Quantitative Measurement of Molecular Permeability to a Synthetic Bacterial Microcompartment Shell SystemClick to copy article linkArticle link copied!
- Eric J. YoungEric J. YoungEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California 94702, United StatesMore by Eric J. Young
- Henning KirstHenning KirstEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California 94702, United StatesDepartamento de Genética, Campus de Excelencia Internacional Agroalimentario ceiA3, Universidad de Córdoba, Córdoba 14071, SpainInstituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba 14004, SpainMore by Henning Kirst
- Matthew E. DwyerMatthew E. DwyerMSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824, United StatesMore by Matthew E. Dwyer
- Josh V. Vermaas*Josh V. Vermaas*Email: [email protected]MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824, United StatesBiochemistry and Molecular Biology Department, Michigan State University, East Lansing, Michigan 48824, United StatesMore by Josh V. Vermaas
- Cheryl A. Kerfeld*Cheryl A. Kerfeld*Email: [email protected]Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California 94702, United StatesMSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824, United StatesBiochemistry and Molecular Biology Department, Michigan State University, East Lansing, Michigan 48824, United StatesMolecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California 94702, United StatesMore by Cheryl A. Kerfeld
Abstract
Naturally evolved and synthetically designed forms of compartmentalization benefit encapsulated function by increasing local concentrations of substrates and protecting cargo from destabilizing environments and inhibitors. Crucial to understanding the fundamental principles of compartmentalization are experimental systems enabling the measurement of the permeability rates of small molecules. Here, we report the experimental measurement of the small-molecule permeability of a 40 nm icosahedral bacterial microcompartment shell. This was accomplished by heterologous loading of light-producing luciferase enzymes and kinetic measurement of luminescence using stopped-flow spectrophotometry. Compared to free enzyme, the luminescence signal kinetics was slower when the luciferase was encapsulated in bacterial microcompartment shells. The results indicate that substrates and products can still exchange across the shell, and modeling of the experimental data suggest that a 50× permeability rate increase occurs when shell vertices were vacant. Overall, our results suggest design considerations for the construction of heterologous bacterial microcompartment shell systems and compartmentalized function at the nanoscale.
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Special Issue
Published as part of ACS Synthetic Biology special issue “Quantitative Synthetic Biology”.
Introduction
Figure 1
Figure 1. Design for evaluating the ATP permeability of a model BMC shell. (A) BMC shells lacking pentamers contain ∼5 nm vertex gaps (i.e., “uncapped” configuration). BMC shell structures were prepared in PyMol from PDB structure 5V74. BMC-H proteins are colored blue, BMC-T1 dark green, BMC-T2 and BMC-T3 light green, and BMC-P yellow. (B) Zoom of area bounded in white in Figure 1A. (C) Fusion of a SpyTag/SnoopTag to BMC-T1 shell protein allows for covalent tethering of reciprocally tagged-catcher cargos to the lumen of the BMC shell to evaluate the permeability of ATP to a BMC shell. (D) Transmission electron microscopy of purified uncapped LucZ-SpyCatcher BMC shells.
Results
Characterization of Firefly Luciferase-Loaded BMC Shells
Measuring ATP Permeability through a BMC Shell
Figure 2
Figure 2. Measuring ATP permeability of LucZ-SpyCatcher BMC shells with stopped-flow assays. (A) Stopped-flow assay comparison of uncapped versus capped shells versus a shell-free LucZ in a luciferin/MgCl2 substrate (each trace represents five readings averaged to one). The concentration of shell samples in solution was 100 nM, while the No Shell sample was 1000 nM. Samples were incubated for 1 h in 1 mM luciferin/MgCl2, 50 mM Tris–HCl, and 200 mM NaCl pH 8.0 buffer with or without 1000 nM BMC-P capping protein. Trends represented are from at least three independent experiments.
Figure 3
Figure 3. Differential equation modeling of stop-flow enzyme kinetics to calculate the permeability rate of capped versus uncapped shells. (A, B) Permeability coefficients are given by the rate of the limiting substrate (Px) and the rate of the intermediate complex (Py). Information regarding the equations, assumptions, and additional calculated values is found in the Supporting Information. (A) Capped shells. (B) Uncapped shells.
Discussion
Materials and Methods
Molecular Biology, Cell Growth, and Protein Purification
Luminescence Measurement of Luciferase Reaction
Dynamic Light Scattering
Transmission Electron Microscopy
Stopped-Flow Spectrophotometry
Data Analysis and Figure Preparation
Permeability Calculations
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.4c00290.
Purification of in vivo covalently loaded SpyCatcher-LucZ-uncapped BMC shells; purification of in vivo covalently loaded LucZ-SpyCatcher-uncapped BMC shells; luminescence activity comparison between LucZ and SpyCatcher fusion orientation; activity assay of His-LucZ-SpyCatcher-loaded uncapped BMC shells; capping pentamer protein purification, and workflow for comparing shell permeability in uncapped versus capped configuration; LucZ-SpyCatcher Shells after incubation in substrate buffer; stopped-flow spectrophotometry of uncapped and capped BMC shells; stopped-flow spectrophotometry of uncapped and capped shells in 10 and 20% PEG6000 buffer; stopped-flow spectrophotometry of MonoQ-purified shells after capping; calculation of kM of uncapped and capped LucZ-SpyCatcher BMC shells; protein expression vectors; coding sequences of expression vectors; differential equations for luciferase activity without shells; and addition of permeability into the model (PDF)
The code for the fitting, and reading in the experimental data, is provided on github (DOI: 10.5281/zenodo.10963263).
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Acknowledgments
We thank the current and former members of the Kerfeld Lab for helpful discussions and initial cloning efforts. This work was supported by the National Institutes of Health, National Institute of Allergy and Infectious Diseases (NIAID), grant 5R01AI114975-08. The permeability modeling by J.V.V. was supported in part by the US Department of Energy, Basic Energy Sciences, DE-SC0023395.
References
This article references 72 other publications.
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- 5Kerfeld, C. A.; Sawaya, M. R.; Tanaka, S.; Nguyen, C. V.; Phillips, M.; Beeby, M.; Yeates, T. O. Protein Structures Forming the Shell of Primitive Bacterial Organelles. Science 2005, 309 (5736), 936– 938, DOI: 10.1126/science.1113397Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXmvFSks7w%253D&md5=9001e7177689f87280fe8c491ea46f49Protein Structures Forming the Shell of Primitive Bacterial OrganellesKerfeld, Cheryl A.; Sawaya, Michael R.; Tanaka, Shiho; Nguyen, Chau V.; Phillips, Martin; Beeby, Morgan; Yeates, Todd O.Science (Washington, DC, United States) (2005), 309 (5736), 936-938CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Bacterial microcompartments are primitive organelles composed entirely of protein subunits. Genomic sequence databases reveal the widespread occurrence of microcompartments across diverse microbes. The prototypical bacterial microcompartment is the carboxysome, a protein shell for sequestering carbon fixation reactions. The authors report three-dimensional crystal structures of multiple carboxysome shell proteins, revealing a hexameric unit as the basic microcompartment building block and showing how these hexamers assemble to form flat facets of the polyhedral shell. The structures suggest how mol. transport across the shell may be controlled and how structural variations might govern the assembly and architecture of these subcellular compartments.
- 6Klein, 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 (2), 319– 333, DOI: 10.1016/j.jmb.2009.03.056Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVKkt77O&md5=5555ea1b2add3c076176135c9c3b0c69Identification and Structural Analysis of a Novel Carboxysome Shell Protein with Implications for Metabolite TransportKlein, 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.
- 7Ferlez, B. H.; Kirst, H.; Greber, B. J.; Nogales, E.; Sutter, M.; Kerfeld, C. A. Heterologous Assembly of Pleomorphic Bacterial Microcompartment Shell Architectures Spanning the Nano- to Microscale. Adv. Mater. 2023, 35 (23), e2212065 DOI: 10.1002/adma.202212065Google ScholarThere is no corresponding record for this reference.
