely permeable propanediol. The results highlight the importance of mol. diffusion and transport in a new biol. context.
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    Shifrin, S.; Hunn, G. Effect of alcohols on the enzymatic activity and subunit association of β-galactosidase. Arch. Biochem. Biophys. 1969, 130, 530– 535,  DOI: 10.1016/0003-9861(69)90066-6
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    Effect of alcohols on the enzymatic activity and subunit association of β-galactosidase
    Shifrin, Sidney; Hunn, Gilbert
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    The catalytic activity of β-galactosidase from Escherichia coli K-12 and the associative properties of its subunits have been studied in solns. of MeOH, EtOH, iso-PrOH and PrOH. All of the alcs. at low concns. (5%) stimulate the rate at which o-nitrophenyl β-D-galactopyranoside is cleaved. The transferase activity of the enzyme was demonstrated by identification of Me β-galactoside as one of the reaction products. Neither MeOH nor EtOH dissoc. the subunits of the active tetramer nor do they induce conformational changes in the protein as measured by sedimentation velocity, uv absorption spectroscopy, and fluorescence. Low concns. of PrOH in the absence of added Mg2+, however, cause the tetramer to dissoc. into inactive dimers.
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    Goldstein, L.; Levin, Y.; Katchalski, E. A Water-Insoluble Polyanionic Derivative of Trypsin. II. Effect of the Polyelectrolyte Carrier on the Kinetic Behavior of the Bound Trypsin*. Biochemistry 1964, 3, 1913– 1919,  DOI: 10.1021/bi00900a022
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    A water-insoluble polyanionic derivative of trypsin. II. Effect of the polyelectrolyte carrier on the kinetic behavior of the bound trypsin
    Goldstein, Leon; Levin, Yehuda; Katchalski, Ephraim
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    The mode of action of IMET, obtained by the covalent binding of trypsin to a copolymer of maleic acid and ethylene (1:1), was investigated at 25°. The pH-activity profile of IMET at low ionic strength (5.8 × 10-3), using benzoyl-L-arginine Et ester as substrate, was displaced by approx. 2.5 pH units toward more alk. pH values, when compared with trypsin under similar conditions. At higher ionic strength, the pH-activity curve of IMET shifted toward more acid pH values, approaching the pH-activity curve of IMET-trypsin at ionic strength 1.0. The Km = 0.2 ± 0.05 × 10-3M measured for the benzoyl-L-arginine amide system at low ionic strength (0.04) and optimal pH (9.5) was approx. 30 times lower than that of the trypsin-benzoyl-L-arginine amide system, at its optimal pH (7.5) at ionic strength 0.04. The Km at high ionic strength (0.5), measured for the IMET-benzoyl-L-arginine amide system at the pH of optimal activity (pH 9.5), approached that for the trypsin-benzoyl-L-arginine amide system (Km = 6.8 ± 1.0 × 10-3M) when measured at its optimal pH (7.5) and the same ionic strength. The effect of the polyanionic carrier on the pH-activity profiles and Km values of the bound enzymes investigated can be explained as resulting from the effect of the electrostatic potential of the polyelectrolyte carrier on the local concn. of H+ and pos. charged substrate mols. in the microenvironment of the bound enzyme mols. Theoretical analysis of the kinetic data allowed a quant. correlation of the displacement in the pH-activity curves and the shifts in the Km values with the electrostatic potential prevailing in the domain of the polyelectrolyte carrier.
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    Goldstein, L. Microenvironmental effects on enzyme catalysis. Kinetic study of polyanionic and polycationic derivatives of chymotrypsin. Biochemistry 1972, 11, 4072– 4084,  DOI: 10.1021/bi00772a009
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    Microenvironmental effects on enzyme catalysis. Kinetic study of polyanionic and polycationic derivatives of chymotrypsin
    Goldstein, Leon
    Biochemistry (1972), 11 (22), 4072-84CODEN: BICHAW; ISSN:0006-2960.
    A series of water-sol. polyanionic and polycationic derivs. of chymotrypsin were prepd. by growing poly(glutamyl) or poly(ornithyl) side chains on the enzyme, by coupling chymotrypsin to an ethylene-maleic acid copolymer (EMA), and by partial succinylation or acetylation. The pH-activity profiles of the polyanionic derivs. of chymotrypsin were displaced toward more alk. pH values as compared to the native enzyme; conversely the pH-activity profiles of the polycationic derivs. were displaced toward more acidic pH values. The kcat values of the charged chymotrypsin derivs. acting on ester, amide, and anilide substrates were displaced symmetrically, relative to the native enzyme-to higher values in the case of the polyanionic derivs. (poly(glutamyl) chymotrypsin, EMA-chymotrypsin, succinylchymotrypsin, and acetylchymotrypsin), and to lower values in the case of the polycationic (poly(ornithyl)chymotrypsin) derivs. The electrostatic effects on kcat were much more pronounced when the substrate was amide or anilide than when it was an ester. Increasing the ionic strength caused an increase in the values of kcat of both native chymotrypsin and the pos. charged derivs. of the enzyme. The kcat values of the neg. charged derivs. were not affected by the ionic strength. With ester substrates, the values of Km(app) of the polycationic derivs. were higher by an order of magnitude in comparison to the native enzyme; the Km(app) values of the polyanionic derivs. were only slightly perturbed. The values of Km(app) of all chymotrypsin derivs. acting on amide and anilide substrates were unperturbed and essentially identical with the value of the Michaelis const. of the native enzyme. These findings are discussed in the light of some recent ideas regarding the mechanism of action of chymotrypsin.
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    Zhang, Y.; Tsitkov, S.; Hess, H. Proximity does not contribute to activity enhancement in the glucose oxidase–horseradish peroxidase cascade. Nat. Commun. 2016, 7, 13982  DOI: 10.1038/ncomms13982
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    Proximity does not contribute to activity enhancement in the glucose oxidase-horseradish peroxidase cascade
    Zhang, Yifei; Tsitkov, Stanislav; Hess, Henry
    Nature Communications (2016), 7 (), 13982CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
    A proximity effect has been invoked to explain the enhanced activity of enzyme cascades on DNA scaffolds. Using the cascade reaction carried out by glucose oxidase and horseradish peroxidase as a model system, here we study the kinetics of the cascade reaction when the enzymes are free in soln., when they are conjugated to each other and when a competing enzyme is present. No proximity effect is found, which is in agreement with models predicting that the rapidly diffusing hydrogen peroxide intermediate is well mixed. We suggest that the reason for the activity enhancement of enzymes localized by DNA scaffolds is that the pH near the surface of the neg. charged DNA nanostructures is lower than that in the bulk soln., creating a more optimal pH environment for the anchored enzymes. Our findings challenge the notion of a proximity effect and provide new insights into the role of DNA scaffolds.
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    Ladero, M.; Santos, A.; García, J. L.; García-Ochoa, F. Activity over lactose and ONPG of a genetically engineered β-galactosidase from Escherichia coli in solution and immobilized: kinetic modelling. Enzyme Microb. Technol. 2001, 29, 181– 193,  DOI: 10.1016/S0141-0229(01)00366-0
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    Activity over lactose and ONPG of a genetically engineered β-galactosidase from Escherichia coli in solution and immobilized: kinetic modelling
    Ladero, M.; Santos, A.; Garcia, J. L.; Garcia-Ochoa, F.
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    The kinetic study of the hydrolysis of lactose and o-nitrophenol-β-D-galactoside (ONPG) with a β-galactosidase from Escherichia coli, both in soln. and covalently immobilized on a silica-alumina, is presented. The enzyme employed in this work had been modified previously by genetic engineering and purified to homogeneity by affinity chromatog. Firstly, the influence of pH and temp. on the activity and the stability of the enzyme, both free and immobilized, have been studied. Secondly, hydrolysis runs of lactose and ONPG with both forms of the enzyme were carried out in a wide exptl. range of temp. and concns. of substrates, products and enzyme. Data obtained were fitted to several kinetic models based on the Michaelis-Menten mechanism by non-linear regression. Finally, the models and their parameters were compared to det. the influence of the immobilization process and the substrate on the activity of the enzyme. In the hydrolysis of lactose and with both forms of the enzyme, acompetitive inhibition due to glucose was obsd. while the most common inhibition by galactose (which is usually a competitive inhibitor of β-galactosidases) was not obsd. Curiously, when the immobilized enzyme was the catalyst employed, lactose was an acompetitive inhibitor of the hydrolysis. When the substrate hydrolyzed was the o-nitrophenol-β-D-galactoside (ONPG), the galactose acted as a competitive inhibitor and the o-nitrophenol (ONP) was an acompetitive inhibitor for the free enzyme, being the immobilization process able to avoid the interaction between the ONP and the enzyme.
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    Noack, C. W.; Dzombak, D. A.; Nakles, D. V.; Hawthorne, S. B.; Heebink, L. V.; Dando, N.; Gershenzon, M.; Ghosh, R. S. Comparison of alkaline industrial wastes for aqueous mineral carbon sequestration through a parallel reactivity study. Waste Manage. 2014, 34, 1815– 1822,  DOI: 10.1016/j.wasman.2014.03.009
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    Comparison of alkaline industrial wastes for aqueous mineral carbon sequestration through a parallel reactivity study
    Noack, Clinton W.; Dzombak, David A.; Nakles, David V.; Hawthorne, Steven B.; Heebink, Loreal V.; Dando, Neal; Gershenzon, Michael; Ghosh, Rajat S.
    Waste Management (Oxford, United Kingdom) (2014), 34 (10), 1815-1822CODEN: WAMAE2; ISSN:0956-053X. (Elsevier Ltd.)
    Thirty-one alk. industrial wastes from a wide range of industrial processes were acquired and screened for application in an aq. carbon sequestration process. The wastes were evaluated for their potential to leach polyvalent cations and base species. Following mixing with a simple sodium bicarbonate soln., chemistries of the aq. and solid phases were analyzed. Exptl. results indicated that the most reactive materials were capable of sequestering between 77% and 93% of the available carbon under exptl. conditions in four hours. These materials - cement kiln dust, spray dryer absorber ash, and circulating dry scrubber ash - are thus good candidates for detailed, process-oriented studies. Chem. equil. modeling indicated that amorphous calcium carbonate is likely responsible for the obsd. sequestration. High variability and low reactive fractions render many other materials less attractive for further pursuit without considering preprocessing or activation techniques.
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    Arregui, L.; Ayala, M.; Gómez-Gil, X.; Gutiérrez-Soto, G.; Hernández-Luna, C. E.; Herrera de los Santos, M.; Levin, L.; Rojo-Domínguez, A.; Romero-Martínez, D.; Saparrat, M. C. N.; Trujillo-Roldán, M. A.; Valdez-Cruz, N. A. Laccases: structure, function, and potential application in water bioremediation. Microb. Cell Fact. 2019, 18, 200  DOI: 10.1186/s12934-019-1248-0
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    Laccases: structure, function, and potential application in water bioremediation
    Arregui, Leticia; Ayala, Marcela; Gomez-Gil, Ximena; Gutierrez-Soto, Guadalupe; Hernandez-Luna, Carlos Eduardo; Herrera de los Santos, Mayra; Levin, Laura; Rojo-Dominguez, Arturo; Romero-Martinez, Daniel; Saparrat, Mario C. N.; Trujillo-Roldan, Mauricio A.; Valdez-Cruz, Norma A.
    Microbial Cell Factories (2019), 18 (1), 200CODEN: MCFICT; ISSN:1475-2859. (BioMed Central Ltd.)
    A review. The global rise in urbanization and industrial activity has led to the prodn. and incorporation of foreign contaminant mols. into ecosystems, distorting them and impacting human and animal health. Phys., chem., and biol. strategies have been adopted to eliminate these contaminants from water bodies under anthropogenic stress. Biotechnol. processes involving microorganisms and enzymes have been used for this purpose; specifically, laccases, which are broad spectrum biocatalysts, have been used to degrade several compds., such as those that can be found in the effluents from industries and hospitals. Laccases have shown high potential in the biotransformation of diverse pollutants using crude enzyme exts. or free enzymes. However, their application in bioremediation and water treatment at a large scale is limited by the complex compn. and high salt concn. and pH values of contaminated media that affect protein stability, recovery and recycling. These issues are also assocd. with operational problems and the necessity of large-scale prodn. of laccase. Hence, more knowledge on the mol. characteristics of water bodies is required to identify and develop new laccases that can be used under complex conditions and to develop novel strategies and processes to achieve their efficient application in treating contaminated water. Recently, stability, efficiency, sepn. and reuse issues have been overcome by the immobilization of enzymes and development of novel biocatalytic materials. This review provides recent information on laccases from different sources, their structures and biochem. properties, mechanisms of action, and application in the bioremediation and biotransformation of contaminant mols. in water. Moreover, we discuss a series of improvements that have been attempted for better org. solvent tolerance, thermo-tolerance, and operational stability of laccases, as per process requirements.
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    Wang, M.; Abad, D.; Kickhoefer, V. A.; Rome, L. H.; Mahendra, S. Vault Nanoparticles Packaged with Enzymes as an Efficient Pollutant Biodegradation Technology. ACS Nano 2015, 9, 10931– 10940,  DOI: 10.1021/acsnano.5b04073
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    Vault Nanoparticles Packaged with Enzymes as an Efficient Pollutant Biodegradation Technology
    Wang, Meng; Abad, Danny; Kickhoefer, Valerie A.; Rome, Leonard H.; Mahendra, Shaily
    ACS Nano (2015), 9 (11), 10931-10940CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    Vault nanoparticles packaged with enzymes were synthesized as agents for efficiently degrading environmental contaminants. Enzymic biodegrdn. is an attractive technol. for in situ cleanup of contaminated environments because enzyme-catalyzed reactions are not constrained by nutrient requirements for microbial growth and often have higher biodegrdn. rates. However, the limited stability of extracellular enzymes remains a major challenge for practical applications. Encapsulation is a recognized method to enhance enzymic stability, but it can increase substrate diffusion resistance, lower catalytic rates, and increase the apparent half-satn. consts. We report an effective approach for boosting enzymic stability by single-step packaging into vault nanoparticles. With hollow core structures, assembled vault nanoparticles can simultaneously contain multiple enzymes. Mn peroxidase (MnP), which is widely used in biodegrdn. of org. contaminants, was chosen as a model enzyme here. MnP was incorporated into vaults via fusion to a packaging domain called INT, which strongly interacts with vaults' interior surface. MnP fused to INT and vaults packaged with the MnP-INT fusion protein maintained peroxidase activity. MnP-INT packaged in vaults displayed stability significantly higher than that of free MnP-INT, with slightly increased Km value. Vault-packaged MnP-INT exhibited 3 times higher phenol biodegrdn. in 24 h than did unpackaged MnP-INT. These results indicate that the packaging of MnP enzymes in vault nanoparticles extends their stability without compromising catalytic activity. This research will serve as the foundation for the development of efficient and sustainable vault-based bioremediation approaches for removing multiple contaminants from drinking water and groundwater.
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    Kaplan, O.; Vejvoda, V.; Plíhal, O.; Pompach, P.; Kavan, D.; Bojarová, P.; Bezouška, K.; Macková, M.; Cantarella, M.; Jirku, V.; Křen, V.; Martínková, L. Purification and characterization of a nitrilase from Aspergillus niger K10. Appl. Microbiol. Biotechnol. 2006, 73, 567– 575,  DOI: 10.1007/s00253-006-0503-6
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    Purification and characterization of a nitrilase from Aspergillus niger K10
    Kaplan, Ondrej; Vejvoda, Vojtech; Plihal, Ondrej; Pompach, Petr; Kavan, Daniel; Bojarova, Pavla; Bezouska, Karel; Mackova, Martina; Cantarella, Maria; Jirku, Vladimir; Kren, Vladimir; Martinkova, Ludmila
    Applied Microbiology and Biotechnology (2006), 73 (3), 567-575CODEN: AMBIDG; ISSN:0175-7598. (Springer)
    Aspergillus niger K10 cultivated on 2-cyanopyridine produced high levels of an intracellular nitrilase, which was partially purified (18.6-fold) with a 24% yield. The N-terminal amino acid sequence of the enzyme was highly homologous with that of a putative nitrilase from Aspergillus fumigatus Af293. The enzyme was copurified with two proteins, the N-terminal amino acid sequences of which revealed high homol. with those of hsp60 and an ubiquitin-conjugating enzyme. The nitrilase exhibited max. activity (91.6 U mg-1) at 45°C and pH 8.0. Its preferred substrates, in the descending order, were 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene, thiophen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile. Formation of amides as byproducts was most intensive, in the descending order, for 2-cyanopyridine, 4-chlorobenzonitrile, 4-cyanopyridine, and 1,4-dicyanobenzene. The enzyme stability was markedly improved in the presence of d-sorbitol or xylitol (20% w/v each). p-Hydroxymercuribenzoate and heavy metal ions were the most powerful inhibitors of the enzyme.
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  • Abstract

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    Figure 1

    Figure 1. H. nea α-carboxysome operon (cso). The dotted line indicates 10 genes between cbbO and csoS1D that are unlikely to be associated with shell assembly or function. Gene lengths and intervening distances are not drawn to scale.

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    Figure 2

    Figure 2. Identifying combinations that produce minimal α-carboxysome-derived shells. (A) Schematic of the combinatorial assembly process used to construct shell-expressing pathways. (B–E) Transmission electron microscopy (TEM) micrographs of combinations that produced capsid-like structures, (B) Cso-PATHA, (C) Cso-PAHA, (D) Cso-PATHC, and (E) Cso-PAHC. Scale bars (bottom right) represent 50 nm. (F, G) SDS-PAGE analysis of shell-containing samples following anion-exchange purification of (F) Cso-PATHA and (G) Cso-PAHA. The protein ladder is indicated by L in kDa.

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    Figure 3

    Figure 3. Identifying S2-CR sequences for mediating cargo encapsulation within the Cso-shell. (A) CsoS2 can be split into three distinct regions based on sequence homology. Regions are not drawn to scale. (B) Schematic of the encapsulation-degradation assay. Encapsulation of GFP cargo within the shell prevents its degradation by the ClpXP protease. The blue and yellow blocks represent CsoS1A (BMC-H) and CsoS4A (BMC-P), respectively. (C) Pathway design for co-expressing UmuD (1−40)-GFP-S2-Cx (S2-Cx represents S2-C variants) with the Cso-PAHA shells. (D) Western blot detection of GFP in purified shell samples. The identities of the proteins in the ladder (L) lane are: His6-GFP-SpyTag (29.7 kDa), His6-GFP-SpyCatcher (38.2 kDa), and His6-GFP-SpyCatcher-SpyTag-GFP (67.9 kDa). Lanes 1–5 are Cso-PAHA co-expressed with the UmuD (1−40)-GFP variant indicated below the lanes. (E) DLS analysis of shells co-expressed with various cargo. The numbers to the right of the spectra correspond to the samples loaded in (D). The baseline is vertically displaced by 0.2 for each subsequent spectrum so that all peaks can be seen in one graph.

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    Figure 4

    Figure 4. Atomic-scale models of the minimal α-carboxysome shells. (A) Operon schematic of Cso-PAHA + GFP-S2-CTP. (B, C) Surface representations of the shells, with the CsoS1A colored blue and CsoS4A in yellow. The blue and red patches over yellow indicate N- and C-termini of a CsoS4A monomer, and the cyan and red patches over blue indicate those of a CsoS1A monomer.

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    Figure 5

    Figure 5. Close examination of the interfacial contacts in Cso-shells reinforcing notions of conserved features in BMC shell assembly. (A) CsoS4A conformations pre-shell (gray) and post-shell integration (yellow). The dashed arrow indicates movement of CsoS4A’s C-terminal loop. W80 is highlighted with dark gray (pre-integration) and orange (post-integration). (B) CsoS1A hexamers (gray) become expanded after shell incorporation (blue). Arrow indicates movement of the helices. (C) Pentamer–hexamer interface, T = 3. (D) Pentamer–hexamer interface, T = 4. (E) Hexamer–hexamer interface, T = 3. (F) Hexamer–hexamer interface, T = 4. For clarity, only half of the polar interactions left to the pseudo-2-fold axis (orange oval) at the hexamer–hexamer interface are indicated in (E) and (F). For (C)–(F), CsoS4A is colored yellow, while CsoS1A chains are colored blue and green. Dotted lines represent interatomic distances in Å.

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    Figure 6

    Figure 6. Investigating a possible molecular mechanism contributing to encapsulation by S2-C-derived peptides in the Cso-shell. (A) Standard free energies of binding, ΔG°, of the S2-C-derived peptides titrated against CsoS1A under various NaCl concentrations. Error bars indicate uncertainties of isotherm fitting. (B) Electrostatic potential map of a cross-sectional view of the Cso-shell lumen. Negative potential is colored red, while positive potential is represented by blue over a ±4 kT/e range. Maps were calculated using APBS. (46) (C) Electrostatic potential map of the Cso-shell exterior. (D) Calculated isoelectric points and net charge at pH 7.4 of the distinct regions on S2-C.

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    Figure 7

    Figure 7. Assessing the tolerance of empty Cso-shell against common denaturing factors for potential stabilization of encapsulated enzymes. (A–D) DLS spectra of empty Cso-shells tested against the conditions indicated. The baseline is vertically displaced by 0.2 for each subsequent spectrum so that all spectra can be seen in one graph. (E) Operon schematic for co-expressing Cso-shells with enzymes.

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    Figure 8

    Figure 8. Investigating the stability of the Cso-shell and scaffolded enzymes in potentially denaturing conditions. Residual enzymes activities of free and encapsulated (+ shell) enzymes were obtained by normalizing the activity to that of the pristine sample, shown as (B) 23 °C, (C) 0% v/v methanol, (D) no freeze–thawing, and (E) pH 8. Error bars represent one standard deviation of the mean.

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  • References

    This article references 71 other publications.