- 8Tan, Y. Q.; Ali, S.; Xue, B.; Teo, W. Z.; Ling, L. H.; Go, M. K.; Lv, H.; Robinson, R. C.; Narita, A.; Yew, W. S. Structure of a Minimal Α-Carboxysome-Derived Shell and Its Utility in Enzyme Stabilization. Biomacromolecules 2021, 22 (10), 4095– 4109, DOI: 10.1021/acs.biomac.1c00533Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhslGrs7nP&md5=658a0654c1640d34ebd1f66251d15fb5Structure of a Minimal α-Carboxysome-Derived Shell and Its Utility in Enzyme StabilizationTan, Yong Quan; Ali, Samson; Xue, Bo; Teo, Wei Zhe; Ling, Lay Hiang; Go, Maybelle Kho; Lv, Hong; Robinson, Robert C.; Narita, Akihiro; Yew, Wen ShanBiomacromolecules (2021), 22 (10), 4095-4109CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)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 at.-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 enzymic activities against heat shock, presence of methanol co-solvent, consecutive freeze-thawing, and alk. 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.
- 9Cesle, E. E.; Filimonenko, A.; Tars, K.; Kalnins, G. Variety of Size and Form of GRM2 Bacterial Microcompartment Particles. Protein Sci. 2021, 30 (5), 1035– 1043, DOI: 10.1002/pro.4069Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXnslGnu7Y%253D&md5=2932e8028787a2d625c5fc54fcd10618Variety of size and form of GRM2 bacterial microcompartment particlesCesle, Eva Emilija; Filimonenko, Anatolij; Tars, Kaspars; Kalnins, GintsProtein Science (2021), 30 (5), 1035-1043CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)Bacterial microcompartments (BMCs) are bacterial organelles involved in enzymic processes, such as carbon fixation, choline, ethanolamine and propanediol degrdn., and others. Formed of a semi-permeable protein shell and an enzymic core, they can enhance enzyme performance and protect the cell from harmful intermediates. With the ability to encapsulate non-native enzymes, BMCs show high potential for applied use. For this goal, a detailed look into shell form variability is significant to predict shell adaptability. Here we present four novel 3D cryo-EM maps of recombinant Klebsiella pneumoniae GRM2 BMC shell particles with the resoln. in range of 9 to 22 Å and nine novel 2D classes corresponding to discrete BMC shell forms. These structures reveal icosahedral, elongated, oblate, multi-layered and polyhedral traits of BMCs, indicating considerable variation in size and form as well as adaptability during shell formation processes.
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- 16Kerfeld, C. A.; Erbilgin, O. Bacterial Microcompartments and the Modular Construction of Microbial Metabolism. Trends Microbiol. 2015, 23 (1), 22– 34, DOI: 10.1016/j.tim.2014.10.003Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsl2ltb7O&md5=96858d72be4d76b3821cb00da17ba412Bacterial microcompartments and the modular construction of microbial metabolismKerfeld, Cheryl A.; Erbilgin, OnurTrends in Microbiology (2015), 23 (1), 22-34CODEN: TRMIEA; ISSN:0966-842X. (Elsevier Ltd.)A review. Bacterial microcompartments (BMCs) are protein-bound organelles predicted to be present across 23 bacterial phyla. BMCs facilitate carbon fixation as well as the aerobic and anaerobic catabolism of a variety of org. compds. These functions have been linked to ecol. nutrient cycling, symbiosis, pathogenesis, and cardiovascular disease. Within bacterial cells, BMCs are metabolic modules that can be further dissocd. into their constituent structural and functional protein domains. Viewing BMCs as genetic, structural, functional, and evolutionary modules provides a framework for understanding both BMC-mediated metab. and for adapting their architectures for applications in synthetic biol.
- 17Liu, L.-N. Advances in the Bacterial Organelles for CO2 Fixation. Trends Microbiol. 2022, 30 (6), 567– 580, DOI: 10.1016/j.tim.2021.10.004Google ScholarThere is no corresponding record for this reference.
- 18Turmo, A.; Gonzalez-Esquer, C. R.; Kerfeld, C. A. Carboxysomes: Metabolic Modules for CO2 Fixation. FEMS Microbiol. Lett. 2017, 364 (18), 1000118, DOI: 10.1093/femsle/fnx176Google ScholarThere is no corresponding record for this reference.
- 19Faulkner, 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 (1), 17501, DOI: 10.1038/s41598-020-74536-5Google ScholarThere is no corresponding record for this reference.
- 20Tsai, 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 (6), e144 DOI: 10.1371/journal.pbio.0050144Google ScholarThere is no corresponding record for this reference.
- 21Mahinthichaichan, 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 (39), 9110– 9118, DOI: 10.1021/acs.jpcb.8b06822Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhs1ygtrjO&md5=fa585e72f333795d11f776b0953f9092Selective Permeability of Carboxysome Shell Pores to Anionic MoleculesMahinthichaichan, Paween; Morris, Dylan M.; Wang, Yi; Jensen, Grant J.; Tajkhorshid, EmadJournal 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.
- 22Trettel, D. S.; Neale, C.; Zhao, M.; Gnanakaran, S.; Gonzalez-Esquer, C. R. Monatomic Ions Influence Substrate Permeation across Bacterial Microcompartment Shells. Sci. Rep. 2023, 13 (1), 15738, DOI: 10.1038/s41598-023-42688-9Google ScholarThere is no corresponding record for this reference.
- 23Park, 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 (34), 8149– 8154, DOI: 10.1021/acs.jpcb.7b07232Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlKisb7L&md5=265fa3d2313ef00e57959feb755c2f14Molecular Dynamics Simulations of Selective Metabolite Transport across the Propanediol Bacterial Microcompartment ShellPark, 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.
- 24Sarkar, D.; Maffeo, C.; Sutter, M.; Aksimentiev, A.; Kerfeld, C. A.; Vermaas, J. V. Atomic View of Photosynthetic Metabolite Permeability Pathways and Confinement in Synthetic Carboxysome Shells. Proc. Natl. Acad. Sci. U. S. A. 2024, 121 (45), e2402277121 DOI: 10.1073/pnas.2402277121Google ScholarThere is no corresponding record for this reference.
- 25Penrod, J. T.; Roth, J. R. Conserving a Volatile Metabolite: A Role for Carboxysome-Like Organelles in Salmonella Enterica. J. Bacteriol. 2006, 188 (8), 2865– 2874, DOI: 10.1128/JB.188.8.2865-2874.2006Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xjslejt78%253D&md5=67c1e4f99f8ce19b258541f34b92f98bConserving a volatile metabolite: a role for carboxysome-like organelles in Salmonella entericaPenrod, Joseph T.; Roth, John R.Journal of Bacteriology (2006), 188 (8), 2865-2874CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)Salmonellae can use ethanolamine (EA) as a sole source of carbon and nitrogen. This ability is encoded by an operon (eut) contg. 17 genes, only 6 of which are required under std. conditions (37°C; pH 7.0). Five of the extra genes (eutM, -N, -L, -K, and -G) become necessary under conditions that favor loss of the volatile intermediate, acetaldehyde, which escapes as a gas during growth on EA and is lost at a higher rate from these mutants. The eutM, -N, -L, and -K genes encode homologs of shell proteins of the carboxysome, an organelle shown (in other organisms) to conc. CO2. We propose that carboxysome-like organelles help bacteria conserve certain volatile metabolites-CO2 or acetaldehyde-perhaps by providing a low-pH compartment. The EutG enzyme converts acetaldehyde to ethanol, which may improve carbon retention by forming acetals; alternatively, EutG may recycle NADH within the carboxysome.