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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXltFagsrY%253D&md5=a0600f39ebaca5b559c6f54e72ed7615
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      Structure of a synthetic β-carboxysome shell
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      Plant Physiology (2019), 181 (3), 1050-1058CODEN: PLPHAY; ISSN:1532-2548. (American Society of Plant Biologists)
      Carboxysomes are capsid-like, CO2-fixing organelles that are present in all cyanobacteria and some chemoautotrophs and that substantially contribute to global primary prodn. They are composed of a selectively permeable protein shell that encapsulates Rubisco, the principal CO2-fixing enzyme, and carbonic anhydrase. As the centerpiece of the carbon-concg. mechanism, by packaging enzymes that collectively enhance catalysis, the carboxysome shell enables the generation of a locally elevated concn. of substrate CO2 and the prevention of CO2 escape. A functional carboxysome consisting of an intact shell and cargo is essential for cyanobacterial growth under ambient CO2 concns. Using cryo-electron microscopy, we have detd. the structure of a recombinantly produced simplified β-carboxysome shell. The structure reveals the sidedness and the specific interactions between the carboxysome shell proteins. The model provides insight into the structural basis of selective permeability of the carboxysome shell and can be used to design modifications to investigate the mechanisms of cargo encapsulation and other physiochem. properties such as permeability. Notably, the permeability properties are of great interest for modeling and evaluating this carbon-concg. mechanism in metabolic engineering. Moreover, we find striking similarity between the carboxysome shell and the structurally characterized, evolutionarily distant metabolosome shell, implying universal architectural principles for bacterial microcompartment shells.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitl2kt73N&md5=fb5f3642ac5a252a91af2096cb82027e
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      Many bacteria contain primitive organelles composed entirely of protein. These bacterial microcompartments share a common architecture of an enzymic core encapsulated in a selectively permeable protein shell; prominent examples include the carboxysome for CO2 fixation and catabolic microcompartments found in many pathogenic microbes. The shell sequesters enzymic reactions from the cytosol, analogous to the lipid-based membrane of eukaryotic organelles. Despite available structural information for single building blocks, the principles of shell assembly have remained elusive. We present the crystal structure of an intact shell from Haliangium ochraceum, revealing the basic principles of bacterial microcompartment shell construction. Given the conservation among shell proteins of all bacterial microcompartments, these principles apply to functionally diverse organelles and can inform the design and engineering of shells with new functionalities.
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      Atomic-Level Models of the Bacterial Carboxysome Shell
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      The carboxysome is a bacterial microcompartment that functions as a simple organelle by sequestering enzymes involved in carbon fixation. The carboxysome shell is roughly 800 to 1400 angstroms in diam. and is assembled from several thousand protein subunits. Previous studies have revealed the three-dimensional structures of hexameric carboxysome shell proteins, which self-assemble into mol. layers that most likely constitute the facets of the polyhedral shell. Here, we report the three-dimensional structures of two proteins of previously unknown function, CcmL and OrfA (or CsoS4A), from the two known classes of carboxysomes, at resolns. of 2.4 and 2.15 angstroms. Both proteins assemble to form pentameric structures whose size and shape are compatible with formation of vertices in an icosahedral shell. Combining these pentamers with the hexamers previously elucidated gives two plausible, preliminary at. models for the carboxysome shell.
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      9
      Structure and mechanisms of a protein-based organelle in Escherichia coli
      Tanaka, Shiho; Sawaya, Michael R.; Yeates, Todd O.
      Science (Washington, DC, United States) (2010), 327 (5961), 81-84CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Many bacterial cells contain proteinaceous microcompartments that act as simple organelles by sequestering specific metabolic processes involving volatile or toxic metabolites. Here, the authors report the three-dimensional (3D) crystal structures, with resolns. between 1.65 and 2.5 angstroms, of the four homologous proteins (EutS, EutL, EutK, and EutM) that are thought to be the major shell constituents of a functionally complex ethanolamine utilization (Eut) microcompartment. The Eut microcompartment is used to sequester the metab. of ethanolamine in bacteria such as Escherichia coli and Salmonella enterica. The four Eut shell proteins share an overall similar 3D fold, but they have distinguishing structural features that help explain the specific roles they play in the microcompartment. For example, EutL undergoes a conformational change that is probably involved in gating mol. transport through shell protein pores, whereas structural evidence suggests that EutK might bind a nucleic acid component.
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      Kalnins, G.; Cesle, E.-E.; Jansons, J.; Liepins, J.; Filimonenko, A.; Tars, K. Encapsulation mechanisms and structural studies of GRM2 bacterial microcompartment particles. Nat. Commun. 2020, 11, 388  DOI: 10.1038/s41467-019-14205-y
      10
      Encapsulation mechanisms and structural studies of GRM2 bacterial microcompartment particles
      Kalnins, Gints; Cesle, Eva-Emilija; Jansons, Juris; Liepins, Janis; Filimonenko, Anatolij; Tars, Kaspars
      Nature Communications (2020), 11 (1), 388CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Bacterial microcompartments (BMCs) are prokaryotic organelles consisting of a protein shell and an encapsulated enzymic core. BMCs are involved in several biochem. processes, such as choline, glycerol and ethanolamine degrdn. and carbon fixation. Since non-native enzymes can also be encapsulated in BMCs, an improved understanding of BMC shell assembly and encapsulation processes could be useful for synthetic biol. applications. Here we report the isolation and recombinant expression of BMC structural genes from the Klebsiella pneumoniae GRM2 locus, the investigation of mechanisms behind encapsulation of the core enzymes, and the characterization of shell particles by cryo-EM. We conclude that the enzymic core is encapsulated in a hierarchical manner and that the CutC choline lyase may play a secondary role as an adaptor protein. We also present a cryo-EM structure of a pT = 4 quasi-sym. icosahedral shell particle at 3.3 Å resoln., and demonstrate variability among the minor shell forms.
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    11. 11
      Chowdhury, C.; Sinha, S.; Chun, S.; Yeates, T. O.; Bobik, T. A. Diverse bacterial microcompartment organelles. Microbiol. Mol. Biol. Rev. 2014, 78, 438– 468,  DOI: 10.1128/MMBR.00009-14
      11
      Diverse bacterial microcompartment organelles
      Chowdhury Chiranjit; Sinha Sharmistha; Bobik Thomas A; Chun Sunny; Yeates Todd O
      Microbiology and molecular biology reviews : MMBR (2014), 78 (3), 438-68 ISSN:.
      Bacterial microcompartments (MCPs) are sophisticated protein-based organelles used to optimize metabolic pathways. They consist of metabolic enzymes encapsulated within a protein shell, which creates an ideal environment for catalysis and facilitates the channeling of toxic/volatile intermediates to downstream enzymes. The metabolic processes that require MCPs are diverse and widely distributed and play important roles in global carbon fixation and bacterial pathogenesis. The protein shells of MCPs are thought to selectively control the movement of enzyme cofactors, substrates, and products (including toxic or volatile intermediates) between the MCP interior and the cytoplasm of the cell using both passive electrostatic/steric and dynamic gated mechanisms. Evidence suggests that specialized shell proteins conduct electrons between the cytoplasm and the lumen of the MCP and/or help rebuild damaged iron-sulfur centers in the encapsulated enzymes. The MCP shell is elaborated through a family of small proteins whose structural core is known as a bacterial microcompartment (BMC) domain. BMC domain proteins oligomerize into flat, hexagonally shaped tiles, which assemble into extended protein sheets that form the facets of the shell. Shape complementarity along the edges allows different types of BMC domain proteins to form mixed sheets, while sequence variation provides functional diversification. Recent studies have also revealed targeting sequences that mediate protein encapsulation within MCPs, scaffolding proteins that organize lumen enzymes and the use of private cofactor pools (NAD/H and coenzyme A [HS-CoA]) to facilitate cofactor homeostasis. Although much remains to be learned, our growing understanding of MCPs is providing a basis for bioengineering of protein-based containers for the production of chemicals/pharmaceuticals and for use as molecular delivery vehicles.
      >> More from SciFinder ®
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    12. 12
      Wheatley, N. M.; Gidaniyan, S. D.; Liu, Y.; Cascio, D.; Yeates, T. O. Bacterial microcompartment shells of diverse functional types possess pentameric vertex proteins. Protein Sci. 2013, 22, 660– 665,  DOI: 10.1002/pro.2246
      12
      Bacterial microcompartment shells of diverse functional types possess pentameric vertex proteins
      Wheatley, Nicole M.; Gidaniyan, Soheil D.; Liu, Yuxi; Cascio, Duilio; Yeates, Todd O.
      Protein Science (2013), 22 (5), 660-665CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)
      Bacterial microcompartments (MCPs) are large proteinaceous structures comprised of a roughly icosahedral shell and a series of encapsulated enzymes. MCPs carrying out three different metabolic functions have been characterized in some detail, while gene expression and bioinformatics studies have implicated other types, including one believed to perform glycyl radical-based metab. of 1,2-propanediol (Grp). Here we report the crystal structure of a protein (GrpN), which is presumed to be part of the shell of a Grp-type MCP in Rhodospirillum rubrum F11. GrpN is homologous to a family of proteins (EutN/PduN/CcmL/CsoS4) whose members have been implicated in forming the vertices of MCP shells. Consistent with that notion, the crystal structure of GrpN revealed a pentameric assembly. That observation revived an outstanding question about the oligomeric state of this protein family: pentameric forms (for CcmL and CsoS4A) and a hexameric form (for EutN) had both been obsd. in previous crystal structures. To clarify these confounding observations, we revisited the case of EutN. We developed a mol. biol.-based method for accurately detg. the no. of subunits in homo-oligomeric proteins, and found unequivocally that EutN is a pentamer in soln. Based on these convergent findings, we propose the name bacterial microcompartment vertex for this special family of MCP shell proteins.
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    13. 13
      Hagen, A. R.; Plegaria, J. S.; Sloan, N.; Ferlez, B.; Aussignargues, C.; Burton, R.; Kerfeld, C. A. In Vitro Assembly of Diverse Bacterial Microcompartment Shell Architectures. Nano Lett. 2018, 18, 7030– 7037,  DOI: 10.1021/acs.nanolett.8b02991
      13
      In Vitro Assembly of Diverse Bacterial Microcompartment Shell Architectures
      Hagen, Andrew R.; Plegaria, Jefferson S.; Sloan, Nancy; Ferlez, Bryan; Aussignargues, Clement; Burton, Rodney; Kerfeld, Cheryl A.
      Nano Letters (2018), 18 (11), 7030-7037CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      Bacterial microcompartments (BMCs) are organelles composed of a selectively permeable protein shell that encapsulates enzymes involved in CO2 fixation (carboxysomes) or carbon catabolism (metabolosomes). Confinement of sequential reactions by the BMC shell presumably increases the efficiency of the pathway by reducing the crosstalk of metabolites, release of toxic intermediates, and accumulation of inhibitory products. Because BMCs are composed entirely of protein and self-assemble, they are an emerging platform for engineering nanoreactors and mol. scaffolds. However, testing designs for assembly and function through in vivo expression is labor-intensive and has limited the potential of BMCs in bioengineering. Here, we developed a new method for in vitro assembly of defined nanoscale BMC architectures: shells and nanotubes. By inserting a "protecting group", a short ubiquitin-like modifier (SUMO) domain, self-assembly of shell proteins in vivo was thwarted, enabling prepn. of concs. of shell building blocks. Addn. of the cognate protease removes the SUMO domain and subsequent mixing of the constituent shell proteins in vitro results in the self-assembly of three types of supramol. architectures: a metabolosome shell, a carboxysome shell, and a BMC protein-based nanotube. We next applied our method to generate a metabolosome shell engineered with a hyper-basic luminal surface, allowing for the encapsulation of biotic or abiotic cargos functionalized with an acidic accessory group. This is the first demonstration of using charge complementarity to encapsulate diverse cargos in BMC shells. Collectively, our work provides a generally applicable method for in vitro assembly of natural and engineered BMC-based architectures.
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    14. 14
      Aussignargues, C.; Pandelia, M.-E.; Sutter, M.; Plegaria, J. S.; Zarzycki, J.; Turmo, A.; Huang, J.; Ducat, D. C.; Hegg, E. L.; Gibney, B. R.; Kerfeld, C. A. Structure and Function of a Bacterial Microcompartment Shell Protein Engineered to Bind a [4Fe-4S] Cluster. J. Am. Chem. Soc. 2016, 138, 5262– 5270,  DOI: 10.1021/jacs.5b11734
      14
      Structure and Function of a Bacterial Microcompartment Shell Protein Engineered to Bind a [4Fe-4S] Cluster
      Aussignargues, Clement; Pandelia, Maria-Eirini; Sutter, Markus; Plegaria, Jefferson S.; Zarzycki, Jan; Turmo, Aiko; Huang, Jingcheng; Ducat, Daniel C.; Hegg, Eric L.; Gibney, Brian R.; Kerfeld, Cheryl A.
      Journal of the American Chemical Society (2016), 138 (16), 5262-5270CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Bacterial microcompartments (BMCs) are self-assembling organelles composed of a selectively permeable protein shell and encapsulated enzymes. They are considered promising templates for the engineering of designed bionanoreactors for biotechnol. In particular, encapsulation of oxidoreductive reactions requiring electron transfer between the lumen of the BMC and the cytosol relies on the ability to conduct electrons across the shell. We detd. the crystal structure of a component protein of a synthetic BMC shell, which informed the rational design of a [4Fe-4S] cluster-binding site in its pore. We also solved the structure of the [4Fe-4S] cluster-bound, engineered protein to 1.8 Å resoln., providing the first structure of a BMC shell protein contg. a metal center. The [4Fe-4S] cluster was characterized by optical and EPR spectroscopies; it has a redn. potential of -370 mV vs the std. hydrogen electrode (SHE) and is stable through redox cycling. This remarkable stability may be attributable to the hydrogen-bonding network provided by the main chain of the protein scaffold. The properties of the [4Fe-4S] cluster resemble those in low-potential bacterial ferredoxins, while its ligation to three cysteine residues is reminiscent of enzymes such as aconitase and radical S-adenosymethionine (SAM) enzymes. This engineered shell protein provides the foundation for conferring electron-transfer functionality to BMC shells.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitVyrurbE&md5=1c5e321a559dbb2f3ab9ccfa43e04c8c
    15. 15
      Lawrence, A. D.; Frank, S.; Newnham, S.; Lee, M. J.; Brown, I. R.; Xue, W.-F.; Rowe, M. L.; Mulvihill, D. P.; Prentice, M. B.; Howard, M. J.; Warren, M. J. Solution Structure of a Bacterial Microcompartment Targeting Peptide and Its Application in the Construction of an Ethanol Bioreactor. ACS Synth. Biol. 2014, 3, 454– 465,  DOI: 10.1021/sb4001118
      15
      Solution Structure of a Bacterial Microcompartment Targeting Peptide and Its Application in the Construction of an Ethanol Bioreactor
      Lawrence, Andrew D.; Frank, Stefanie; Newnham, Sarah; Lee, Matthew J.; Brown, Ian R.; Xue, Wei-Feng; Rowe, Michelle L.; Mulvihill, Daniel P.; Prentice, Michael B.; Howard, Mark J.; Warren, Martin J.
      ACS Synthetic Biology (2014), 3 (7), 454-465CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      Targeting of proteins to bacterial microcompartments (BMCs) is mediated by an 18-amino-acid peptide sequence. Herein, we report the soln. structure of the N-terminal targeting peptide (P18) of PduP, the aldehyde dehydrogenase assocd. with the 1,2-propanediol utilization metabolosome from Citrobacter freundii. The soln. structure reveals the peptide to have a well-defined helical conformation along its whole length. Satn. transfer difference and transferred NOE NMR has highlighted the obsd. interaction surface on the peptide with its main interacting shell protein, PduK. By tagging both a pyruvate decarboxylase and an alc. dehydrogenase with targeting peptides, it has been possible to direct these enzymes to empty BMCs in vivo and to generate an ethanol bioreactor. Not only are the purified, redesigned BMCs able to transform pyruvate into ethanol efficiently, but the strains contg. the modified BMCs produce elevated levels of alc.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVOjurk%253D&md5=f82c98604ef70551c623a4cfb008b0cc
    16. 16
      Cai, F.; Bernstein, S. L.; Wilson, S. C.; Kerfeld, C. A. Production and Characterization of Synthetic Carboxysome Shells with Incorporated Luminal Proteins. Plant Physiol. 2016, 170, 1868– 1877,  DOI: 10.1104/pp.15.01822
      16
      Production and characterization of synthetic carboxysome shells with incorporated luminal proteins
      Cai, Fei; Bernstein, Susan L.; Wilson, Steven C.; Kerfeld, Cheryl A.
      Plant Physiology (2016), 170 (3), 1868-1877CODEN: PLPHAY; ISSN:1532-2548. (American Society of Plant Biologists)
      Spatial segregation of metab., such as cellular-localized CO2 fixation in C4 plants or in the cyanobacterial carboxysome, enhances the activity of inefficient enzymes by selectively concg. them with their substrates. The carboxysome and other bacterial microcompartments (BMCs) have drawn particular attention for bioengineering of nanoreactors because they are selfassembling proteinaceous organelles. All BMCs share an architecturally similar, selectively permeable shell that encapsulates enzymes. Fundamental to engineering carboxysomes and other BMCs for applications in plant synthetic biol. and metabolic engineering is understanding the structural determinants of cargo packaging and shell permeability. Here we describe the expression of a synthetic operon in Escherichia coli that produces carboxysome shells. Protein domains native to the carboxysome core were used to encapsulate foreign cargo into the synthetic shells. These synthetic shells can be purified to homogeneity with or without luminal proteins. Our results not only further the understanding of protein-protein interactions governing carboxysome assembly, but also establish a platform to study shell permeability and the structural basis of the function of intact BMC shells both in vivo and in vitro. This system will be esp. useful for developing synthetic carboxysomes for plant engineering.
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    17. 17
      Hagen, A.; Sutter, M.; Sloan, N.; Kerfeld, C. A. Programmed loading and rapid purification of engineered bacterial microcompartment shells. Nat. Commun. 2018, 9, 2881  DOI: 10.1038/s41467-018-05162-z
      17
      Programmed loading and rapid purification of engineered bacterial microcompartment shells
      Hagen Andrew; Sutter Markus; Sloan Nancy; Kerfeld Cheryl A; Sutter Markus; Kerfeld Cheryl A; Kerfeld Cheryl A
      Nature communications (2018), 9 (1), 2881 ISSN:.
      Bacterial microcompartments (BMCs) are selectively permeable proteinaceous organelles which encapsulate segments of metabolic pathways across bacterial phyla. They consist of an enzymatic core surrounded by a protein shell composed of multiple distinct proteins. Despite great potential in varied biotechnological applications, engineering efforts have been stymied by difficulties in their isolation and characterization and a dearth of robust methods for programming cores and shell permeability. We address these challenges by functionalizing shell proteins with affinity handles, enabling facile complementation-based affinity purification (CAP) and specific cargo docking sites for efficient encapsulation via covalent-linkage (EnCo). These shell functionalizations extend our knowledge of BMC architectural principles and enable the development of minimal shell systems of precisely defined structure and composition. The generalizability of CAP and EnCo will enable their application to functionally diverse microcompartment systems to facilitate both characterization of natural functions and the development of bespoke shells for selectively compartmentalizing proteins.
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    18. 18
      Fan, C.; Cheng, S.; Liu, Y.; Escobar, C. M.; Crowley, C. S.; Jefferson, R. E.; Yeates, T. O.; Bobik, T. A. Short N-terminal sequences package proteins into bacterial microcompartments. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 7509– 7514,  DOI: 10.1073/pnas.0913199107
      18
      Short N-terminal sequences package proteins into bacterial microcompartments
      Fan, Chenguang; Cheng, Shouqiang; Liu, Yu; Escobar, Cristina M.; Crowley, Christopher S.; Jefferson, Robert E.; Yeates, Todd O.; Bobik, Thomas A.
      Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (16), 7509-7514, S7509/1-S7509/327CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Hundreds of bacterial species produce proteinaceous microcompartments (MCPs) that act as simple organelles by confining the enzymes of metabolic pathways that have toxic or volatile intermediates. A fundamental unanswered question about bacterial MCPs is how enzymes are packaged within the protein shell that forms their outer surface. Here, we report that a short N-terminal peptide is necessary and sufficient for packaging enzymes into the lumen of an MCP involved in B12-dependent 1,2-propanediol utilization (Pdu MCP). Deletion of 10 or 14 amino acids from the N terminus of the propionaldehyde dehydrogenase (PduP) enzyme, which is normally found within the Pdu MCP, substantially impaired packaging, with minimal effects on its enzymic activity. Fusion of the 18 N-terminal amino acids from PduP to GFP, GST, or maltose-binding protein resulted in their encapsulation within MCPs. Bioinformatic analyses revealed N-terminal extensions in two addnl. Pdu proteins and three proteins from two unrelated MCPs, suggesting that N-terminal peptides may be used to package proteins into diverse MCPs. The potential uses of MCP assembly principles in nature and in biotechnol. are discussed.
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    19. 19
      Bonacci, W.; Teng, P. K.; Afonso, B.; Niederholtmeyer, H.; Grob, P.; Silver, P. A.; Savage, D. F. Modularity of a carbon-fixing protein organelle. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 478– 483,  DOI: 10.1073/pnas.1108557109
      19
      Modularity of a carbon-fixing protein organelle
      Bonacci, Walter; Teng, Poh K.; Afonso, Bruno; Niederholtmeyer, Henrike; Grob, Patricia; Silver, Pamela A.; Savage, David F.
      Proceedings of the National Academy of Sciences of the United States of America (2012), 109 (2), 478-483, S478/1-S478/4CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Bacterial microcompartments are proteinaceous complexes that catalyze metabolic pathways in a manner reminiscent of organelles. Although microcompartment structure is well understood, much less is known about their assembly and function in vivo. We show here that carboxysomes, CO2-fixing microcompartments encoded by 10 genes, can be heterologously produced in Escherichia coli. Expression of carboxysomes in E. coli resulted in the prodn. of icosahedral complexes similar to those from the native host. In vivo, the complexes were capable of both assembling with carboxysomal proteins and fixing CO2. Characterization of purified synthetic carboxysomes indicated that they were well formed in structure, contained the expected mol. components, and were capable of fixing CO2 in vitro. In addn., we verify assocn. of the postulated pore-forming protein CsoS1D with the carboxysome and show how it may modulate function. We have developed a genetic system capable of producing modular carbon-fixing microcompartments in a heterologous host. In doing so, we lay the groundwork for understanding these elaborate protein complexes and for the synthetic biol. engineering of self-assembling mol. structures.
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    20. 20
      Chaijarasphong, T.; Nichols, R. J.; Kortright, K. E.; Nixon, C. F.; Teng, P. K.; Oltrogge, L. M.; Savage, D. F. Programmed Ribosomal Frameshifting Mediates Expression of the Alpha-Carboxysome. J. Mol. Biol. 2016, 428, 153– 164,  DOI: 10.1016/j.jmb.2015.11.017
      20
      Programmed ribosomal frameshifting mediates expression of the α-carboxysome
      Chaijarasphong, Thawatchai; Nichols, Robert J.; Kortright, Kaitlyn E.; Nixon, Charlotte F.; Teng, Poh K.; Oltrogge, Luke M.; Savage, David F.
      Journal of Molecular Biology (2016), 428 (1), 153-164CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      Many bacteria employ a protein organelle, the carboxysome, to catalyze carbon dioxide fixation in the Calvin Cycle. Only 10 genes from Halothiobacillus neapolitanus are sufficient for heterologous expression of carboxysomes in Escherichia coli, opening the door to detailed mechanistic anal. of the assembly process of this complex (more than 200 MDa). One of these genes, csoS2, has been implicated in assembly but ascribing a mol. function is confounded by the observation that the single csoS2 gene yields expression of two gene products and both display an apparent mol. wt. incongruent with the predicted amino acid sequence. Here, we elucidate the co-translational mechanism responsible for the expression of the two protein isoforms. Specifically, csoS2 was found to possess - 1 frameshifting elements that lead to the prodn. of the full-length protein, CsoS2B, and a truncated protein, CsoS2A, which possesses a C-terminus translated from the alternate frame. The frameshifting elements comprise both a ribosomal slippery sequence and a 3' secondary structure, and ablation of either sequence is sufficient to eliminate the slip. Using these mutants, we investigated the individual roles of CsoS2B and CsoS2A on carboxysome formation. In this in vivo formation assay, cells expressing only the CsoS2B isoform were capable of producing intact carboxysomes, while those with only CsoS2A were not. Thus, we have answered a long-standing question about the nature of CsoS2 in this model microcompartment and demonstrate that CsoS2B is functionally distinct from CsoS2A in the assembly of α-carboxysomes.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvFantrjO&md5=9f14c9a7162630363c9062f9e5b50771
    21. 21
      Cai, F.; Dou, Z.; Bernstein, S. L.; Leverenz, R.; Williams, E. B.; Heinhorst, S.; Shively, J.; Cannon, G. C.; Kerfeld, C. A. Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component. Life 2015, 5, 1141– 1171,  DOI: 10.3390/life5021141
      21
      Advances in understanding carboxysome assembly in Prochlorococcus and Synechococcus implicate CsoS2 as a critical component
      Cai, Fei; Dou, Zhicheng; Bernstein, Susan L.; Leverenz, Ryan; Williams, Eric B.; Heinhorst, Sabine; Shively, Jessup; Cannon, Gordon C.; Kerfeld, Cheryl A.
      Life (Basel, Switzerland) (2015), 5 (2), 1141-1171CODEN: LBSIB7; ISSN:2075-1729. (MDPI AG)
      The marine Synechococcus and Prochlorococcus are the numerically dominant cyanobacteria in the ocean and important in global carbon fixation. They have evolved a CO2-concg.-mechanism, of which the central component is the carboxysome, a self-assembling proteinaceous organelle. Two types of carboxysome, α and β, encapsulating form IA and form IB D-ribulose-1,5-bisphosphate carboxylase/oxygenase, resp.,differ in gene organization and assocd. proteins. In contrast to the β-carboxysome, the assembly process of the α-carboxysome is enigmatic. Moreover, an absolutely conserved α-carboxysome protein, CsoS2, is of unknown function and has proven recalcitrant to crystn. Here, we present studies on the CsoS2 protein in three model organisms and show that CsoS2 is vital for α-carboxysome biogenesis. The primary structure of CsoS2 appears tripartite, composed of an N-terminal, middle (M)-, and C-terminal region. Repetitive motifs can be identified in the N- and M-regions. Multiple lines of evidence suggest CsoS2 is highly flexible, possibly an intrinsically disordered protein. Based on our results from bioinformatic, biophys., genetic and biochem. approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-carboxysome. Analogies between the pathway for β-carboxysome biogenesis and our model for α-carboxysome assembly are discussed.
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    22. 22
      Li, T.; Jiang, Q.; Huang, J.; Aitchison, C. M.; Huang, F.; Yang, M.; Dykes, G. F.; He, H.-L.; Wang, Q.; Sprick, R. S.; Cooper, A. I.; Liu, L.-N. Reprogramming bacterial protein organelles as a nanoreactor for hydrogen production. Nat. Commun. 2020, 11, 5448  DOI: 10.1038/s41467-020-19280-0
      22
      Reprogramming bacterial protein organelles as a nanoreactor for hydrogen production
      Li, Tianpei; Jiang, Qiuyao; Huang, Jiafeng; Aitchison, Catherine M.; Huang, Fang; Yang, Mengru; Dykes, Gregory F.; He, Hai-Lun; Wang, Qiang; Sprick, Reiner Sebastian; Cooper, Andrew I.; Liu, Lu-Ning
      Nature Communications (2020), 11 (1), 5448CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Abstr.: Compartmentalization is a ubiquitous building principle in cells, which permits segregation of biol. elements and reactions. The carboxysome is a specialized bacterial organelle that encapsulates enzymes into a virus-like protein shell and plays essential roles in photosynthetic carbon fixation. The naturally designed architecture, semi-permeability, and catalytic improvement of carboxysomes have inspired rational design and engineering of new nanomaterials to incorporate desired enzymes into the protein shell for enhanced catalytic performance. Here, we build large, intact carboxysome shells (over 90 nm in diam.) in the industrial microorganism Escherichia coli by expressing a set of carboxysome protein-encoding genes. We develop strategies for enzyme activation, shell self-assembly, and cargo encapsulation to construct a robust nanoreactor that incorporates catalytically active [FeFe]-hydrogenases and functional partners within the empty shell for the prodn. of hydrogen. We show that shell encapsulation and the internal microenvironment of the new catalyst facilitate hydrogen prodn. of the encapsulated oxygen-sensitive hydrogenases. The study provides insights into the assembly and formation of carboxysomes and paves the way for engineering carboxysome shell-based nanoreactors to recruit specific enzymes for diverse catalytic reactions.
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    23. 23
      Kerfeld, C. A.; Melnicki, M. R. Assembly, function and evolution of cyanobacterial carboxysomes. Curr. Opin. Plant Biol. 2016, 31, 66– 75,  DOI: 10.1016/j.pbi.2016.03.009
      23
      Assembly, function and evolution of cyanobacterial carboxysomes
      Kerfeld, Cheryl A.; Melnicki, Matthew R.
      Current Opinion in Plant Biology (2016), 31 (), 66-75CODEN: COPBFZ; ISSN:1369-5266. (Elsevier Ltd.)
      All cyanobacteria contain carboxysomes, RuBisCO-encapsulating bacterial microcompartments that function as prokaryotic organelles. The two carboxysome types, alpha and beta, differ fundamentally in components, assembly, and species distribution. Alpha carboxysomes share a highly-conserved gene organization, with evidence of horizontal gene transfer from chemoautotrophic proteobacteria to the picocyanobacteria, and seem to co-assemble shells concomitantly with aggregation of cargo enzymes. In contrast, beta carboxysomes assemble an enzymic core first, with an encapsulation peptide playing a crit. role in formation of the surrounding shell. Based on similarities in assembly, and phylogenetic anal. of the pentameric shell protein conserved across all bacterial microcompartments, beta carboxysomes appear to be more closely related to the microcompartments of heterotrophic bacteria (metabolosomes) than to alpha carboxysomes, which appear deeply divergent. Beta carboxysomes can be found in the basal cyanobacterial clades that diverged before the ancestor of the chloroplast and have recently been shown to be able to encapsulate functional RuBisCO enzymes resurrected from ancestrally-reconstructed sequences, consistent with an ancient origin. Alpha and beta carboxysomes are not only distinct units of evolution, but are now emerging as genetic/metabolic modules for synthetic biol.; heterologous expression and redesign of both the shell and the enzymic core have recently been achieved.
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    24. 24
      Plegaria, J. S.; Kerfeld, C. A. Engineering nanoreactors using bacterial microcompartment architectures. Curr. Opin. Biotechnol. 2018, 51, 1– 7,  DOI: 10.1016/j.copbio.2017.09.005
      24
      Engineering nanoreactors using bacterial microcompartment architectures
      Plegaria, Jefferson S.; Kerfeld, Cheryl A.
      Current Opinion in Biotechnology (2018), 51 (), 1-7CODEN: CUOBE3; ISSN:0958-1669. (Elsevier B.V.)
      Bacterial microcompartments (BMCs) are organelles that encapsulate enzymes involved in CO2 fixation or carbon catabolism in a selectively permeable protein shell. Here, we highlight recent advances in the bioengineering of these protein-based nanoreactors in heterologous systems, including transfer and expression of BMC gene clusters, the prodn. of template empty shells, and the encapsulation of non-native enzymes.
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    25. 25
      Didovyk, A.; Tonooka, T.; Tsimring, L.; Hasty, J. Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression. ACS Synth. Biol. 2017, 6, 2198– 2208,  DOI: 10.1021/acssynbio.7b00253
      25
      Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression
      Didovyk, Andriy; Tonooka, Taishi; Tsimring, Lev; Hasty, Jeff
      ACS Synthetic Biology (2017), 6 (12), 2198-2208CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      Cell-free gene expression systems are emerging as an important platform for a diverse range of synthetic biol. and biotechnol. applications, including prodn. of robust field-ready biosensors. Here, the authors combine programmed cellular autolysis with a freeze-thaw or freeze-dry cycle to create a practical, reproducible, and a labor- and cost-effective approach for rapid prodn. of bacterial lysates for cell-free gene expression. Using this method, robust and highly active bacterial cell lysates can be produced without specialized equipment at a wide range of scales, making cell-free gene expression easily and broadly accessible. Moreover, live autolysis strain can be freeze-dried directly and subsequently lysed upon rehydration to produce active lysate. The authors demonstrate the utility of autolyzates for synthetic biol. by regulating protein prodn. and degrdn., implementing quorum sensing, and showing quant. protection of linear DNA templates by GamS protein. To allow versatile and sensitive β-galactosidase (LacZ) based readout the authors produce autolyzates with no detectable background LacZ activity and use them to produce sensitive mercury(II) biosensors with LacZ-mediated colorimetric and fluorescent outputs. The autolysis approach can facilitate wider adoption of cell-free technol. for cell-free gene expression as well as other synthetic biol. and biotechnol. applications, such as metabolic engineering, natural product biosynthesis, or proteomics.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlSitLvN&md5=9e772abe1adb3af657f6d9fb7d93d2fd
    26. 26
      Guo, Y.; Dong, J.; Zhou, T.; Auxillos, J.; Li, T.; Zhang, W.; Wang, L.; Shen, Y.; Luo, Y.; Zheng, Y.; Lin, J.; Chen, G. Q.; Wu, Q.; Cai, Y.; Dai, J. YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae. Nucleic Acids Res. 2015, 43, e88  DOI: 10.1093/nar/gkv464
      26
      YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae
      Guo, Yakun; Dong, Junkai; Zhou, Tong; Auxillos, Jamie; Li, Tianyi; Zhang, Weimin; Wang, Lihui; Shen, Yue; Luo, Yisha; Zheng, Yijing; Lin, Jiwei; Chen, Guo-Qiang; Wu, Qingyu; Cai, Yizhi; Dai, Junbiao
      Nucleic Acids Research (2015), 43 (13), e88/1-e88/14CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      It is a routine task in metabolic engineering to introduce multicomponent pathways into a heterologous host for prodn. of metabolites. However, this process sometimes may take weeks to months due to the lack of standardized genetic tools. Here, we present a method for the design and construction of biol. parts based on the native genes and regulatory elements in Saccharomyces cerevisiae. We have developed highly efficient protocols (termed YeastFab Assembly) to synthesize these genetic elements as standardized biol. parts, which can be used to assemble transcriptional units in a single-tube reaction. In addn., standardized characterization assays are developed using reporter constructs to calibrate the function of promoters. Furthermore, the assembled transcription units can be either assayed individually or applied to construct multi-gene metabolic pathways, which targets a genomic locus or a receiving plasmid effectively, through a simple in vitro reaction. Finally, using β-carotene biosynthesis pathway as an example, we demonstrate that our method allows us not only to construct and test a metabolic pathway in several days, but also to optimize the prodn. through combinatorial assembly of a pathway using hundreds of regulatory biol. parts.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhs1Ols7%252FP&md5=24625e6abdccf5459ee689e5a5aaf341
    27. 27