- 26Havemann, G. D.; Sampson, E. M.; Bobik, T. A. PduA Is a Shell Protein of Polyhedral Organelles Involved in Coenzyme B 12 -Dependent Degradation of 1,2-Propanediol in Salmonella Enterica Serovar Typhimurium LT2. J. Bacteriol. 2002, 184 (5), 1253– 1261, DOI: 10.1128/JB.184.5.1253-1261.2002Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XhtlKnsbo%253D&md5=f830432faeeed6d38718dfb5df33a7abPduA is a shell protein of polyhedral organelles involved in coenzyme B12-dependent degradation of 1,2-propanediol in Salmonella enterica serovar typhimurium LT2Havemann, Gregory D.; Sampson, Edith M.; Bobik, Thomas A.Journal of Bacteriology (2002), 184 (5), 1253-1261CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)Salmonella enterica forms polyhedral organelles involved in coenzyme B12-dependent 1,2-propanediol degrdn. These organelles are thought to consist of a proteinaceous shell that encases coenzyme B12-dependent diol dehydratase and perhaps other enzymes involved in 1,2-propanediol degrdn. The function of these organelles is unknown, and no detailed studies of their structure have been reported. Genes needed for organelle formation and for 1,2-propanediol degrdn. are located at the 1,2-propanediol utilization (pdu) locus, but the specific genes involved in organelle formation have not been identified. Here, we show that the pduA gene encodes a shell protein required for the formation of polyhedral organelles involved in coenzyme B12-dependent 1,2-propanediol degrdn. A His6-PduA fusion protein was purified from a recombinant Escherichia coli strain and used for the prepn. of polyclonal antibodies. The anti-PduA antibodies obtained were partially purified by a subtraction procedure and used to demonstrate that the PduA protein localized to the shell of the polyhedral organelles. In addn., electron microscopy studies established that strains with nonpolar pduA mutations were unable to form organelles. These results show that the pduA gene is essential for organelle formation and indicate that the PduA protein is a structural component of the shell of these organelles. Physiol. studies of nonpolar pduA mutants were also conducted. Such mutants grew similarly to the wild-type strain at low concns. of 1,2-propanediol but exhibited a period of interrupted growth in the presence of higher concns. of this growth substrate. Growth tests also showed that a nonpolar pduA deletion mutant grew faster than the wild-type strain at low vitamin B12 concns. These results suggest that the polyhedral organelles formed by S. enterica during growth on 1,2-propanediol are not involved in the concn. of 1,2-propanediol or coenzyme B12, but are consistent with the hypothesis that these organelles moderate aldehyde prodn. to minimize toxicity.
- 27Chowdhury, C.; Chun, S.; Pang, A.; Sawaya, M. R.; Sinha, S.; Yeates, T. O.; Bobik, T. A. Selective Molecular Transport through the Protein Shell of a Bacterial Microcompartment Organelle. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (10), 2990– 2995, DOI: 10.1073/pnas.1423672112Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjtFemtbk%253D&md5=ba014ac2a2ef0b2e41aeaf9f7f4af6b6Selective molecular transport through the protein shell of a bacterial microcompartment organelleChowdhury, Chiranjit; Chun, Sunny; Pang, Allan; Sawaya, Michael R.; Sinha, Sharmistha; Yeates, Todd O.; Bobik, Thomas A.Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (10), 2990-2995CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Bacterial microcompartments are widespread prokaryotic organelles that have important and diverse roles ranging from carbon fixation to enteric pathogenesis. Current models for microcompartment function propose that their outer protein shell is selectively permeable to small mols., but whether a protein shell can mediate selective permeability and how this occurs are unresolved questions. Here, biochem. and physiol. studies of structure-guided mutants are used to show that the hexameric PduA shell protein of the 1,2-propanediol utilization (Pdu) microcompartment forms a selectively permeable pore tailored for the influx of 1,2-propanediol (the substrate of the Pdu microcompartment) while restricting the efflux of propionaldehyde, a toxic intermediate of 1,2-propanediol catabolism. Crystal structures of various PduA mutants provide a foundation for interpreting the obsd. biochem. and phenotypic data in terms of mol. diffusion across the shell. Overall, these studies provide a basis for understanding a class of selectively permeable channels formed by nonmembrane proteins.
- 28Chowdhury, C.; Chun, S.; Sawaya, M. R.; Yeates, T. O.; Bobik, T. A. The Function of the PduJ Microcompartment Shell Protein Is Determined by the Genomic Position of Its Encoding Gene. Mol. Microbiol. 2016, 101 (5), 770– 783, DOI: 10.1111/mmi.13423Google ScholarThere is no corresponding record for this reference.
- 29Chowdhury, C.; Bobik, T. A. Engineering the PduT Shell Protein to Modify the Permeability of the 1,2-Propanediol Microcompartment of Salmonella. Microbiology 2019, 165 (12), 1355– 1364, DOI: 10.1099/mic.0.000872Google ScholarThere is no corresponding record for this reference.
- 30Cai, F.; Menon, B. B.; Cannon, G. C.; Curry, K. J.; Shively, J. M.; Heinhorst, S. The Pentameric Vertex Proteins Are Necessary for the Icosahedral Carboxysome Shell to Function as a CO2 Leakage Barrier. PLoS One 2009, 4 (10), e7521 DOI: 10.1371/journal.pone.0007521Google ScholarThere is no corresponding record for this reference.
- 31Lee, M. F. S.; Jakobson, C. M.; Tullman-Ercek, D. Evidence for Improved Encapsulated Pathway Behavior in a Bacterial Microcompartment through Shell Protein Engineering. ACS Synth. Biol. 2017, 6 (10), 1880– 1891, DOI: 10.1021/acssynbio.7b00042Google ScholarThere is no corresponding record for this reference.
- 32Huang, J.; Jiang, Q.; Yang, M.; Dykes, G. F.; Weetman, S. L.; Xin, W.; He, H.-L.; Liu, L.-N. Probing the Internal PH and Permeability of a Carboxysome Shell. Biomacromolecules 2022, 23 (10), 4339– 4348, DOI: 10.1021/acs.biomac.2c00781Google ScholarThere is no corresponding record for this reference.
- 33Rae, B. D.; Long, B. M.; Badger, M. R.; Price, G. D. Structural Determinants of the Outer Shell of β-Carboxysomes in Synechococcus Elongatus PCC 7942: Roles for CcmK2, K3-K4, CcmO, and CcmL. PLoS One 2012, 7 (8), e43871 DOI: 10.1371/journal.pone.0043871Google ScholarThere is no corresponding record for this reference.
- 34Cai, F.; Sutter, M.; Bernstein, S. L.; Kinney, J. N.; Kerfeld, C. A. Engineering Bacterial Microcompartment Shells: Chimeric Shell Proteins and Chimeric Carboxysome Shells. ACS Synth. Biol. 2015, 4 (4), 444– 453, DOI: 10.1021/sb500226jGoogle Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlalu7bL&md5=b8df8cf38136dbffd8a57cace90fcb85Engineering Bacterial Microcompartment Shells: Chimeric Shell Proteins and Chimeric Carboxysome ShellsCai, Fei; Sutter, Markus; Bernstein, Susan L.; Kinney, James N.; Kerfeld, Cheryl A.ACS Synthetic Biology (2015), 4 (4), 444-453CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Bacterial microcompartments (BMCs) are self-assembling organelles composed entirely of protein. Depending on the enzymes they encapsulate, BMCs function in either inorg. carbon fixation (carboxysomes) or org. carbon use (metabolosomes). The hallmark feature of all BMCs is a selectively permeable shell formed by multiple paralogous proteins, each proposed to confer specific flux characteristics. Gene clusters encoding diverse BMCs are distributed broadly across bacterial phyla, providing a rich variety of building blocks with a predicted range of permeability properties. In theory, shell permeability can be engineered by modifying residues flanking the pores (symmetry axes) of hexameric shell proteins or by combining shell proteins from different types of BMCs into chimeric shells. The authors undertook both approaches to altering shell properties using the carboxysome as a model system. There are two types of carboxysomes, α and β. In both, the predominant shell protein(s) contain a single copy of the BMC domain (pfam00936), but they are significantly different in primary structure. Indeed, phylogenetic anal. shows that the two types of carboxysome shell proteins are more similar to their counterparts in metabolosomes than to each other. The authors solved high resoln. crystal structures of the major shell proteins, CsoS1 and CcmK2, and the presumed minor shell protein CcmK4, representing both types of cyanobacterial carboxysomes and then tested the interchangeability. The in vivo study presented here confirms that both engineering pores to mimic those of other shell proteins and the construction of chimeric shells is feasible.
- 35Cameron, J. C.; Wilson, S. C.; Bernstein, S. L.; Kerfeld, C. A. Biogenesis of a Bacterial Organelle: The Carboxysome Assembly Pathway. Cell 2013, 155 (5), 1131– 1140, DOI: 10.1016/j.cell.2013.10.044Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFWjtbjI&md5=04e529a8a8a3a5ad3af30d9d7c887b9bBiogenesis of a bacterial organelle: The carboxysome assembly pathwayCameron, Jeffrey C.; Wilson, Steven C.; Bernstein, Susan L.; Kerfeld, Cheryl A.Cell (Cambridge, MA, United States) (2013), 155 (5), 1131-1140CODEN: CELLB5; ISSN:0092-8674. (Cell Press)The carboxysome is a protein-based organelle for carbon fixation in cyanobacteria, keystone organisms in the global carbon cycle. It is composed of thousands of subunits including hexameric and pentameric proteins that form a shell to encapsulate the enzymes ribulose 1,5-bisphosphate carboxylase/oxygenase and carbonic anhydrase. Here, the authors describe the stages of carboxysome assembly and the requisite gene products necessary for progression through each. Unlike membrane-bound organelles of eukaryotes, in carboxysomes the interior of the compartment forms first, at a distinct site within the cell. Subsequently, shell proteins encapsulate this procarboxysome, inducing budding and distribution of functional organelles within the cell. The authors propose that the principles of carboxysome assembly that we have uncovered extend to diverse bacterial microcompartments.