      Waterhouse, A. M.; Procter, J. B.; Martin, D. M. A.; Clamp, M.; Barton, G. J. Jalview Version 2─a multiple sequence alignment editor and analysis workbench. Bioinformatics 2009, 25, 1189– 1191,  DOI: 10.1093/bioinformatics/btp033
      27
      Jalview Version 2-a multiple sequence alignment editor and analysis workbench
      Waterhouse, Andrew M.; Procter, James B.; Martin, David M. A.; Clamp, Michele; Barton, Geoffrey J.
      Bioinformatics (2009), 25 (9), 1189-1191CODEN: BOINFP; ISSN:1367-4803. (Oxford University Press)
      Summary: Jalview Version 2 is a system for interactive WYSIWYG editing, anal. and annotation of multiple sequence alignments. Core features include keyboard and mouse-based editing, multiple views and alignment overviews, and linked structure display with Jmol. Jalview 2 is available in two forms: a lightwt. Java applet for use in web applications, and a powerful desktop application that employs web services for sequence alignment, secondary structure prediction and the retrieval of alignments, sequences, annotation and structures from public databases and any DAS 1.53 compliant sequence or annotation server. Availability: The Jalview 2 Desktop application and JalviewLite applet are made freely available under the GPL, and can be downloaded from www.jalview.org.
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    28. 28
      Sievers, F.; Higgins, D. G. Clustal Omega, accurate alignment of very large numbers of sequences. Methods Mol. Biol. 2014, 1079, 105– 116,  DOI: 10.1007/978-1-62703-646-7_6
      28
      Clustal Omega, Accurate Alignment of Very Large Numbers of Sequences
      Sievers, Fabian; Higgins, Desmond G.
      Methods in Molecular Biology (New York, NY, United States) (2014), 1079 (Multiple Sequence Alignment Methods), 105-116CODEN: MMBIED; ISSN:1940-6029. (Springer)
      Clustal Omega is a completely rewritten and revised version of the widely used Clustal series of programs for multiple sequence alignment. It can deal with very large nos. (many tens of thousands) of DNA/RNA or protein sequences due to its use of the mBED algorithm for calcg. guide trees. This algorithm allows very large alignment problems to be tackled very quickly, even on personal computers. The accuracy of the program has been considerably improved over earlier Clustal programs, through the use of the HHalign method for aligning profile hidden Markov models. The program currently is used from the command line or can be run on line.
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    29. 29
      Nichols, T. M.; Kennedy, N. W.; Tullman-Ercek, D. Cargo encapsulation in bacterial microcompartments: Methods and analysis. Methods Enzymol. 2019, 617, 155– 186,  DOI: 10.1016/bs.mie.2018.12.009
      29
      Cargo encapsulation in bacterial microcompartments: methods and analysis
      Nichols, Taylor M.; Kennedy, Nolan W.; Tullman-Ercek, Danielle
      Methods in Enzymology (2019), 617 (Metabolons and Supramolecular Enzyme Assemblies), 155-186CODEN: MENZAU; ISSN:0076-6879. (Elsevier Inc.)
      A review. Metabolic engineers seek to produce high-value products from inexpensive starting materials in a sustainable and cost-effective manner by using microbes as cellular factories. However, pathway development and optimization can be arduous tasks, complicated by pathway bottlenecks and toxicity. Pathway organization has emerged as a potential soln. to these issues, and the use of protein- or DNA-based scaffolds has successfully increased the prodn. of several industrially relevant compds. These efforts demonstrate the usefulness of pathway colocalization and spatial organization for metabolic engineering applications. In particular, scaffolding within an enclosed, subcellular compartment shows great promise for pathway optimization, offering benefits such as increased local enzyme and substrate concns., sequestration of toxic or volatile intermediates, and alleviation of cofactor and resource competition with the host. Here, we describe the 1,2-propanediol utilization (Pdu) bacterial microcompartment (MCP) as an enclosed scaffold for pathway sequestration and organization. We first describe methods for controlling Pdu MCP formation, expressing and encapsulating heterologous cargo, and tuning cargo loading levels. We further describe assays for analyzing Pdu MCPs and assessing encapsulation levels. These methods will enable the repurposing of MCPs as tunable nanobioreactors for heterologous pathway encapsulation.
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    30. 30
      Lassila, J. K.; Bernstein, S. L.; Kinney, J. N.; Axen, S. D.; Kerfeld, C. A. Assembly of robust bacterial microcompartment shells using building blocks from an organelle of unknown function. J. Mol. Biol. 2014, 426, 2217– 2228,  DOI: 10.1016/j.jmb.2014.02.025
      30
      Assembly of Robust Bacterial Microcompartment Shells Using Building Blocks from an Organelle of Unknown Function
      Lassila, Jonathan K.; Bernstein, Susan L.; Kinney, James N.; Axen, Seth D.; Kerfeld, Cheryl A.
      Journal of Molecular Biology (2014), 426 (11), 2217-2228CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      Bacterial microcompartments (BMCs) sequester enzymes from the cytoplasmic environment by encapsulation inside a selectively permeable protein shell. Bioinformatic analyses indicate that many bacteria encode BMC clusters of unknown function and with diverse combinations of shell proteins. The genome of the halophilic myxobacterium Haliangium ochraceum encodes one of the most atypical sets of shell proteins in terms of compn. and primary structure. We found that microcompartment shells could be purified in high yield when all seven H. ochraceum BMC shell genes were expressed from a synthetic operon in Escherichia coli. These shells differ substantially from previously isolated shell systems in that they are considerably smaller and more homogeneous, with measured diams. of 39±2 nm. The size and nearly uniform geometry allowed the development of a structural model for the shells composed of 260 hexagonal units and 13 hexagons per icosahedral face. We found that new proteins could be recruited to the shells by fusion to a predicted targeting peptide sequence, setting the stage for the use of these remarkably homogeneous shells for applications such as three-dimensional scaffolding and the construction of synthetic BMCs. Our results demonstrate the value of selecting from the diversity of BMC shell building blocks found in genomic sequence data for the construction of novel compartments.
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    31. 31
      Anderson, J. C. Anderson Promoter Library Registry of Standard Biological Parts. http://parts.igem.org/Promoters/Catalog/Anderson (accessed 1 May, 2019).
      There is no corresponding record for this reference.
    32. 32
      Yadav, V. G.; De Mey, M.; Lim, C. G.; Ajikumar, P. K.; Stephanopoulos, G. The future of metabolic engineering and synthetic biology: towards a systematic practice. Metab. Eng. 2012, 14, 233– 241,  DOI: 10.1016/j.ymben.2012.02.001
      32
      The future of metabolic engineering and synthetic biology: Towards a systematic practice
      Yadav, Vikramaditya G.; De Mey, Marjan; Giaw Lim, Chin; Kumaran Ajikumar, Parayil; Stephanopoulos, Gregory
      Metabolic Engineering (2012), 14 (3), 233-241CODEN: MEENFM; ISSN:1096-7176. (Elsevier B. V.)
      Industrial biotechnol. promises to revolutionize conventional chem. manufg. in the years ahead, largely owing to the excellent progress in our ability to re-engineer cellular metab. However, most successes of metabolic engineering have been confined to over-producing natively synthesized metabolites in E. coli and S. cerevisiae. A major reason for this development has been the descent of metabolic engineering, particularly secondary metabolic engineering, to a collection of demonstrations rather than a systematic practice with generalizable tools. Synthetic biol., a more recent development, faces similar criticisms. Herein, we attempt to lay down a framework around which bioreaction engineering can systematize itself just like chem. reaction engineering. Central to this undertaking is a new approach to engineering secondary metab. known as 'multivariate modular metabolic engineering' (MMME), whose novelty lies in its assessment and elimination of regulatory and pathway bottlenecks by re-defining the metabolic network as a collection of distinct modules. After introducing the core principles of MMME, we shall then present a no. of recent developments in secondary metabolic engineering that could potentially serve as its facilitators. It is hoped that the ever-declining costs of de novo gene synthesis; the improved use of bioinformatic tools to mine, sort and analyze biol. data; and the increasing sensitivity and sophistication of investigational tools will make the maturation of microbial metabolic engineering an autocatalytic process. Encouraged by these advances, research groups across the world would take up the challenge of secondary metabolite prodn. in simple hosts with renewed vigor, thereby adding to the range of products synthesized using metabolic engineering.
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    33. 33
      Baneyx, F. Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 1999, 10, 411– 421,  DOI: 10.1016/S0958-1669(99)00003-8
      33
      Recombinant protein expression in Escherichia coli
      Baneyx, Francois
      Current Opinion in Biotechnology (1999), 10 (5), 411-421CODEN: CUOBE3; ISSN:0958-1669. (Current Biology Publications)
      A review with 69 refs. Escherichia coli is one of the most widely used hosts for the prodn. of heterologous proteins and its genetics are far better characterized than those of any other microorganism. Recent progress in the fundamental understanding of transcription, translation, and protein folding in E. coli, together with serendipitous discoveries and the availability of improved genetic tools are making this bacterium more valuable than ever for the expression of complex eukaryotic proteins.
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    34. 34
      Oltrogge, L. M.; Chaijarasphong, T.; Chen, A. W.; Bolin, E. R.; Marqusee, S.; Savage, D. F. Multivalent interactions between CsoS2 and Rubisco mediate α-carboxysome formation. Nat. Struct. Mol. Biol. 2020, 27, 281– 287,  DOI: 10.1038/s41594-020-0387-7
      34
      Multivalent interactions between CsoS2 and Rubisco mediate α-carboxysome formation
      Oltrogge, Luke M.; Chaijarasphong, Thawatchai; Chen, Allen W.; Bolin, Eric R.; Marqusee, Susan; Savage, David F.
      Nature Structural & Molecular Biology (2020), 27 (3), 281-287CODEN: NSMBCU; ISSN:1545-9993. (Nature Research)
      Abstr.: Carboxysomes are bacterial microcompartments that function as the centerpiece of the bacterial CO2-concg. mechanism by facilitating high CO2 concns. near the carboxylase Rubisco. The carboxysome self-assembles from thousands of individual proteins into icosahedral-like particles with a dense enzyme cargo encapsulated within a proteinaceous shell. In the case of the α-carboxysome, there is little mol. insight into protein-protein interactions that drive the assembly process. Here, studies on the α-carboxysome from Halothiobacillus neapolitanus demonstrate that Rubisco interacts with the N terminus of CsoS2, a multivalent, intrinsically disordered protein. X-ray structural anal. of the CsoS2 interaction motif bound to Rubisco reveals a series of conserved electrostatic interactions that are only made with properly assembled hexadecameric Rubisco. Although biophys. measurements indicate that this single interaction is weak, its implicit multivalency induces high-affinity binding through avidity. Taken together, our results indicate that CsoS2 acts as an interaction hub to condense Rubisco and enable efficient α-carboxysome formation.
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    35. 35
      Gonzalez, M.; Frank, E. G.; Levine, A. S.; Woodgate, R. Lon-mediated proteolysis of the Escherichia coli UmuD mutagenesis protein: in vitro degradation and identification of residues required for proteolysis. Genes Dev. 1998, 12, 3889– 3899,  DOI: 10.1101/gad.12.24.3889
      35
      Lon-mediated proteolysis of the Escherichia coli UmuD mutagenesis protein: in vitro degradation and identification of residues required for proteolysis
      Gonzalez, Martin; Frank, Ekaterina G.; Levine, Arthur S.; Woodgate, Roger
      Genes & Development (1998), 12 (24), 3889-3899CODEN: GEDEEP; ISSN:0890-9369. (Cold Spring Harbor Laboratory Press)
      Most SOS mutagenesis in Escherichia coli is dependent on the UmuD and UmuC proteins. Perhaps as a consequence, the activity of these proteins is exquisitely regulated. The intracellular level of UmuD and UmuC is normally quite low but increases dramatically in Ion- strains, suggesting that both proteins are substrates of the Lon protease. We report here that the highly purified UmuD protein is specifically degraded in vitro by Lon in an ATP-dependent manner. To identify the regions of UmuD necessary for Lon-mediated proteolysis, we performed 'alanine-stretch' mutagenesis on umuD and followed the stability of the mutant protein in vivo. Such an approach allowed us to localize the site(s) within UmuD responsible for Lon-mediated proteolysis. The primary signal is located between residues 15 and 18 (FPLF), with an auxiliary site between residues 26 and 29 (FPSP), of the amino terminus of UmuD. Transfer of the amino terminus of UmuD (residues 1-40) to an otherwise stable protein imparts Lon-mediated proteolysis, thereby indicating that the amino terminus of UmuD is sufficient for Lon recognition and the ensuing degrdn. of the protein.
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    36. 36
      Neher, S. B.; Sauer, R. T.; Baker, T. A. Distinct peptide signals in the UmuD and UmuD′ subunits of UmuD/D′ mediate tethering and substrate processing by the ClpXP protease. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13219– 13224,  DOI: 10.1073/pnas.2235804100
      36
      Distinct peptide signals in the UmuD and UmuD' subunits of UmuD/D' mediate tethering and substrate processing by the ClpXP protease
      Neher, Saskia B.; Sauer, Robert T.; Baker, Tania A.
      Proceedings of the National Academy of Sciences of the United States of America (2003), 100 (23), 13219-13224CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The Escherichia coli UmuD' protein is a component of DNA polymerase V, an error-prone polymerase that carries out translesion synthesis on damaged DNA templates. The intracellular concn. of UmuD' is strictly controlled by regulated transcription, by posttranslational processing of UmuD to UmuD', and by ClpXP degrdn. UmuD' is a substrate for the ClpXP protease but must form a heterodimer with its unabbreviated precursor, UmuD, for efficient degrdn. to occur. Here, we show that UmuD functions as a UmuD' delivery protein for ClpXP. UmuD can also deliver a UmuD partner for degrdn. UmuD resembles SspB, a well-characterized substrate-delivery protein for ClpX, in that both proteins use related peptide motifs to bind to the N-terminal domain of ClpX, thereby tethering substrate complexes to ClpXP. The combined use of a weak substrate recognition signal and a delivery factor that tethers the substrate to the protease allows regulated proteolysis of UmuD/D' in the cell. Dual recognition strategies of this type may be a relatively common feature of intracellular protein turnover.
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    37. 37
      Ferlez, B.; Sutter, M.; Kerfeld, C. A. A designed bacterial microcompartment shell with tunable composition and precision cargo loading. Metab. Eng. 2019, 54, 286– 291,  DOI: 10.1016/j.ymben.2019.04.011
      37
      A designed bacterial microcompartment shell with tunable composition and precision cargo loading
      Ferlez, Bryan; Sutter, Markus; Kerfeld, Cheryl A.
      Metabolic Engineering (2019), 54 (), 286-291CODEN: MEENFM; ISSN:1096-7176. (Elsevier B.V.)
      Microbes often augment their metab. by conditionally constructing proteinaceous organelles, known as bacterial microcompartments (BMCs), that encapsulate enzymes to degrade org. compds. or assimilate CO2. BMCs self-assemble and are spatially delimited by a semi-permeable shell made up of hexameric, trimeric, and pentameric shell proteins. Bioengineers aim to recapitulate the organization and efficiency of these complex biol. architectures by redesigning the shell to incorporate non-native enzymes from biotechnol. relevant pathways. To meet this challenge, a diverse set of synthetic biol. tools are required, including methods to manipulate the properties of the shell as well as target and organize cargo encapsulation. We designed and detd. the crystal structure of a synthetic shell protein building block with an inverted sidedness of its N- and C-terminal residues relative to its natural counterpart; the inversion targets genetically fused protein cargo to the lumen of the shell. Moreover, the titer of fluorescent protein cargo encapsulated using this strategy is controllable using an inducible tetracycline promoter. These results expand the available set of building blocks for precision engineering of BMC-based nanoreactors and are compatible with orthogonal methods which will facilitate the installation and organization of multi-enzyme pathways.
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    38. 38
      Tsai, Y.; Sawaya, M. R.; Cannon, G. C.; Cai, F.; Williams, E. B.; Heinhorst, S.; Kerfeld, C. A.; Yeates, T. O. Structural Analysis of CsoS1A and the Protein Shell of the Halothiobacillus neapolitanus Carboxysome. PLoS Biol. 2007, 5, e144  DOI: 10.1371/journal.pbio.0050144
      There is no corresponding record for this reference.
    39. 39
      Klein, M. G.; Zwart, P.; Bagby, S. C.; Cai, F.; Chisholm, S. W.; Heinhorst, S.; Cannon, G. C.; Kerfeld, C. A. Identification and structural analysis of a novel carboxysome shell protein with implications for metabolite transport. J. Mol. Biol. 2009, 392, 319– 333,  DOI: 10.1016/j.jmb.2009.03.056
      39
      Identification and Structural Analysis of a Novel Carboxysome Shell Protein with Implications for Metabolite Transport
      Klein, Michael G.; Zwart, Peter; Bagby, Sarah C.; Cai, Fei; Chisholm, Sallie W.; Heinhorst, Sabine; Cannon, Gordon C.; Kerfeld, Cheryl A.
      Journal of Molecular Biology (2009), 392 (2), 319-333CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      Bacterial microcompartments (BMCs) are polyhedral bodies, composed entirely of proteins, that function as organelles in bacteria; they promote subcellular processes by encapsulating and co-localizing targeted enzymes with their substrates. The best-characterized BMC is the carboxysome, a central part of the carbon-concg. mechanism that greatly enhances carbon fixation in cyanobacteria and some chemoautotrophs. Here we report the first structural insights into the carboxysome of Prochlorococcus, the numerically dominant cyanobacterium in the world's oligotrophic oceans. Bioinformatic methods, substantiated by anal. of gene expression data, were used to identify a new carboxysome shell component, CsoS1D, in the genome of Prochlorococcus strain MED4; orthologs were subsequently found in all cyanobacteria. Two independent crystal structures of Prochlorococcus MED4 CsoS1D reveal three features not seen in any BMC-domain protein structure solved to date. First, CsoS1D is composed of a fused pair of BMC domains. Second, this double-domain protein trimerizes to form a novel pseudohexameric building block for incorporation into the carboxysome shell, and the trimers further dimerize, forming a two-tiered shell building block. Third, and most strikingly, the large pore formed at the 3-fold axis of symmetry appears to be gated. Each dimer of trimers contains one trimer with an open pore and one whose pore is obstructed due to side-chain conformations of two residues that are invariant among all CsoS1D orthologs. This is the first evidence of the potential for gated transport across the carboxysome shell and reveals a new type of building block for BMC shells.
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    40. 40
      Mohajerani, F.; Sayer, E.; Neil, C.; Inlow, K.; Hagan, M. F. Mechanisms of Scaffold-Mediated Microcompartment Assembly and Size Control. ACS Nano 2021, 15, 4197– 4212,  DOI: 10.1021/acsnano.0c05715
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      Mechanisms of Scaffold-Mediated Microcompartment Assembly and Size Control
      Mohajerani, Farzaneh; Sayer, Evan; Neil, Christopher; Inlow, Koe; Hagan, Michael F.
      ACS Nano (2021), 15 (3), 4197-4212CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      This article describes a theor. and computational study of the dynamical assembly of a protein shell around a complex consisting of many cargo mols. and long, flexible scaffold mols. Our study is motivated by bacterial microcompartments, which are proteinaceous organelles that assemble around a condensed droplet of enzymes and reactants. As in many examples of cytoplasmic liq.-liq. phase sepn., condensation of the microcompartment interior cargo is driven by flexible scaffold proteins that have weak multivalent interactions with the cargo. Our results predict that the shell size, amt. of encapsulated cargo, and assembly pathways depend sensitively on properties of the scaffold, including its length and valency of scaffold-cargo interactions. Moreover, the ability of self-assembling protein shells to change their size to accommodate scaffold mols. of different lengths depends crucially on whether the spontaneous curvature radius of the protein shell is smaller or larger than a characteristic elastic length scale of the shell. Beyond natural microcompartments, these results have important implications for synthetic biol. efforts to target alternative mols. for encapsulation by microcompartments or viral shells. More broadly, the results elucidate how cells exploit coupling between self-assembly and liq.-liq. phase sepn. to organize their interiors.
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    41. 41
      Sinha, S.; Cheng, S.; Sung, Y. W.; McNamara, D. E.; Sawaya, M. R.; Yeates, T. O.; Bobik, T. A. Alanine scanning mutagenesis identifies an asparagine-arginine-lysine triad essential to assembly of the shell of the Pdu microcompartment. J. Mol. Biol. 2014, 426, 2328– 2345,  DOI: 10.1016/j.jmb.2014.04.012
      41
      Alanine Scanning Mutagenesis Identifies an Asparagine-Arginine-Lysine Triad Essential to Assembly of the Shell of the Pdu Microcompartment
      Sinha, Sharmistha; Cheng, Shouqiang; Sung, Yea Won; McNamara, Dan E.; Sawaya, Michael R.; Yeates, Todd O.; Bobik, Thomas A.
      Journal of Molecular Biology (2014), 426 (12), 2328-2345CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      Bacterial microcompartments (MCPs) are the simplest organelles known. They function to enhance metabolic pathways by confining several related enzymes inside an all-protein envelope called the shell. In this study, we investigated the factors that govern MCP assembly by performing scanning mutagenesis on the surface residues of PduA, a major shell protein of the MCP used for 1,2-propanediol degrdn. Biochem., genetic, and structural anal. of 20 mutants allowed us to det. that PduA K26, N29, and R79 are crucial residues that stabilize the shell of the 1,2-propanediol MCP. In addn., we identify two PduA mutants (K37A and K55A) that impair MCP function most likely by altering the permeability of its protein shell. These are the first studies to examine the phenotypic effects of shell protein structural mutations in an MCP system. The findings reported here may be applicable to engineering protein containers with improved stability for biotechnol. applications.
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    42. 42
      Cai, F.; Sutter, M.; Cameron, J. C.; Stanley, D. N.; Kinney, J. N.; Kerfeld, C. A. The structure of CcmP, a tandem bacterial microcompartment domain protein from the β-carboxysome, forms a subcompartment within a microcompartment. J. Biol. Chem. 2013, 288, 16055– 16063,  DOI: 10.1074/jbc.M113.456897
      42
      The Structure of CcmP, a Tandem Bacterial Microcompartment Domain Protein from the β-Carboxysome, Forms a Subcompartment Within a Microcompartment
      Cai, Fei; Sutter, Markus; Cameron, Jeffrey C.; Stanley, Desiree N.; Kinney, James N.; Kerfeld, Cheryl A.
      Journal of Biological Chemistry (2013), 288 (22), 16055-16063CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The carboxysome is a bacterial organelle found in all cyanobacteria; it encapsulates CO2 fixation enzymes within a protein shell. The most abundant carboxysome shell protein contains a single bacterial microcompartment (BMC) domain. We present in vivo evidence that a hypothetical protein (dubbed CcmP) encoded in all β-cyanobacterial genomes is part of the carboxysome. We show that CcmP is a tandem BMC domain protein, the first to be structurally characterized from a β-carboxysome. CcmP forms a dimer of tightly stacked trimers, resulting in a nanocompartment-contg. shell protein that may weakly bind 3-phosphoglycerate, the product of CO2 fixation. The trimers have a large central pore through which metabolites presumably pass into the carboxysome. Conserved residues surrounding the pore have alternate side-chain conformations suggesting that it can be open or closed. Furthermore, CcmP and its orthologs in α-cyanobacterial genomes form a distinct clade of shell proteins. Members of this subgroup are also found in numerous heterotrophic BMC-assocd. gene clusters encoding functionally diverse bacterial organelles, suggesting that the potential to form a nanocompartment within a microcompartment shell is widespread. Given that carboxysomes and architecturally related bacterial organelles are the subject of intense interest for applications in synthetic biol./metabolic engineering, our results describe a new type of building block with which to functionalize BMC shells.
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    43. 43
      Szatmári, D.; Sárkány, P.; Kocsis, B.; Nagy, T.; Miseta, A.; Barkó, S.; Longauer, B.; Robinson, R. C.; Nyitrai, M. Intracellular ion concentrations and cation-dependent remodelling of bacterial MreB assemblies. Sci. Rep. 2020, 10, 12002  DOI: 10.1038/s41598-020-68960-w
      43
      Intracellular ion concentrations and cation-dependent remodelling of bacterial MreB assemblies
      Szatmari, David; Sarkany, Peter; Kocsis, Bela; Nagy, Tamas; Miseta, Attila; Barko, Szilvia; Longauer, Beata; Robinson, Robert C.; Nyitrai, Miklos
      Scientific Reports (2020), 10 (1), 12002CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
      Abstr.: Here, we measured the concns. of several ions in cultivated Gram-neg. and Gram-pos. bacteria, and analyzed their effects on polymer formation by the actin homolog MreB. We measured potassium, sodium, chloride, calcium and magnesium ion concns. in Leptospira interrogans, Bacillus subtilis and Escherichia coli. Intracellular ionic strength contributed from these ions varied within the 130-273 mM range. The intracellular sodium ion concn. range was between 122 and 296 mM and the potassium ion concn. range was 5 and 38 mM. However, the levels were significantly influenced by extracellular ion levels. L. interrogans, Rickettsia rickettsii and E. coli MreBs were heterologously expressed and purified from E. coli using a novel filtration method to prep. MreB polymers. The structures and stability of Alexa-488 labeled MreB polymers, under varying ionic strength conditions, were investigated by confocal microscopy and MreB polymn. rates were assessed by measuring light scattering. MreB polymn. was fastest in the presence of monovalent cations in the 200-300 mM range. MreB filaments showed high stability in this concn. range and formed large assemblies of tape-like bundles that transformed to extensive sheets at higher ionic strengths. Changing the calcium concn. from 0.2 to 0 mM and then to 2 mM initialized rapid remodelling of MreB polymers.
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    44. 44
      Mahinthichaichan, P.; Morris, D. M.; Wang, Y.; Jensen, G. J.; Tajkhorshid, E. Selective Permeability of Carboxysome Shell Pores to Anionic Molecules. J. Phys. Chem. B 2018, 122, 9110– 9118,  DOI: 10.1021/acs.jpcb.8b06822
      44
      Selective Permeability of Carboxysome Shell Pores to Anionic Molecules
      Mahinthichaichan, Paween; Morris, Dylan M.; Wang, Yi; Jensen, Grant J.; Tajkhorshid, Emad
      Journal of Physical Chemistry B (2018), 122 (39), 9110-9118CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
      Carboxysomes are closed polyhedral cellular microcompartments that increase the efficiency of carbon fixation in autotrophic bacteria. Carboxysome shells consist of small proteins that form hexameric units with semi-permeable central pores contg. binding sites for anions. This feature is thought to selectively allow access to RuBisCO enzymes inside the carboxysome by HCO3- (the dominant form of CO2 in the aq. soln. at pH 7.4) but not O2, which leads to a non-productive reaction. To test this hypothesis, here we use mol. dynamics simulations to characterize the energetics and permeability of CO2, O2, and HCO3- through the central pores of two different shell proteins, namely, CsoS1A of α-carboxysome and CcmK4 of β-carboxysome shells. We find that the central pores are in fact selectively permeable to anions such as HCO3-, as predicted by the model.
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    45. 45
      Marsh, J. A.; Forman-Kay, J. D. Sequence determinants of compaction in intrinsically disordered proteins. Biophys. J. 2010, 98, 2383– 2390,  DOI: 10.1016/j.bpj.2010.02.006
      45
      Sequence determinants of compaction in intrinsically disordered proteins
      Marsh, Joseph A.; Forman-Kay, Julie D.
      Biophysical Journal (2010), 98 (10), 2383-2390CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      Intrinsically disordered proteins (IDPs), which lack folded structure and are disordered under nondenaturing conditions, have been shown to perform important functions in a large no. of cellular processes. These proteins have interesting structural properties that deviate from the random-coil-like behavior exhibited by chem. denatured proteins. In particular, IDPs are often obsd. to exhibit significant compaction. In this study, we have analyzed the hydrodynamic radii of a no. of IDPs to investigate the sequence determinants of this compaction. Net charge and proline content are obsd. to be strongly correlated with increased hydrodynamic radii, suggesting that these are the dominant contributors to compaction. Hydrophobicity and secondary structure, on the other hand, appear to have negligible effects on compaction, which implies that the determinants of structure in folded and intrinsically disordered proteins are profoundly different. Finally, we observe that polyhistidine tags seem to increase IDP compaction, which suggests that these tags have significant perturbing effects and thus should be removed before any structural characterizations of IDPs. Using the relationships obsd. in this anal., we have developed a sequence-based predictor of hydrodynamic radius for IDPs that shows substantial improvement over a simple model based upon chain length alone.
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    46. 46
      Baker, N. A.; Sept, D.; Joseph, S.; Holst, M. J.; McCammon, J. A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 10037– 10041,  DOI: 10.1073/pnas.181342398
      46
      Electrostatics of nanosystems: application to microtubules and the ribosome
      Baker, Nathan A.; Sept, David; Joseph, Simpson; Holst, Michael J.; McCammon, J. Andrew
      Proceedings of the National Academy of Sciences of the United States of America (2001), 98 (18), 10037-10041CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Evaluation of the electrostatic properties of biomols. has become a std. practice in mol. biophysics. Foremost among the models used to elucidate the electrostatic potential is the Poisson-Boltzmann equation; however, existing methods for solving this equation have limited the scope of accurate electrostatic calcns. to relatively small biomol. systems. Here we present the application of numerical methods to enable the trivially parallel soln. of the Poisson-Boltzmann equation for supramol. structures that are orders of magnitude larger in size. As a demonstration of this methodol., electrostatic potentials have been calcd. for large microtubule and ribosome structures. The results point to the likely role of electrostatics in a variety of activities of these structures.
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    47. 47
      Silva, C.; Martins, M.; Jing, S.; Fu, J.; Cavaco-Paulo, A. Practical insights on enzyme stabilization. Crit. Rev. Biotechnol. 2018, 38, 335– 350,  DOI: 10.1080/07388551.2017.1355294
      47
      Practical insights on enzyme stabilization
      Silva, Carla; Martins, Madalena; Jing, Su; Fu, Jiajia; Cavaco-Paulo, Artur
      Critical Reviews in Biotechnology (2018), 38 (3), 335-350CODEN: CRBTE5; ISSN:0738-8551. (Taylor & Francis Ltd.)
      Enzymes are efficient catalysts designed by nature to work in physiol. environments of living systems. The best operational conditions to access and convert substrates at the industrial level are different from nature and normally extreme. Strategies to isolate enzymes from extremophiles can redefine new operational conditions, however not always solving all industrial requirements. The stability of enzymes is therefore a key issue on the implementation of the catalysts in industrial processes which require the use of extreme environments that can undergo enzyme instability. Strategies for enzyme stabilization have been exhaustively reviewed, however they lack a practical approach. This review intends to compile and describe the most used approaches for enzyme stabilization highlighting case studies in a practical point of view.
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    48. 48
      Tan, Y. Q.; Xue, B.; Yew, W. S. Genetically Encodable Scaffolds for Optimizing Enzyme Function. Molecules 2021, 26, 1389  DOI: 10.3390/molecules26051389
      48
      Genetically encodable scaffolds for optimizing enzyme function
      Tan, Yong Quan; Xue, Bo; Yew, Wen Shan
      Molecules (2021), 26 (5), 1389CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
      Enzyme engineering is an indispensable tool in the field of synthetic biol., where enzymes are challenged to carry out novel or improved functions. Achieving these goals sometimes goes beyond modifying the primary sequence of the enzyme itself. The use of protein or nucleic acid scaffolds to enhance enzyme properties has been reported for applications such as microbial prodn. of chems., biosensor development and bioremediation. Key advantages of using these assemblies include optimizing reaction conditions, improving metabolic flux and increasing enzyme stability. This review summarizes recent trends in utilizing genetically encodable scaffolds, developed in line with synthetic biol. methodologies, to complement the purposeful deployment of enzymes. Current mol. tools for constructing these synthetic enzyme-scaffold systems are also highlighted.
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    49. 49
      Küchler, A.; Yoshimoto, M.; Luginbühl, S.; Mavelli, F.; Walde, P. Enzymatic reactions in confined environments. Nat. Nanotechnol. 2016, 11, 409– 420,  DOI: 10.1038/nnano.2016.54
      49
      Enzymatic reactions in confined environments
      Kuchler, Andreas; Yoshimoto, Makoto; Luginbuhl, Sandra; Mavelli, Fabio; Walde, Peter
      Nature Nanotechnology (2016), 11 (5), 409-420CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
      A review. Within each biol. cell, surface- and vol.-confined enzymes control a highly complex network of chem. reactions. These reactions are efficient, timely, and spatially defined. Efforts to transfer such appealing features to in vitro systems have led to several successful examples of chem. reactions catalyzed by isolated and immobilized enzymes. In most cases, these enzymes are either bound or adsorbed to an insol. support, phys. trapped in a macromol. network, or encapsulated within compartments. Advanced applications of enzymic cascade reactions with immobilized enzymes include enzymic fuel cells and enzymic nanoreactors, both for in vitro and possible in vivo applications. Here, the authors discuss some of the general principles of enzymic reactions confined on surfaces, at interfaces, and inside small vols. The authors also highlight the similarities and differences between the in vivo and in vitro cases and attempt to critically evaluate some of the necessary future steps to improve the fundamental understanding of these systems.
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    50. 50
      Das, S.; Zhao, L.; Elofson, K.; Finn, M. G. Enzyme Stabilization by Virus-Like Particles. Biochemistry 2020, 59, 2870– 2881,  DOI: 10.1021/acs.biochem.0c00435
      50
      Enzyme Stabilization by Virus-Like Particles
      Das, Soumen; Zhao, Liangjun; Elofson, Kristen; Finn, M. G.
      Biochemistry (2020), 59 (31), 2870-2881CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The properties of enzymes packaged within the coat protein shell of virus-like particles (VLPs) were studied to provide a comprehensive assessment of such factors. Such entrainment did not seem to perturb enzyme function, but it did significantly enhance enzyme stability against several denaturing stimuli such as heat, org. solvents, and chaotropic agents. This improvement in performance is general and independent of the no. of independent subunits required and of the no. of catalytically active enzymes packaged. Packaged enzymes were found by measurements of intrinsic tryptophan fluorescence to retain some of their native folded structure even longer than their catalytic activity, suggesting that protein folding is a significant component of the obsd. catalytic benefits. While the authors are unable to distinguish between kinetic and thermodn. effects - including inhibition of enzyme unfolding, acceleration of refolding, and biasing of folding equil. - VLP packaging appears to represent a useful general strategy for the stabilization of enzymes that operate on diffusible substrates and products.
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    51. 51
      Patterson, D. P.; Schwarz, B.; El-Boubbou, K.; van der Oost, J.; Prevelige, P. E.; Douglas, T. Virus-like particle nanoreactors: programmed encapsulation of the thermostable CelB glycosidase inside the P22 capsid. Soft Matter 2012, 8, 10158– 10166,  DOI: 10.1039/c2sm26485d
      51
      Virus-like particle nanoreactors: programmed encapsulation of the thermostable CelB glycosidase inside the P22 capsid