- 36Yang, M.; Wenner, N.; Dykes, G. F.; Li, Y.; Zhu, X.; Sun, Y.; Huang, F.; Hinton, J. C. D.; Liu, L.-N. Biogenesis of a Bacterial Metabolosome for Propanediol Utilization. Nat. Commun. 2022, 13 (1), 2920, DOI: 10.1038/s41467-022-30608-wGoogle ScholarThere is no corresponding record for this reference.
- 37Huseby, D. L.; Roth, J. R. Evidence That a Metabolic Microcompartment Contains and Recycles Private Cofactor Pools. J. Bacteriol. 2013, 195 (12), 2864– 2879, DOI: 10.1128/JB.02179-12Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXpsVartbc%253D&md5=c859df086c8e90e4f6664e2d49bec440Evidence that a metabolic microcompartment contains and recycles private cofactor poolsHuseby, Douglas L.; Roth, John R.Journal of Bacteriology (2013), 195 (12), 2864-2879CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)Microcompartments are loose protein cages that encapsulate enzymes for particular bacterial metabolic pathways. These structures are thought to retain and perhaps conc. pools of small, uncharged intermediates that would otherwise diffuse from the cell. In Salmonella enterica, a microcompartment encloses enzymes for ethanolamine catabolism. The cage has been thought to retain the volatile intermediate acetaldehyde but allow diffusion of the much larger cofactors NAD and CoA (CoA). Genetic tests support an alternative idea that the microcompartment contains and recycles private pools of the large cofactors NAD and CoA. Two central enzymes convert ethanolamine to acetaldehyde (EutBC) and then to acetyl-CoA (EutE). Two seemingly peripheral redundant enzymes encoded by the eut operon proved to be essential for ethanolamine utilization, when subjected to sufficiently stringent tests. These are EutD (acetyl-CoA to acetyl phosphate) and EutG (acetaldehyde to ethanol). Obligatory recycling of cofactors couples the three reactions and drives acetaldehyde consumption. Loss and toxic effects of acetaldehyde are minimized by accelerating its consumption. In a eutD mutant, acetyl-CoA cannot escape the compartment but is released by mutations that disrupt the structure. The model predicts that EutBC (ethanolamine-ammonia lyase) lies outside the compartment, using external coenzyme B12 and injecting its product, acetaldehyde, into the lumen, where it is degraded by the EutE, EutD, and EutG enzymes using private pools of CoA and NAD. The compartment appears to allow free diffusion of the intermediates ethanol and acetyl-PO4 but (to our great surprise) restricts diffusion of acetaldehyde.
- 38Sommer, M.; Sutter, M.; Gupta, S.; Kirst, H.; Turmo, A.; Lechno-Yossef, S.; Burton, R. L.; Saechao, C.; Sloan, N. B.; Cheng, X.; Chan, L.-J. G.; Petzold, C. J.; Fuentes-Cabrera, M.; Ralston, C. Y.; Kerfeld, C. A. Heterohexamers Formed by CcmK3 and CcmK4 Increase the Complexity of Beta Carboxysome Shells. Plant Physiol. 2019, 179 (1), 156– 167, DOI: 10.1104/pp.18.01190Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXotFeru70%253D&md5=879e497ec69aa657f0c5395107665404Heterohexamers formed by CcmK3 and CcmK4 increase the complexity of beta carboxysome shellsSommer, Manuel; Sutter, Markus; Gupta, Sayan; Kirst, Henning; Turmo, Aiko; Lechno-Yossef, Sigal; Burton, Rodney L.; Saechao, Christine; Sloan, Nancy B.; Cheng, Xiaolin; Chan, Leanne-Jade G.; Petzold, Christopher J.; Fuentes-Cabrera, Miguel; Ralston, Corie Y.; Kerfeld, Cheryl A.Plant Physiology (2019), 179 (1), 156-167CODEN: PLPHAY; ISSN:1532-2548. (American Society of Plant Biologists)Bacterial microcompartments (BMCs) encapsulate enzymes within a selectively permeable, proteinaceous shell. Carboxysomes are BMCs contg. ribulose-1,5-bisphosphate carboxylase oxygenase and carbonic anhydrase that enhance carbon dioxide fixation. The carboxysome shell consists of three structurally characterized protein types, each named after the oligomer they form: BMC-H (hexamer), BMC-P (pentamer), and BMC-T (trimer). These three protein types form cyclic homooligomers with pores at the center of symmetry that enable metabolite transport across the shell. Carboxysome shells contain multiple BMC-H paralogs, each with distinctly conserved residues surrounding the pore, which are assumed to be assocd. with specific metabolites. We studied the regulation of β-carboxysome shell compn. by investigating the BMC-H genes ccmK3 and ccmK4 situated in a locus remote from other carboxysome genes. We made single and double deletion mutants of ccmK3 and ccmK4 in Synechococcus elongatus PCC7942 and show that, unlike CcmK3, CcmK4 is necessary for optimal growth. In contrast to other CcmK proteins, CcmK3 does not form homohexamers; instead CcmK3 forms heterohexamers with CcmK4 with a 1:2 stoichiometry. The CcmK3-CcmK4 heterohexamers form stacked dodecamers in a pH-dependent manner.
- 39Garcia-Alles, L. F.; Root, K.; Maveyraud, L.; Aubry, N.; Lesniewska, E.; Mourey, L.; Zenobi, R.; Truan, G. Occurrence and Stability of Hetero-Hexamer Associations Formed by β-Carboxysome CcmK Shell Components. PLoS One 2019, 14 (10), e0223877 DOI: 10.1371/journal.pone.0223877Google ScholarThere is no corresponding record for this reference.
- 40Sun, Y.; Wollman, A. J. M.; Huang, F.; Leake, M. C.; Liu, L.-N. Single-Organelle Quantification Reveals Stoichiometric and Structural Variability of Carboxysomes Dependent on the Environment. Plant Cell 2019, 31 (7), 1648– 1664, DOI: 10.1105/tpc.18.00787Google ScholarThere is no corresponding record for this reference.
- 41Sommer, M.; Cai, F.; Melnicki, M.; Kerfeld, C. A. β-Carboxysome Bioinformatics: Identification and Evolution of New Bacterial Microcompartment Protein Gene Classes and Core Locus Constraints. J. Exp. Bot. 2017, 68 (14), 3841– 3855, DOI: 10.1093/jxb/erx115Google ScholarThere is no corresponding record for this reference.
- 42Hagen, A.; Sutter, M.; Sloan, N.; Kerfeld, C. A. Programmed Loading and Rapid Purification of Engineered Bacterial Microcompartment Shells. Nat. Commun. 2018, 9 (1), 2881, DOI: 10.1038/s41467-018-05162-zGoogle Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3c7htF2nug%253D%253D&md5=1486d359a87968f84608690e5ec53d7dProgrammed loading and rapid purification of engineered bacterial microcompartment shellsHagen Andrew; Sutter Markus; Sloan Nancy; Kerfeld Cheryl A; Sutter Markus; Kerfeld Cheryl A; Kerfeld Cheryl ANature 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.
- 43Trettel, D. S.; Resager, W.; Ueberheide, B. M.; Jenkins, C. C.; Winkler, W. C. Chemical Probing Provides Insight into the Native Assembly State of a Bacterial Microcompartment. Structure 2022, 30 (4), 537– 550, DOI: 10.1016/j.str.2022.02.002Google ScholarThere is no corresponding record for this reference.