      Patterson, Dustin P.; Schwarz, Benjamin; El-Boubbou, Kheireddine; van der Oost, John; Prevelige, Peter E.; Douglas, Trevor
      Soft Matter (2012), 8 (39), 10158-10166CODEN: SMOABF; ISSN:1744-683X. (Royal Society of Chemistry)
      Self-assembling biol. systems hold great potential for the synthetic construction of new active soft nanomaterials. Here we demonstrate the hierarchical bottom-up assembly of bacteriophage P22 virus-like particles (VLPs) that encapsulate the thermostable CelB glycosidase creating catalytically active nanoreactors. The in vivo assembly and encapsulation produces P22 VLPs with a high packaging d. of the tetrameric CelB, but without loss of enzyme activity or the ability of the P22 VLP to undergo unique morphol. transitions that modify the VLPs internal vol. and shell porosity. The P22 VLPs encapsulating CelB are also shown to retain a high percentage of the enzyme activity upon being embedded and immobilized in a polymeric matrix.
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    52. 52
      Patterson, D. P.; Prevelige, P. E.; Douglas, T. Nanoreactors by Programmed Enzyme Encapsulation Inside the Capsid of the Bacteriophage P22. ACS Nano 2012, 6, 5000– 5009,  DOI: 10.1021/nn300545z
      52
      Nanoreactors by Programmed Enzyme Encapsulation Inside the Capsid of the Bacteriophage P22
      Patterson, Dustin P.; Prevelige, Peter E.; Douglas, Trevor
      ACS Nano (2012), 6 (6), 5000-5009CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      The virus like particle (VLP) derived from bacteriophage P22 presents a unique platform for constructing catalytically functional nanomaterials by encapsulation of enzymes into its interior. Encapsulation has been engineered to be genetically programmed allowing "one pot" synthesis and incorporation of desired enzymes. The unique characteristic that separates P22 from other VLP systems is the ability to modulate the overall vol. and porosity of the VLP structure, thus controlling substrate access to the encapsulated enzyme. The present study demonstrates incorporation of an enzyme, alc. dehydrogenase D, with the highest internal loading for an active enzyme by any VLP described thus far. In addn., we show that not only does encapsulating AdhD inside P22 affect its kinetic parameters in comparison with the "free" enzyme, but transformation of P22 to different morphol. states, which changes the internal vol. of the VLP, yields changes in the overall activity of the encapsulated enzyme as well. The findings reported here clearly illustrate that P22 holds potential for synthetic approaches to create nanoreactors, by design, using the power of highly evolved enzymes for chem. transformations.
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    53. 53
      Sánchez-Sánchez, L.; Tapia-Moreno, A.; Juarez-Moreno, K.; Patterson, D. P.; Cadena-Nava, R. D.; Douglas, T.; Vazquez-Duhalt, R. Design of a VLP-nanovehicle for CYP450 enzymatic activity delivery. J. Nanobiotechnol. 2015, 13, 66  DOI: 10.1186/s12951-015-0127-z
      53
      Design of a VLP-nanovehicle for CYP450 enzymatic activity delivery
      Sanchez-Sanchez, Lorena; Tapia-Moreno, Alejandro; Juarez-Moreno, Karla; Patterson, Dustin P.; Cadena-Nava, Ruben D.; Douglas, Trevor; Vazquez-Duhalt, Rafael
      Journal of Nanobiotechnology (2015), 13 (), 66/1-66/10CODEN: JNOAAO; ISSN:1477-3155. (BioMed Central Ltd.)
      Background: The intracellular delivery of enzymes for therapeutic use has a promising future for the treatment of several diseases such as genetic disorders and cancer. Virus-like particles offer an interesting platform for enzymic delivery to targeted cells because of their great cargo capacity and the enhancement of the biocatalyst stability towards several factors important in the practical application of these nanoparticles. Results: We have designed a nano-bioreactor based on the encapsulation of a cytochrome P 450 (CYP) inside the capsid derived from the bacteriophage P22. An enhanced peroxigenase, CYPBM3, was selected as a model enzyme because of its potential in enzyme prodrug therapy. A total of 109 enzymes per capsid were encapsulated with a 70 % retention of activity for cytochromes with the correct incorporation of the heme cofactor. Upon encapsulation, the stability of the enzyme towards protease degrdn. and acidic pH was increased. Cytochrome P 450 activity was delivered into Human cervix carcinoma cells via transfecting P22-CYP nanoparticles with lipofectamine. Conclusion: This work provides a clear demonstration of the potential of biocatalytic virus-like particles as medical relevant enzymic delivery vehicles for clin. applications.
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    54. 54
      Azuma, Y.; Zschoche, R.; Tinzl, M.; Hilvert, D. Quantitative Packaging of Active Enzymes into a Protein Cage. Angew. Chem., Int. Ed 2016, 55, 1531– 1534,  DOI: 10.1002/anie.201508414
      54
      Quantitative Packaging of Active Enzymes into a Protein Cage
      Azuma, Yusuke; Zschoche, Reinhard; Tinzl, Matthias; Hilvert, Donald
      Angewandte Chemie, International Edition (2016), 55 (4), 1531-1534CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Genetic fusion of cargo proteins to a pos. supercharged variant of green fluorescent protein enables their quant. encapsulation by engineered lumazine synthase capsids possessing a neg. charged lumenal surface. This simple tagging system provides a robust and versatile means of creating hierarchically ordered protein assemblies for use as nanoreactors. The generality of the encapsulation strategy and its effect on enzyme function were investigated with eight structurally and mechanistically distinct catalysts.
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    55. 55
      Yu, Z.; Reid, J. C.; Yang, Y.-P. Utilizing Dynamic Light Scattering as a Process Analytical Technology for Protein Folding and Aggregation Monitoring in Vaccine Manufacturing. J. Pharm. Sci. 2013, 102, 4284– 4290,  DOI: 10.1002/jps.23746
      55
      Utilizing Dynamic Light Scattering as a Process Analytical Technology for Protein Folding and Aggregation Monitoring in Vaccine Manufacturing
      Yu, Zhou; Reid, Jennifer C.; Yang, Yan-Ping
      Journal of Pharmaceutical Sciences (2013), 102 (12), 4284-4290CODEN: JPMSAE; ISSN:0022-3549. (John Wiley & Sons, Inc.)
      Protein aggregation is a common challenge in the manufg. of biol. products. It is possible to minimize the extent of aggregation through timely measurement and in-depth characterization of aggregation. In this study, we demonstrated the use of dynamic light scattering (DLS) to monitor inclusion body (IB) solubilization, protein refolding, and aggregation near the prodn. line of a recombinant protein-based vaccine candidate. Our results were in good agreement with those measured by size-exclusion chromatog. DLS was also used to characterize the mechanism of aggregation. As DLS is a quick, nonperturbing technol., it can potentially be used as an at-line process anal. technol. to ensure complete IB solubilization and aggregate-free refolding. pr 2013 Wiley Periodicals, Inc. and the American Pharmacists Assocn. J Pharm Sci.
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    56. 56
      Lam, S. S.; Martell, J. D.; Kamer, K. J.; Deerinck, T. J.; Ellisman, M. H.; Mootha, V. K.; Ting, A. Y. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat. Methods 2015, 12, 51– 54,  DOI: 10.1038/nmeth.3179
      56
      Directed evolution of APEX2 for electron microscopy and proximity labeling
      Lam, Stephanie S.; Martell, Jeffrey D.; Kamer, Kimberli J.; Deerinck, Thomas J.; Ellisman, Mark H.; Mootha, Vamsi K.; Ting, Alice Y.
      Nature Methods (2015), 12 (1), 51-54CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      APEX is an engineered peroxidase that functions as an electron microscopy tag and a promiscuous labeling enzyme for live-cell proteomics. Because limited sensitivity precludes applications requiring low APEX expression, the authors used yeast-display evolution to improve its catalytic efficiency. APEX2 is far more active in cells, enabling the use of electron microscopy to resolve the submitochondrial localization of calcium uptake regulatory protein MICU1. APEX2 also permits superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins.
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    57. 57
      Golan, R.; Zehavi, U.; Naim, M.; Patchornik, A.; Smirnoff, P. Inhibition of Escherichia coli beta-galactosidase by 2-nitro-1-(4,5-dimethoxy-2-nitrophenyl) ethyl, a photoreversible thiol label. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1996, 1293, 238– 242,  DOI: 10.1016/0167-4838(95)00254-5
      57
      Inhibition of Escherichia coli β-galactosidase by 2-nitro-1-(4,5-dimethoxy-2-nitrophenyl)ethyl, a photoreversible thiol label
      Golan, Rachel; Zehavi, Uri; Naim, Michael; Patchornik, Abraham; Smirnoff, Patricia
      Biochimica et Biophysica Acta, Protein Structure and Molecular Enzymology (1996), 1293 (2), 238-42CODEN: BBAEDZ; ISSN:0167-4838. (Elsevier B.V.)
      1-Nitro-2-phenylethene (β-nitrostyrene, 1), which is a thiol-protecting reagent (Jung, G., Fouad, H. and Heusel, G. (1975) Angew. Chem. Int. Ed. Engl. 14, 817-818) was demonstrated in this work to be an irreversible inhibitor of β-galactosidase (EC 3.2.1.23), an enzyme known to be inhibited by some thiol reagents or through modifying a methionine residue at the active site. No reversal of the inhibition was obsd. upon subsequent incubation with mercaptoethanol or irradn. (350 nm). 1-(4,5-Dimethoxy-2-nitrophenyl)-2-Nitroethene (2) was also shown to be an irreversible inhibitor (94% inhibition, pH 8.3) of the enzyme. Kcat values of β-galactosidase at pH 8.3 with o-nitrophenyl β-D-galactopyranoside (ONPG) as the substrate and at the highest inhibitor concns. employed for compd. 1 (4.06·10-4 M) ranged from 1.67·104 s-1 after 30 min of preincubation to <0.07·104 s-1 after 180 min preincubation. For compd. 2 (9.5·10-5 M) Kcat values ranged from 2.70·104 s-1 following 30 min preincubation to 1.15·104 s-1 after 180 min of preincubation; the changes in Kmapp, however, were small. The activity was not recovered following incubation with mercaptoethanol. Since compd. 2 and the inhibited enzyme are 2-nitrobenzyl derivs., they are expected to be photosensitive and indeed, irradn. of the inhibited enzyme in the presence of mercaptoethanol resulted in recovery (89%, pH 8.3) of the enzyme activity.
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    58. 58
      Juers, D. H.; Hakda, S.; Matthews, B. W.; Huber, R. E. Structural Basis for the Altered Activity of Gly794 Variants of Escherichia coli β-Galactosidase. Biochemistry 2003, 42, 13505– 13511,  DOI: 10.1021/bi035506j
      58
      Structural Basis for the Altered Activity of Gly794 Variants of Escherichia coli β-Galactosidase
      Juers, Douglas H.; Hakda, Shamina; Matthews, Brian W.; Huber, Reuben E.
      Biochemistry (2003), 42 (46), 13505-13511CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The open-closed conformational switch in the active site of Escherichia coli β-galactosidase was studied by X-ray crystallog. and enzyme kinetics. Replacement of Gly794 by alanine causes the apoenzyme to adopt the closed rather than the open conformation. Binding of the competitive inhibitor iso-Pr thio-β-D-galactoside (IPTG) requires the mutant enzyme to adopt its less favored open conformation, weakening affinity relative to wild type. In contrast, transition-state inhibitors bind to the enzyme in the closed conformation, which is favored for the mutant, and display increased affinity relative to wild type. Changes in affinity suggest that the free energy difference between the closed and open forms is 1-2 kcal/mol. By favoring the closed conformation, the substitution moves the resting state of the enzyme along the reaction coordinate relative to the native enzyme and destabilizes the ground state relative to the first transition state. The result is that the rate const. for galactosylation is increased but degalactosylation is slower. The covalent intermediate may be better stabilized than the second transition state. The substitution also results in better binding of glucose to both the free and the galactosylated enzyme. However, transgalactosylation with glucose to produce allolactose (the inducer of the lac operon) is slower with the mutant than with the native enzyme. This suggests either that the glucose is misaligned for the reaction or that the galactosylated enzyme with glucose bound is stabilized relative to the transition state for transgalactosylation.
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    59. 59
      Zhou, H.-X.; Rivas, G.; Minton, A. P. Macromolecular Crowding and Confinement: Biochemical, Biophysical, and Potential Physiological Consequences. Annu. Rev. Biophys. 2008, 37, 375– 397,  DOI: 10.1146/annurev.biophys.37.032807.125817
      59
      Macromolecular crowding and confinement: Biochemical, biophysical, and potential physiological consequences
      Zhou, Huan-Xiang; Rivas, German; Minton, Allen P.
      Annual Review of Biophysics (2008), 37 (), 375-397CODEN: ARBNCV ISSN:. (Annual Reviews Inc.)
      A review. Expected and obsd. effects of vol. exclusion on the free energy of rigid and flexible macromols. in crowded and confined systems, and consequent effects of crowding and confinement on macromol. reaction rates and equil. are summarized. Findings from relevant theor./simulation and exptl. literature published from 2004 onward are reviewed. Addnl. complexity arising from the heterogeneity of local environments in biol. media, and the presence of nonspecific interactions between macromols. over and above steric repulsion, are discussed. Theor. and exptl. approaches to the characterization of crowding- and confinement-induced effects in systems approaching the complexity of living organisms are suggested.
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    60. 60
      Comellas-Aragonès, M.; Engelkamp, H.; Claessen, V. I.; Sommerdijk, N. A. J. M.; Rowan, A. E.; Christianen, P. C. M.; Maan, J. C.; Verduin, B. J. M.; Cornelissen, J. J. L. M.; Nolte, R. J. M. A virus-based single-enzyme nanoreactor. Nat. Nanotechnol. 2007, 2, 635– 639,  DOI: 10.1038/nnano.2007.299
      60
      A virus-based single-enzyme nanoreactor
      Comellas-Aragones, Marta; Engelkamp, Hans; Claessen, Victor I.; Sommerdijk, Nico A. J. M.; Rowan, Alan E.; Christianen, Peter C. M.; Maan, Jan C.; Verduin, Benedictus J. M.; Cornelissen, Jeroen J. L. M.; Nolte, Roeland J. M.
      Nature Nanotechnology (2007), 2 (10), 635-639CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
      Most enzyme studies are carried out in bulk aq. soln., at the so-called ensemble level, but more recently studies have appeared in which enzyme activity is measured at the level of a single mol., revealing previously unseen properties. To this end, enzymes have been chem. or phys. anchored to a surface, which is often disadvantageous because it may lead to denaturation. In a natural environment, enzymes are present in a confined reaction space, which inspired us to develop a generic method to carry out single-enzyme expts. in the restricted spatial environment of a virus capsid. We report here the incorporation of individual horseradish peroxidase enzymes in the inner cavity of a virus, and describe single-mol. studies on their enzymic behavior. These show that the virus capsid is permeable for substrate and product and that this permeability can be altered by changing pH.
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    61. 61
      Faulkner, M.; Szabó, I.; Weetman, S. L.; Sicard, F.; Huber, R. G.; Bond, P. J.; Rosta, E.; Liu, L.-N. Molecular simulations unravel the molecular principles that mediate selective permeability of carboxysome shell protein. Sci. Rep. 2020, 10, 17501  DOI: 10.1038/s41598-020-74536-5
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      Molecular simulations unravel molecular principles that mediate selective permeability of carboxysome shell protein
      Faulkner, Matthew; Szabo, Istvan; Weetman, Samantha L.; Sicard, Francois; Huber, Roland G.; Bond, Peter J.; Rosta, Edina; Liu, Lu-Ning
      Scientific Reports (2020), 10 (1), 17501CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
      Bacterial microcompartments (BMCs) are nanoscale proteinaceous organelles that encapsulate enzymes from the cytoplasm using an icosahedral protein shell that resembles viral capsids. Of particular interest are the carboxysomes (CBs), which sequester the CO2-fixing enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to enhance carbon assimilation. The carboxysome shell serves as a semi-permeable barrier for passage of metabolites in and out of the carboxysome to enhance CO2 fixation. How the protein shell directs influx and efflux of mols. in an effective manner has remained elusive. Here we use mol. dynamics and umbrella sampling calcns. to det. the free-energy profiles of the metabolic substrates, bicarbonate, CO2 and ribulose bisphosphate and the product 3-phosphoglycerate assocd. with their transition through the major carboxysome shell protein CcmK2. We elucidate the electrostatic charge-based permeability and key amino acid residues of CcmK2 functioning in mediating mol. transit through the central pore. Conformational changes of the loops forming the central pore may also be required for transit of specific metabolites. The importance of these in-silico findings is validated exptl. by site-directed mutagenesis of the key CcmK2 residue Serine 39. This study provides insight into the mechanism that mediates mol. transport through the shells of carboxysomes, applicable to other BMCs. It also offers a predictive approach to investigate and manipulate the shell permeability, with the intent of engineering BMC-based metabolic modules for new functions in synthetic biol.
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    62. 62
      Park, J.; Chun, S.; Bobik, T. A.; Houk, K. N.; Yeates, T. O. Molecular Dynamics Simulations of Selective Metabolite Transport across the Propanediol Bacterial Microcompartment Shell. J. Phys. Chem. B 2017, 121, 8149– 8154,  DOI: 10.1021/acs.jpcb.7b07232
      62
      Molecular Dynamics Simulations of Selective Metabolite Transport across the Propanediol Bacterial Microcompartment Shell
      Park, Jiyong; Chun, Sunny; Bobik, Thomas A.; Houk, Kendall N.; Yeates, Todd O.
      Journal of Physical Chemistry B (2017), 121 (34), 8149-8154CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
      Bacterial microcompartments are giant protein-based organelles that encapsulate special metabolic pathways in diverse bacteria. Structural and genetic studies indicate that metabolic substrates enter these microcompartments by passing through the central pores in hexameric assemblies of shell proteins. Limiting the escape of toxic metabolic intermediates created inside the microcompartments would confer a selective advantage for the host organism. Here, we report the first mol. dynamics (MD) simulation studies to analyze small-mol. transport across a microcompartment shell. PduA is a major shell protein in a bacterial microcompartment that metabolizes 1,2-propanediol via a toxic aldehyde intermediate, propionaldehyde. Using both metadynamics and replica-exchange umbrella sampling, we find that the pore of the PduA hexamer has a lower energy barrier for passage of the propanediol substrate compared to the toxic propionaldehyde generated within the microcompartment. The energetic effect is consistent with a lower capacity of a serine side chain, which protrudes into the pore at a point of constriction, to form hydrogen bonds with propionaldehyde relative to the more freely permeable propanediol. The results highlight the importance of mol. diffusion and transport in a new biol. context.