- 44Jiang, Q.; Li, T.; Yang, J.; Aitchison, C. M.; Huang, J.; Chen, Y.; Huang, F.; Wang, Q.; Cooper, A. I.; Liu, L.-N. Synthetic Engineering of a New Biocatalyst Encapsulating [NiFe]-Hydrogenases for Enhanced Hydrogen Production. J. Mater. Chem. B 2023, 11 (12), 2684– 2692, DOI: 10.1039/D2TB02781JGoogle ScholarThere is no corresponding record for this reference.
- 45Nguyen, N. D.; Pulsford, S. B.; Hee, W. Y.; Rae, B. D.; Rourke, L. M.; Price, G. D.; Long, B. M. Towards Engineering a Hybrid Carboxysome. Photosynth. Res. 2023, 156 (2), 265– 277, DOI: 10.1007/s11120-023-01009-xGoogle ScholarThere is no corresponding record for this reference.
- 46Kirst, H.; Ferlez, B. H.; Lindner, S. N.; Cotton, C. A. R.; Bar-Even, A.; Kerfeld, C. A. Toward a Glycyl Radical Enzyme Containing Synthetic Bacterial Microcompartment to Produce Pyruvate from Formate and Acetate. Proc. Natl. Acad. Sci. U. S. A. 2022, 119 (8), e2116871119 DOI: 10.1073/pnas.2116871119Google ScholarThere is no corresponding record for this reference.
- 47Li, 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 (1), 5448, DOI: 10.1038/s41467-020-19280-0Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit1Ojur%252FK&md5=f9a4fcfafcb7204877e067c4fbbb1152Reprogramming bacterial protein organelles as a nanoreactor for hydrogen productionLi, 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-NingNature 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.
- 48Wagner, H. J.; Capitain, C. C.; Richter, K.; Nessling, M.; Mampel, J. Engineering Bacterial Microcompartments with Heterologous Enzyme Cargos. Eng. Life Sci. 2017, 17 (1), 36– 46, DOI: 10.1002/elsc.201600107Google ScholarThere is no corresponding record for this reference.
- 49Lawrence, 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 (7), 454– 465, DOI: 10.1021/sb4001118Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVOjurk%253D&md5=f82c98604ef70551c623a4cfb008b0ccSolution Structure of a Bacterial Microcompartment Targeting Peptide and Its Application in the Construction of an Ethanol BioreactorLawrence, 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.
- 50Choudhary, S.; Quin, M. B.; Sanders, M. A.; Johnson, E. T.; Schmidt-Dannert, C. Engineered Protein Nano-Compartments for Targeted Enzyme Localization. PLoS One 2012, 7 (3), e33342 DOI: 10.1371/journal.pone.0033342Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XksVGgsLs%253D&md5=0b56b21e0cbad66c0be1d4ecbbc53988Engineered protein nano-compartments for targeted enzyme localizationChoudhary, Swati; Quin, Maureen B.; Sanders, Mark A.; Johnson, Ethan T.; Schmidt-Dannert, ClaudiaPLoS One (2012), 7 (3), e33342CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Compartmentalized co-localization of enzymes and their substrates represents an attractive approach for multi-enzymic synthesis in engineered cells and biocatalysis. Sequestration of enzymes and substrates would greatly increase reaction efficiency while also protecting engineered host cells from potentially toxic reaction intermediates. Several bacteria form protein-based polyhedral microcompartments which sequester functionally related enzymes and regulate their access to substrates and other small metabolites. Such bacterial microcompartments may be engineered into protein-based nano-bioreactors, provided that they can be assembled in a non-native host cell, and that heterologous enzymes and substrates can be targeted into the engineered compartments. Here, we report that recombinant expression of Salmonella enterica ethanolamine utilization (eut) bacterial microcompartment shell proteins in E. coli results in the formation of polyhedral protein shells. Purified recombinant shells are morphol. similar to the native Eut microcompartments purified from S. enterica. Surprisingly, recombinant expression of only one of the shell proteins (EutS) is sufficient and necessary for creating properly delimited compartments. Co-expression with EutS also facilitates the encapsulation of EGFP fused with a putative Eut shell-targeting signal sequence. We also demonstrate the functional localization of a heterologous enzyme (β-galactosidase) targeted to the recombinant shells. Together our results provide proof-of-concept for the engineering of protein nano-compartments for biosynthesis and biocatalysis.
- 51Li, T.; Chang, P.; Chen, W.; Shi, Z.; Xue, C.; Dykes, G. F.; Huang, F.; Wang, Q.; Liu, L.-N. Nanoengineering Carboxysome Shells for Protein Cages with Programmable Cargo Targeting. ACS Nano 2024, 18, 7473, DOI: 10.1021/acsnano.3c11559Google ScholarThere is no corresponding record for this reference.
- 52Lassila, 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 (11), 2217– 2228, DOI: 10.1016/j.jmb.2014.02.025Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXkslGgu7o%253D&md5=0dfa6cfb708e477d860aa712e53ab221Assembly of Robust Bacterial Microcompartment Shells Using Building Blocks from an Organelle of Unknown FunctionLassila, 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.
- 53Ferlez, 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.2019.04.011Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXps1Gltbc%253D&md5=a952be90a5194ff9fdd5003de0968f8aA designed bacterial microcompartment shell with tunable composition and precision cargo loadingFerlez, 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.
- 54Dale, R.; Ohmuro-Matsuyama, Y.; Ueda, H.; Kato, N. Mathematical Model of the Firefly Luciferase Complementation Assay Reveals a Non-Linear Relationship between the Detected Luminescence and the Affinity of the Protein Pair Being Analyzed. PLoS One 2016, 11 (2), e0148256 DOI: 10.1371/journal.pone.0148256Google ScholarThere is no corresponding record for this reference.
- 55Dale, R.; Ohmuro-Matsuyama, Y.; Ueda, H.; Kato, N. Non-Steady State Analysis of Enzyme Kinetics in Real Time Elucidates Substrate Association and Dissociation Rates: Demonstration with Analysis of Firefly Luciferase Mutants. Biochemistry 2019, 58 (23), 2695– 2702, DOI: 10.1021/acs.biochem.9b00272Google ScholarThere is no corresponding record for this reference.
- 56Zakeri, B.; Fierer, J. O.; Celik, E.; Chittock, E. C.; Schwarz-Linek, U.; Moy, V. T.; Howarth, M. Peptide Tag Forming a Rapid Covalent Bond to a Protein, through Engineering a Bacterial Adhesin. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (12), E690– E697, DOI: 10.1073/pnas.1115485109Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XkvFGhtr4%253D&md5=eef3f7f046b9c6595b75d460294ba2c5Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesinZakeri, Bijan; Fierer, Jacob O.; Celik, Emrah; Chittock, Emily C.; Schwarz-Linek, Ulrich; Moy, Vincent T.; Howarth, MarkProceedings of the National Academy of Sciences of the United States of America (2012), 109 (12), E690-E697, SE690/1-SE690/20CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Protein interactions with peptides generally have low thermodn. and mech. stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (Spy-Catcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temp., and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-mol. dynamic force spectroscopy showed that SpyTag did not sep. from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures.
- 57Ribeiro, C.; Esteves da Silva, J. C. G. Kinetics of Inhibition of Firefly Luciferase by Oxyluciferin and Dehydroluciferyl-Adenylate. Photochem. Photobiol. Sci. 2008, 7 (9), 1085– 1090, DOI: 10.1039/b809935aGoogle ScholarThere is no corresponding record for this reference.
- 58Yang, N. J.; Hinner, M. J. Site-Specific Protein Labeling, Methods and Protocols. Methods Mol. Biol. 2015, 1266, 29– 53, DOI: 10.1007/978-1-4939-2272-7_3Google Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xls1Siur4%253D&md5=4002e2e8ab2fcc86d9ea400e1aa2a29bGetting Across the Cell Membrane: An Overview for Small Molecules, Peptides, and ProteinsYang, Nicole J.; Hinner, Marlon J.Methods in Molecular Biology (New York, NY, United States) (2015), 1266 (Site-Specific Protein Labeling), 29-53CODEN: MMBIED; ISSN:1940-6029. (Springer)The ability to efficiently access cytosolic proteins is desired in both biol. research and medicine. However, targeting intracellular proteins is often challenging, because to reach the cytosol, exogenous mols. must first traverse the cell membrane. This review provides a broad overview of how certain mols. are thought to cross this barrier, and what kinds of approaches are being made to enhance the intracellular delivery of those that are impermeable. We first discuss rules that govern the passive permeability of small mols. across the lipid membrane, and mechanisms of membrane transport that have evolved in nature for certain metabolites, peptides, and proteins. Then, we introduce design strategies that have emerged in the development of small mols. and peptides with improved permeability. Finally, intracellular delivery systems that have been engineered for protein payloads are surveyed. Viewpoints from varying disciplines have been brought together to provide a cohesive overview of how the membrane barrier is being overcome.
- 59Jakobson, C. M.; Tullman-Ercek, D.; Slininger, M. F.; Mangan, N. M. A Systems-Level Model Reveals That 1,2-Propanediol Utilization Microcompartments Enhance Pathway Flux through Intermediate Sequestration. PLoS Comput. Biol. 2017, 13 (5), e1005525 DOI: 10.1371/journal.pcbi.1005525Google Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1CitrnN&md5=c502a788a472a729bfad09ba0d5138aaA systems-level model reveals that 1,2-propanediol utilization microcompartments enhance pathway flux through intermediate sequestrationJakobson, Christopher M.; Tullman-Ercek, Danielle; Slininger, Marilyn F.; Mangan, Niall M.PLoS Computational Biology (2017), 13 (5), e1005525/1-e1005525/24CODEN: PCBLBG; ISSN:1553-7358. (Public Library of Science)The spatial organization of metab. is common to all domains of life. Enteric and other bacteria use subcellular organelles known as bacterial microcompartments to spatially organize the metab. of pathogenicity-relevant carbon sources, such as 1,2-propanediol. The organelles are thought to sequester a private cofactor pool, minimize the effects of toxic intermediates, and enhance flux through the encapsulated metabolic pathways. We develop a math. model of the function of the 1,2-propanediol utilization microcompartment of Salmonella enterica and use it to analyze the function of the microcompartment organelles in detail. Our model makes accurate ests. of doubling times based on an optimized compartment shell permeability detd. by maximizing metabolic flux in the model. The compartments function primarily to decouple cytosolic intermediate concns. from the concns. in the microcompartment, allowing significant enhancement in pathway flux by the generation of large concn. gradients across the microcompartment shell. We find that selective permeability of the microcompartment shell is not absolutely necessary, but is often beneficial in establishing this intermediate-trapping function. Our findings also implicate active transport of the 1,2-propanediol substrate under conditions of low external substrate concn., and we present a math. bound, in terms of external 1,2-propanediol substrate concn. and diffusive rates, on when active transport of the substrate is advantageous. By allowing us to predict exptl. inaccessible aspects of microcompartment function, such as intra-microcompartment metabolite concns., our model presents avenues for future research and underscores the importance of carefully considering changes in external metabolite concns. and other conditions during batch cultures. Our results also suggest that the encapsulation of heterologous pathways in bacterial microcompartments might yield significant benefits for pathway flux, as well as for toxicity mitigation.
- 60Long, B. M.; Förster, B.; Pulsford, S. B.; Price, G. D.; Badger, M. R. Rubisco Proton Production Can Drive the Elevation of CO2 within Condensates and Carboxysomes. Proc. Natl. Acad. Sci. U. S. A. 2021, 118 (18), e2014406118 DOI: 10.1073/pnas.2014406118Google ScholarThere is no corresponding record for this reference.
- 61Mangan, N. M.; Flamholz, A.; Hood, R. D.; Milo, R.; Savage, D. F. PH Determines the Energetic Efficiency of the Cyanobacterial CO2 Concentrating Mechanism. Proc. Natl. Acad. Sci. U. S. A. 2016, 113 (36), E5354– E5362, DOI: 10.1073/pnas.1525145113Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhtl2mtL3I&md5=c99a9f8f5381655436f418c0e99132bcpH Determines the energetic efficiency of the cyanobacterial CO2 concentrating mechanismMangan, Niall M.; Flamholz, Avi; Hood, Rachel D.; Milo, Ron; Savage, David F.Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (36), E5354-E5362CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Many carbon-fixing bacteria rely on a CO2 concg. mechanism (CCM) to elevate the CO2 concn. around the carboxylating enzyme ribulose bisphosphate carboxylase/oxygenase (RuBisCO). The CCM is postulated to simultaneously enhance the rate of carboxylation and minimize oxygenation, a competitive reaction with O2 also catalyzed by RuBisCO. To achieve this effect, the CCM combines 2 features: active transport of inorg. carbon into the cell and colocalization of carbonic anhydrase and RuBisCO inside proteinaceous microcompartments called carboxysomes. Understanding the significance of the various CCM components requires reconciling biochem. intuition with a quant. description of the system. To this end, we have developed a math. model of the CCM to analyze its energetic costs and the inherent intertwining of physiol. and pH. We find that intracellular pH greatly affects the cost of inorg. carbon accumulation. At low pH the inorg. carbon pool contains more of the highly cell-permeable H2CO3, necessitating a substantial expenditure of energy on transport to maintain internal inorg. carbon levels. An intracellular pH ≈8 reduces leakage, making the CCM significantly more energetically efficient. This pH prediction coincides well with our measurement of intracellular pH in a model cyanobacterium. We also demonstrate that CO2 retention in the carboxysome is necessary, whereas selective uptake of HCO3- into the carboxysome would not appreciably enhance energetic efficiency. Altogether, integration of pH produces a model that is quant. consistent with cyanobacterial physiol., emphasizing that pH cannot be neglected when describing biol. systems interacting with inorg. carbon pools.
- 62Vermaas, J. V.; Dixon, R. A.; Chen, F.; Mansfield, S. D.; Boerjan, W.; Ralph, J.; Crowley, M. F.; Beckham, G. T. Passive Membrane Transport of Lignin-Related Compounds. Proc. Natl. Acad. Sci. U. S. A. 2019, 116 (46), 23117– 23123, DOI: 10.1073/pnas.1904643116Google Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitFGqsrnM&md5=bc9ec48cc3af2ae7f17ebeb7d23687a9Passive membrane transport of lignin-related compoundsVermaas, Josh V.; Dixon, Richard A.; Chen, Fang; Mansfield, Shawn D.; Boerjan, Wout; Ralph, John; Crowley, Michael F.; Beckham, Gregg T.Proceedings of the National Academy of Sciences of the United States of America (2019), 116 (46), 23117-23123CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Lignin is an abundant arom. polymer found in plant secondary cell walls. In recent years, lignin has attracted renewed interest as a feedstock for bio-based chems. via catalytic and biol. approaches and has emerged as a target for genetic engineering to improve lignocellulose digestibility by altering its compn. In lignin biosynthesis and microbial conversion, small phenolic lignin precursors or degrdn. products cross membrane bilayers through an unidentified translocation mechanism prior to incorporation into lignin polymers (synthesis) or catabolism (bioconversion), with both passive and transporter-assisted mechanisms postulated. To test the passive permeation potential of these phenolics, we performed mol. dynamics simulations for 69 monomeric and dimeric lignin-related phenolics with 3 model membranes to det. the membrane partitioning and permeability coeffs. for each compd. The results support an accessible passive permeation mechanism for most compds., including monolignols, dimeric phenolics, and the flavonoid, tricin. Computed lignin partition coeffs. are consistent with concn. enrichment near lipid carbonyl groups, and permeability coeffs. are sufficient to keep pace with cellular metab. Interactions between methoxy and hydroxy groups are found to reduce membrane partitioning and improve permeability. Only carboxylate-modified or glycosylated lignin phenolics are predicted to require transporters for membrane translocation. Overall, the results suggest that most lignin-related compds. can passively traverse plant and microbial membranes on timescales commensurate with required biol. activities, with any potential transport regulation mechanism in lignin synthesis, catabolism, or bioconversion requiring compd. functionalization.