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    63. 63
      Shifrin, S.; Hunn, G. Effect of alcohols on the enzymatic activity and subunit association of β-galactosidase. Arch. Biochem. Biophys. 1969, 130, 530– 535,  DOI: 10.1016/0003-9861(69)90066-6
      63
      Effect of alcohols on the enzymatic activity and subunit association of β-galactosidase
      Shifrin, Sidney; Hunn, Gilbert
      Archives of Biochemistry and Biophysics (1969), 130 (1), 530-5CODEN: ABBIA4; ISSN:0003-9861.
      The catalytic activity of β-galactosidase from Escherichia coli K-12 and the associative properties of its subunits have been studied in solns. of MeOH, EtOH, iso-PrOH and PrOH. All of the alcs. at low concns. (5%) stimulate the rate at which o-nitrophenyl β-D-galactopyranoside is cleaved. The transferase activity of the enzyme was demonstrated by identification of Me β-galactoside as one of the reaction products. Neither MeOH nor EtOH dissoc. the subunits of the active tetramer nor do they induce conformational changes in the protein as measured by sedimentation velocity, uv absorption spectroscopy, and fluorescence. Low concns. of PrOH in the absence of added Mg2+, however, cause the tetramer to dissoc. into inactive dimers.
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    64. 64
      Goldstein, L.; Levin, Y.; Katchalski, E. A Water-Insoluble Polyanionic Derivative of Trypsin. II. Effect of the Polyelectrolyte Carrier on the Kinetic Behavior of the Bound Trypsin*. Biochemistry 1964, 3, 1913– 1919,  DOI: 10.1021/bi00900a022
      64
      A water-insoluble polyanionic derivative of trypsin. II. Effect of the polyelectrolyte carrier on the kinetic behavior of the bound trypsin
      Goldstein, Leon; Levin, Yehuda; Katchalski, Ephraim
      Biochemistry (1964), 3 (12), 1913-19CODEN: BICHAW; ISSN:0006-2960.
      The mode of action of IMET, obtained by the covalent binding of trypsin to a copolymer of maleic acid and ethylene (1:1), was investigated at 25°. The pH-activity profile of IMET at low ionic strength (5.8 × 10-3), using benzoyl-L-arginine Et ester as substrate, was displaced by approx. 2.5 pH units toward more alk. pH values, when compared with trypsin under similar conditions. At higher ionic strength, the pH-activity curve of IMET shifted toward more acid pH values, approaching the pH-activity curve of IMET-trypsin at ionic strength 1.0. The Km = 0.2 ± 0.05 × 10-3M measured for the benzoyl-L-arginine amide system at low ionic strength (0.04) and optimal pH (9.5) was approx. 30 times lower than that of the trypsin-benzoyl-L-arginine amide system, at its optimal pH (7.5) at ionic strength 0.04. The Km at high ionic strength (0.5), measured for the IMET-benzoyl-L-arginine amide system at the pH of optimal activity (pH 9.5), approached that for the trypsin-benzoyl-L-arginine amide system (Km = 6.8 ± 1.0 × 10-3M) when measured at its optimal pH (7.5) and the same ionic strength. The effect of the polyanionic carrier on the pH-activity profiles and Km values of the bound enzymes investigated can be explained as resulting from the effect of the electrostatic potential of the polyelectrolyte carrier on the local concn. of H+ and pos. charged substrate mols. in the microenvironment of the bound enzyme mols. Theoretical analysis of the kinetic data allowed a quant. correlation of the displacement in the pH-activity curves and the shifts in the Km values with the electrostatic potential prevailing in the domain of the polyelectrolyte carrier.
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    65. 65
      Goldstein, L. Microenvironmental effects on enzyme catalysis. Kinetic study of polyanionic and polycationic derivatives of chymotrypsin. Biochemistry 1972, 11, 4072– 4084,  DOI: 10.1021/bi00772a009
      65
      Microenvironmental effects on enzyme catalysis. Kinetic study of polyanionic and polycationic derivatives of chymotrypsin
      Goldstein, Leon
      Biochemistry (1972), 11 (22), 4072-84CODEN: BICHAW; ISSN:0006-2960.
      A series of water-sol. polyanionic and polycationic derivs. of chymotrypsin were prepd. by growing poly(glutamyl) or poly(ornithyl) side chains on the enzyme, by coupling chymotrypsin to an ethylene-maleic acid copolymer (EMA), and by partial succinylation or acetylation. The pH-activity profiles of the polyanionic derivs. of chymotrypsin were displaced toward more alk. pH values as compared to the native enzyme; conversely the pH-activity profiles of the polycationic derivs. were displaced toward more acidic pH values. The kcat values of the charged chymotrypsin derivs. acting on ester, amide, and anilide substrates were displaced symmetrically, relative to the native enzyme-to higher values in the case of the polyanionic derivs. (poly(glutamyl) chymotrypsin, EMA-chymotrypsin, succinylchymotrypsin, and acetylchymotrypsin), and to lower values in the case of the polycationic (poly(ornithyl)chymotrypsin) derivs. The electrostatic effects on kcat were much more pronounced when the substrate was amide or anilide than when it was an ester. Increasing the ionic strength caused an increase in the values of kcat of both native chymotrypsin and the pos. charged derivs. of the enzyme. The kcat values of the neg. charged derivs. were not affected by the ionic strength. With ester substrates, the values of Km(app) of the polycationic derivs. were higher by an order of magnitude in comparison to the native enzyme; the Km(app) values of the polyanionic derivs. were only slightly perturbed. The values of Km(app) of all chymotrypsin derivs. acting on amide and anilide substrates were unperturbed and essentially identical with the value of the Michaelis const. of the native enzyme. These findings are discussed in the light of some recent ideas regarding the mechanism of action of chymotrypsin.
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    66. 66
      Zhang, Y.; Tsitkov, S.; Hess, H. Proximity does not contribute to activity enhancement in the glucose oxidase–horseradish peroxidase cascade. Nat. Commun. 2016, 7, 13982  DOI: 10.1038/ncomms13982
      66
      Proximity does not contribute to activity enhancement in the glucose oxidase-horseradish peroxidase cascade
      Zhang, Yifei; Tsitkov, Stanislav; Hess, Henry
      Nature Communications (2016), 7 (), 13982CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
      A proximity effect has been invoked to explain the enhanced activity of enzyme cascades on DNA scaffolds. Using the cascade reaction carried out by glucose oxidase and horseradish peroxidase as a model system, here we study the kinetics of the cascade reaction when the enzymes are free in soln., when they are conjugated to each other and when a competing enzyme is present. No proximity effect is found, which is in agreement with models predicting that the rapidly diffusing hydrogen peroxide intermediate is well mixed. We suggest that the reason for the activity enhancement of enzymes localized by DNA scaffolds is that the pH near the surface of the neg. charged DNA nanostructures is lower than that in the bulk soln., creating a more optimal pH environment for the anchored enzymes. Our findings challenge the notion of a proximity effect and provide new insights into the role of DNA scaffolds.
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    67. 67
      Ladero, M.; Santos, A.; García, J. L.; García-Ochoa, F. Activity over lactose and ONPG of a genetically engineered β-galactosidase from Escherichia coli in solution and immobilized: kinetic modelling. Enzyme Microb. Technol. 2001, 29, 181– 193,  DOI: 10.1016/S0141-0229(01)00366-0
      67
      Activity over lactose and ONPG of a genetically engineered β-galactosidase from Escherichia coli in solution and immobilized: kinetic modelling
      Ladero, M.; Santos, A.; Garcia, J. L.; Garcia-Ochoa, F.
      Enzyme and Microbial Technology (2001), 29 (2-3), 181-193CODEN: EMTED2; ISSN:0141-0229. (Elsevier Science Ireland Ltd.)
      The kinetic study of the hydrolysis of lactose and o-nitrophenol-β-D-galactoside (ONPG) with a β-galactosidase from Escherichia coli, both in soln. and covalently immobilized on a silica-alumina, is presented. The enzyme employed in this work had been modified previously by genetic engineering and purified to homogeneity by affinity chromatog. Firstly, the influence of pH and temp. on the activity and the stability of the enzyme, both free and immobilized, have been studied. Secondly, hydrolysis runs of lactose and ONPG with both forms of the enzyme were carried out in a wide exptl. range of temp. and concns. of substrates, products and enzyme. Data obtained were fitted to several kinetic models based on the Michaelis-Menten mechanism by non-linear regression. Finally, the models and their parameters were compared to det. the influence of the immobilization process and the substrate on the activity of the enzyme. In the hydrolysis of lactose and with both forms of the enzyme, acompetitive inhibition due to glucose was obsd. while the most common inhibition by galactose (which is usually a competitive inhibitor of β-galactosidases) was not obsd. Curiously, when the immobilized enzyme was the catalyst employed, lactose was an acompetitive inhibitor of the hydrolysis. When the substrate hydrolyzed was the o-nitrophenol-β-D-galactoside (ONPG), the galactose acted as a competitive inhibitor and the o-nitrophenol (ONP) was an acompetitive inhibitor for the free enzyme, being the immobilization process able to avoid the interaction between the ONP and the enzyme.
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    68. 68
      Noack, C. W.; Dzombak, D. A.; Nakles, D. V.; Hawthorne, S. B.; Heebink, L. V.; Dando, N.; Gershenzon, M.; Ghosh, R. S. Comparison of alkaline industrial wastes for aqueous mineral carbon sequestration through a parallel reactivity study. Waste Manage. 2014, 34, 1815– 1822,  DOI: 10.1016/j.wasman.2014.03.009
      68
      Comparison of alkaline industrial wastes for aqueous mineral carbon sequestration through a parallel reactivity study
      Noack, Clinton W.; Dzombak, David A.; Nakles, David V.; Hawthorne, Steven B.; Heebink, Loreal V.; Dando, Neal; Gershenzon, Michael; Ghosh, Rajat S.
      Waste Management (Oxford, United Kingdom) (2014), 34 (10), 1815-1822CODEN: WAMAE2; ISSN:0956-053X. (Elsevier Ltd.)
      Thirty-one alk. industrial wastes from a wide range of industrial processes were acquired and screened for application in an aq. carbon sequestration process. The wastes were evaluated for their potential to leach polyvalent cations and base species. Following mixing with a simple sodium bicarbonate soln., chemistries of the aq. and solid phases were analyzed. Exptl. results indicated that the most reactive materials were capable of sequestering between 77% and 93% of the available carbon under exptl. conditions in four hours. These materials - cement kiln dust, spray dryer absorber ash, and circulating dry scrubber ash - are thus good candidates for detailed, process-oriented studies. Chem. equil. modeling indicated that amorphous calcium carbonate is likely responsible for the obsd. sequestration. High variability and low reactive fractions render many other materials less attractive for further pursuit without considering preprocessing or activation techniques.
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    69. 69
      Arregui, L.; Ayala, M.; Gómez-Gil, X.; Gutiérrez-Soto, G.; Hernández-Luna, C. E.; Herrera de los Santos, M.; Levin, L.; Rojo-Domínguez, A.; Romero-Martínez, D.; Saparrat, M. C. N.; Trujillo-Roldán, M. A.; Valdez-Cruz, N. A. Laccases: structure, function, and potential application in water bioremediation. Microb. Cell Fact. 2019, 18, 200  DOI: 10.1186/s12934-019-1248-0
      69
      Laccases: structure, function, and potential application in water bioremediation
      Arregui, Leticia; Ayala, Marcela; Gomez-Gil, Ximena; Gutierrez-Soto, Guadalupe; Hernandez-Luna, Carlos Eduardo; Herrera de los Santos, Mayra; Levin, Laura; Rojo-Dominguez, Arturo; Romero-Martinez, Daniel; Saparrat, Mario C. N.; Trujillo-Roldan, Mauricio A.; Valdez-Cruz, Norma A.
      Microbial Cell Factories (2019), 18 (1), 200CODEN: MCFICT; ISSN:1475-2859. (BioMed Central Ltd.)
      A review. The global rise in urbanization and industrial activity has led to the prodn. and incorporation of foreign contaminant mols. into ecosystems, distorting them and impacting human and animal health. Phys., chem., and biol. strategies have been adopted to eliminate these contaminants from water bodies under anthropogenic stress. Biotechnol. processes involving microorganisms and enzymes have been used for this purpose; specifically, laccases, which are broad spectrum biocatalysts, have been used to degrade several compds., such as those that can be found in the effluents from industries and hospitals. Laccases have shown high potential in the biotransformation of diverse pollutants using crude enzyme exts. or free enzymes. However, their application in bioremediation and water treatment at a large scale is limited by the complex compn. and high salt concn. and pH values of contaminated media that affect protein stability, recovery and recycling. These issues are also assocd. with operational problems and the necessity of large-scale prodn. of laccase. Hence, more knowledge on the mol. characteristics of water bodies is required to identify and develop new laccases that can be used under complex conditions and to develop novel strategies and processes to achieve their efficient application in treating contaminated water. Recently, stability, efficiency, sepn. and reuse issues have been overcome by the immobilization of enzymes and development of novel biocatalytic materials. This review provides recent information on laccases from different sources, their structures and biochem. properties, mechanisms of action, and application in the bioremediation and biotransformation of contaminant mols. in water. Moreover, we discuss a series of improvements that have been attempted for better org. solvent tolerance, thermo-tolerance, and operational stability of laccases, as per process requirements.
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    70. 70
      Wang, M.; Abad, D.; Kickhoefer, V. A.; Rome, L. H.; Mahendra, S. Vault Nanoparticles Packaged with Enzymes as an Efficient Pollutant Biodegradation Technology. ACS Nano 2015, 9, 10931– 10940,  DOI: 10.1021/acsnano.5b04073
      70
      Vault Nanoparticles Packaged with Enzymes as an Efficient Pollutant Biodegradation Technology
      Wang, Meng; Abad, Danny; Kickhoefer, Valerie A.; Rome, Leonard H.; Mahendra, Shaily
      ACS Nano (2015), 9 (11), 10931-10940CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      Vault nanoparticles packaged with enzymes were synthesized as agents for efficiently degrading environmental contaminants. Enzymic biodegrdn. is an attractive technol. for in situ cleanup of contaminated environments because enzyme-catalyzed reactions are not constrained by nutrient requirements for microbial growth and often have higher biodegrdn. rates. However, the limited stability of extracellular enzymes remains a major challenge for practical applications. Encapsulation is a recognized method to enhance enzymic stability, but it can increase substrate diffusion resistance, lower catalytic rates, and increase the apparent half-satn. consts. We report an effective approach for boosting enzymic stability by single-step packaging into vault nanoparticles. With hollow core structures, assembled vault nanoparticles can simultaneously contain multiple enzymes. Mn peroxidase (MnP), which is widely used in biodegrdn. of org. contaminants, was chosen as a model enzyme here. MnP was incorporated into vaults via fusion to a packaging domain called INT, which strongly interacts with vaults' interior surface. MnP fused to INT and vaults packaged with the MnP-INT fusion protein maintained peroxidase activity. MnP-INT packaged in vaults displayed stability significantly higher than that of free MnP-INT, with slightly increased Km value. Vault-packaged MnP-INT exhibited 3 times higher phenol biodegrdn. in 24 h than did unpackaged MnP-INT. These results indicate that the packaging of MnP enzymes in vault nanoparticles extends their stability without compromising catalytic activity. This research will serve as the foundation for the development of efficient and sustainable vault-based bioremediation approaches for removing multiple contaminants from drinking water and groundwater.
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    71. 71
      Kaplan, O.; Vejvoda, V.; Plíhal, O.; Pompach, P.; Kavan, D.; Bojarová, P.; Bezouška, K.; Macková, M.; Cantarella, M.; Jirku, V.; Křen, V.; Martínková, L. Purification and characterization of a nitrilase from Aspergillus niger K10. Appl. Microbiol. Biotechnol. 2006, 73, 567– 575,  DOI: 10.1007/s00253-006-0503-6
      71
      Purification and characterization of a nitrilase from Aspergillus niger K10
      Kaplan, Ondrej; Vejvoda, Vojtech; Plihal, Ondrej; Pompach, Petr; Kavan, Daniel; Bojarova, Pavla; Bezouska, Karel; Mackova, Martina; Cantarella, Maria; Jirku, Vladimir; Kren, Vladimir; Martinkova, Ludmila
      Applied Microbiology and Biotechnology (2006), 73 (3), 567-575CODEN: AMBIDG; ISSN:0175-7598. (Springer)
      Aspergillus niger K10 cultivated on 2-cyanopyridine produced high levels of an intracellular nitrilase, which was partially purified (18.6-fold) with a 24% yield. The N-terminal amino acid sequence of the enzyme was highly homologous with that of a putative nitrilase from Aspergillus fumigatus Af293. The enzyme was copurified with two proteins, the N-terminal amino acid sequences of which revealed high homol. with those of hsp60 and an ubiquitin-conjugating enzyme. The nitrilase exhibited max. activity (91.6 U mg-1) at 45°C and pH 8.0. Its preferred substrates, in the descending order, were 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene, thiophen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile. Formation of amides as byproducts was most intensive, in the descending order, for 2-cyanopyridine, 4-chlorobenzonitrile, 4-cyanopyridine, and 1,4-dicyanobenzene. The enzyme stability was markedly improved in the presence of d-sorbitol or xylitol (20% w/v each). p-Hydroxymercuribenzoate and heavy metal ions were the most powerful inhibitors of the enzyme.
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biomac.1c00533.

    • Detailed procedures on data collection and analysis of structural models obtained from cryo-electron microscopy and X-ray crystallography; adopting and adapting an established Golden Gate-based genetic toolkit for combinatorial assembly of synthetic BMC pathways; list of constitutively active promoters from the Anderson collection; summary of recombinant combinatorial expression of Cso-shell components; multiple sequence alignment of 100 CsoS2 orthologs, focusing on the C-terminal region; and comparison of pore sizes of minimal BMC-derived shells with known atomic-scale structures and sequences of genetic constructs (PDF)


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