- 63Branchini, B. R.; Magyar, R. A.; Murtiashaw, M. H.; Anderson, S. M.; Zimmer, M. Site-Directed Mutagenesis of Histidine 245 in Firefly Luciferase: A Proposed Model of the Active Site †. Biochemistry 1998, 37 (44), 15311– 15319, DOI: 10.1021/bi981150dGoogle Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXmsFChsr8%253D&md5=0cc5cae21ff14d0777b453df4c9ac4aaSite-directed mutagenesis of histidine 245 in firefly luciferase: A proposed model of the active siteBranchini, Bruce R.; Magyar, Rachelle A.; Murtiashaw, Martha H.; Anderson, Shannon M.; Zimmer, MarcBiochemistry (1998), 37 (44), 15311-15319CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Firefly luciferase (I) catalyzes the highly efficient emission of yellow-green light from the substrate, luciferin, by a sequence of reactions that require Mg-ATP and O2. The authors previously reported that 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a firefly luciferin analog, was a potent photoinactivation reagent for I. A I tetrapeptide [244HHGF247] was identified, the degrdn. of which was directly correlated to the photooxidn. process. Here, the authors report the construction and purifn. of wild-type (WT) I and mutants H244F, H245F, H245A, and H245D. The results of photoinactivation and kinetic and bioluminescence studies with these proteins were consistent with His-245 being the primary functional target of BPTC-catalyzed enzyme inactivation. The possibility that His-245 is oxidized to Asp during the photooxidn. reaction was supported by the extremely low specific activity (∼300-fold lower than WT I) of the H245D mutant. Using the previously reported crystal structures of I without substrates and the functionally related phenylalanine-activating subunit of gramicidin synthetase 1 as a starting point, the authors performed mol. modeling studies and propose here a model for the I active site with substrates, luciferin and Mg-ATP, bound. This model was used to provide a structure-based interpretation of the role of peptide 244HHGF247 in firefly bioluminescence.
- 64Schenkmayerova, A.; Pinto, G. P.; Toul, M.; Marek, M.; Hernychova, L.; Planas-Iglesias, J.; Liskova, V. D.; Pluskal, D.; Vasina, M.; Emond, S.; Dörr, M.; Chaloupkova, R.; Bednar, D.; Prokop, Z.; Hollfelder, F.; Bornscheuer, U. T.; Damborsky, J. Engineering the Protein Dynamics of an Ancestral Luciferase. Nat. Commun. 2021, 12 (1), 3616, DOI: 10.1038/s41467-021-23450-zGoogle Scholar64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsVOqtLjK&md5=9ee528ee46ef28cdd9fbd0c6cef11e72Engineering the protein dynamics of an ancestral luciferaseSchenkmayerova, Andrea; Pinto, Gaspar P.; Toul, Martin; Marek, Martin; Hernychova, Lenka; Planas-Iglesias, Joan; Daniel Liskova, Veronika; Pluskal, Daniel; Vasina, Michal; Emond, Stephane; Dorr, Mark; Chaloupkova, Radka; Bednar, David; Prokop, Zbynek; Hollfelder, Florian; Bornscheuer, Uwe T.; Damborsky, JiriNature Communications (2021), 12 (1), 3616CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Abstr.: Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein AncHLD-RLuc which catalyzes both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of AncHLD-RLuc challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallog. B-factors, hydrogen exchange, and mol. dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins.
- 65Melnicki, M. R.; Sutter, M.; Kerfeld, C. A. Evolutionary Relationships among Shell Proteins of Carboxysomes and Metabolosomes. Curr. Opin. Microbiol. 2021, 63, 1– 9, DOI: 10.1016/j.mib.2021.05.011Google ScholarThere is no corresponding record for this reference.
- 66Du, P.; Xu, S.; Xu, Z.; Wang, Z. Bioinspired Self-Assembling Materials for Modulating Enzyme Functions. Adv. Funct. Mater. 2021, 31 (38), 2104819, DOI: 10.1002/adfm.202104819Google ScholarThere is no corresponding record for this reference.
- 67Küchler, A.; Yoshimoto, M.; Luginbühl, S.; Mavelli, F.; Walde, P. Enzymatic Reactions in Confined Environments. Nat. Nanotechnol. 2016, 11 (5), 409– 420, DOI: 10.1038/nnano.2016.54Google Scholar67https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XnsVWruro%253D&md5=f476bcda9da7f4fccdb11cb2cd11ccd2Enzymatic reactions in confined environmentsKuchler, Andreas; Yoshimoto, Makoto; Luginbuhl, Sandra; Mavelli, Fabio; Walde, PeterNature 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.
- 68Caparco, A. A.; Dautel, D. R.; Champion, J. A. Protein Mediated Enzyme Immobilization. Small 2022, 18 (19), e2106425 DOI: 10.1002/smll.202106425Google ScholarThere is no corresponding record for this reference.
- 69Schindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.; Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.; Schmid, B.; Tinevez, J.-Y.; White, D. J.; Hartenstein, V.; Eliceiri, K.; Tomancak, P.; Cardona, A. Fiji: An Open-Source Platform for Biological-Image Analysis. Nat. Methods 2012, 9 (7), 676– 682, DOI: 10.1038/nmeth.2019Google Scholar69https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtVKnurbJ&md5=ad150521a33367d37a800bee853dd9dbFiji: an open-source platform for biological-image analysisSchindelin, Johannes; Arganda-Carreras, Ignacio; Frise, Erwin; Kaynig, Verena; Longair, Mark; Pietzsch, Tobias; Preibisch, Stephan; Rueden, Curtis; Saalfeld, Stephan; Schmid, Benjamin; Tinevez, Jean-Yves; White, Daniel James; Hartenstein, Volker; Eliceiri, Kevin; Tomancak, Pavel; Cardona, AlbertNature Methods (2012), 9 (7_part1), 676-682CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Fiji is a distribution of the popular open-source software ImageJ focused on biol.-image anal. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biol. research communities.
- 70Mirdita, M.; Schütze, K.; Moriwaki, Y.; Heo, L.; Ovchinnikov, S.; Steinegger, M. ColabFold: Making Protein Folding Accessible to All. Nat. Methods 2022, 19 (6), 679– 682, DOI: 10.1038/s41592-022-01488-1Google Scholar70https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsVWjsLfL&md5=3b7437a490bd18770450c773d23bd6f6ColabFold: making protein folding accessible to allMirdita, Milot; Schuetze, Konstantin; Moriwaki, Yoshitaka; Heo, Lim; Ovchinnikov, Sergey; Steinegger, MartinNature Methods (2022), 19 (6), 679-682CODEN: NMAEA3; ISSN:1548-7091. (Nature Portfolio)ColabFold offers accelerated prediction of protein structures and complexes by combining the fast homol. search of MMseqs2 with AlphaFold2 or RoseTTAFold. ColabFold's 40-60-fold faster search and optimized model utilization enables prediction of close to 1,000 structures per day on a server with one graphics processing unit. Coupled with Google Colab., ColabFold becomes a free and accessible platform for protein folding. ColabFold is open-source software.
- 71Virtanen, P.; Gommers, R.; Oliphant, T. E.; Haberland, M.; Reddy, T.; Cournapeau, D.; Burovski, E.; Peterson, P.; Weckesser, W.; Bright, J.; van der Walt, S. J.; Brett, M.; Wilson, J.; Millman, K. J.; Mayorov, N.; Nelson, A. R. J.; Jones, E.; Kern, R.; Larson, E.; Carey, C. J.; Polat, İ.; Feng, Y.; Moore, E. W.; VanderPlas, J.; Laxalde, D.; Perktold, J.; Cimrman, R.; Henriksen, I.; Quintero, E. A.; Harris, C. R.; Archibald, A. M.; Ribeiro, A. H.; Pedregosa, F.; van Mulbregt, P.; Contributors, S.; Vijaykumar, A.; Bardelli, A. P.; Rothberg, A.; Hilboll, A.; Kloeckner, A.; Scopatz, A.; Lee, A.; Rokem, A.; Woods, C. N.; Fulton, C.; Masson, C.; Häggström, C.; Fitzgerald, C.; Nicholson, D. A.; Hagen, D. R.; Pasechnik, D. V.; Olivetti, E.; Martin, E.; Wieser, E.; Silva, F.; Lenders, F.; Wilhelm, F.; Young, G.; Price, G. A.; Ingold, G.-L.; Allen, G. E.; Lee, G. R.; Audren, H.; Probst, I.; Dietrich, J. P.; Silterra, J.; Webber, J. T.; Slavič, J.; Nothman, J.; Buchner, J.; Kulick, J.; Schönberger, J. L.; Cardoso, J. V. de M.; Reimer, J.; Harrington, J.; Rodríguez, J. L. C.; Nunez-Iglesias, J.; Kuczynski, J.; Tritz, K.; Thoma, M.; Newville, M.; Kümmerer, M.; Bolingbroke, M.; Tartre, M.; Pak, M.; Smith, N. J.; Nowaczyk, N.; Shebanov, N.; Pavlyk, O.; Brodtkorb, P. A.; Lee, P.; McGibbon, R. T.; Feldbauer, R.; Lewis, S.; Tygier, S.; Sievert, S.; Vigna, S.; Peterson, S.; More, S.; Pudlik, T.; Oshima, T.; Pingel, T. J.; Robitaille, T. P.; Spura, T.; Jones, T. R.; Cera, T.; Leslie, T.; Zito, T.; Krauss, T.; Upadhyay, U.; Halchenko, Y. O.; Vázquez-Baeza, Y. SciPy 1.0: Fundamental Algorithms for Scientific Computing in Python. Nat. Methods 2020, 17 (3), 261– 272, DOI: 10.1038/s41592-019-0686-2Google Scholar71https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXislCjuro%253D&md5=f007632188adeb57a43469157898e0a8SciPy 1.0: fundamental algorithms for scientific computing in PythonVirtanen, Pauli; Gommers, Ralf; Oliphant, Travis E.; Haberland, Matt; Reddy, Tyler; Cournapeau, David; Burovski, Evgeni; Peterson, Pearu; Weckesser, Warren; Bright, Jonathan; van der Walt, Stefan J.; Brett, Matthew; Wilson, Joshua; Millman, K. Jarrod; Mayorov, Nikolay; Nelson, Andrew R. J.; Jones, Eric; Kern, Robert; Larson, Eric; Carey, C. J.; Polat, Ilhan; Feng, Yu; Moore, Eric W.; Vander Plas, Jake; Laxalde, Denis; Perktold, Josef; Cimrman, Robert; Henriksen, Ian; Quintero, E. A.; Harris, Charles R.; Archibald, Anne M.; Ribeiro, Antonio H.; Pedregosa, Fabian; van Mulbregt, PaulNature Methods (2020), 17 (3), 261-272CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)Abstr.: SciPy is an open-source scientific computing library for the Python programming language. Since its initial release in 2001, SciPy has become a de facto std. for leveraging scientific algorithms in Python, with over 600 unique code contributors, thousands of dependent packages, over 100,000 dependent repositories and millions of downloads per yr. In this work, we provide an overview of the capabilities and development practices of SciPy 1.0 and highlight some recent tech. developments.
- 72Marquardt, D. W. An Algorithm for Least-Squares Estimation of Nonlinear Parameters. J. Soc. Ind. Appl. Math. 1963, 11 (2), 431– 441, DOI: 10.1137/0111030Google ScholarThere is no corresponding record for this reference.
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Abstract
Figure 1
Figure 1. Design for evaluating the ATP permeability of a model BMC shell. (A) BMC shells lacking pentamers contain ∼5 nm vertex gaps (i.e., “uncapped” configuration). BMC shell structures were prepared in PyMol from PDB structure 5V74. BMC-H proteins are colored blue, BMC-T1 dark green, BMC-T2 and BMC-T3 light green, and BMC-P yellow. (B) Zoom of area bounded in white in Figure 1A. (C) Fusion of a SpyTag/SnoopTag to BMC-T1 shell protein allows for covalent tethering of reciprocally tagged-catcher cargos to the lumen of the BMC shell to evaluate the permeability of ATP to a BMC shell. (D) Transmission electron microscopy of purified uncapped LucZ-SpyCatcher BMC shells.
Figure 2
Figure 2. Measuring ATP permeability of LucZ-SpyCatcher BMC shells with stopped-flow assays. (A) Stopped-flow assay comparison of uncapped versus capped shells versus a shell-free LucZ in a luciferin/MgCl2 substrate (each trace represents five readings averaged to one). The concentration of shell samples in solution was 100 nM, while the No Shell sample was 1000 nM. Samples were incubated for 1 h in 1 mM luciferin/MgCl2, 50 mM Tris–HCl, and 200 mM NaCl pH 8.0 buffer with or without 1000 nM BMC-P capping protein. Trends represented are from at least three independent experiments.
Figure 3
Figure 3. Differential equation modeling of stop-flow enzyme kinetics to calculate the permeability rate of capped versus uncapped shells. (A, B) Permeability coefficients are given by the rate of the limiting substrate (Px) and the rate of the intermediate complex (Py). Information regarding the equations, assumptions, and additional calculated values is found in the Supporting Information. (A) Capped shells. (B) Uncapped shells.
References
This article references 72 other publications.
- 1Kerfeld, C. A.; Aussignargues, C.; Zarzycki, J.; Cai, F.; Sutter, M. Bacterial Microcompartments. Nat. Rev. Microbiol. 2018, 16 (5), 277– 290, DOI: 10.1038/nrmicro.2018.101https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjslOgu7g%253D&md5=b87d0fb5d75108ba37f8b7f65841d524Bacterial microcompartmentsKerfeld, Cheryl A.; Aussignargues, Clement; Zarzycki, Jan; Cai, Fei; Sutter, MarkusNature Reviews Microbiology (2018), 16 (5), 277-290CODEN: NRMACK; ISSN:1740-1526. (Nature Research)A review. Bacterial microcompartments (BMCs) are self-assembling organelles that consist of an enzymic core that is encapsulated by a selectively permeable protein shell. The potential to form BMCs is widespread and found across the kingdom Bacteria. BMCs have crucial roles in carbon dioxide fixation in autotrophs and the catabolism of org. substrates in heterotrophs. They contribute to the metabolic versatility of bacteria, providing a competitive advantage in specific environmental niches. Although BMCs were first visualized more than 60 years ago, it is mainly in the past decade that progress has been made in understanding their metabolic diversity and the structural basis of their assembly and function. This progress has not only heightened our understanding of their role in microbial metab. but is also beginning to enable their use in a variety of applications in synthetic biol. In this Review, we focus on recent insights into the structure, assembly, diversity and function of BMCs.
- 2Sutter, M.; Melnicki, M. R.; Schulz, F.; Woyke, T.; Kerfeld, C. A. A Catalog of the Diversity and Ubiquity of Bacterial Microcompartments. Nat. Commun. 2021, 12 (1), 3809, DOI: 10.1038/s41467-021-24126-42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsFKnsL7L&md5=ee885729fe15bf1e733b2a90919c9d91A catalog of the diversity and ubiquity of bacterial microcompartmentsSutter, Markus; Melnicki, Matthew R.; Schulz, Frederik; Woyke, Tanja; Kerfeld, Cheryl A.Nature Communications (2021), 12 (1), 3809CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Bacterial microcompartments (BMCs) are organelles that segregate segments of metabolic pathways which are incompatible with surrounding metab. BMCs consist of a selectively permeable shell, composed of three types of structurally conserved proteins, together with sequestered enzymes that vary among functionally distinct BMCs. Genes encoding shell proteins are typically clustered with those for the encapsulated enzymes. Here, we report that the no. of identifiable BMC loci has increased twenty-fold since the last comprehensive census of 2014, and the no. of distinct BMC types has doubled. The new BMC types expand the range of compartmentalized catalysis and suggest that there is more BMC biochem. yet to be discovered. Our comprehensive catalog of BMCs provides a framework for their identification, correlation with bacterial niche adaptation, exptl. characterization, and development of BMC-based nanoarchitectures for biomedical and bioengineering applications.
- 3Steele, J. F. C.; Kerfeld, C. A. Encyclopedia of Biological Chemistry 2021, III, 108– 122, DOI: 10.1016/B978-0-12-819460-7.00005-0There is no corresponding record for this reference.
- 4Sutter, M.; Greber, B.; Aussignargues, C.; Kerfeld, C. A. Assembly Principles and Structure of a 6.5-MDa Bacterial Microcompartment Shell. Science 2017, 356 (6344), 1293– 1297, DOI: 10.1126/science.aan32894https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVWrsbvI&md5=f80671688ebdeb94bd1483da23ac2450Assembly principles and structure of a 6.5-MDa bacterial microcompartment shellSutter, Markus; Greber, Basil; Aussignargues, Clement; Kerfeld, Cheryl A.Science (Washington, DC, United States) (2017), 356 (6344), 1293-1297CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)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.
- 5Kerfeld, C. A.; Sawaya, M. R.; Tanaka, S.; Nguyen, C. V.; Phillips, M.; Beeby, M.; Yeates, T. O. Protein Structures Forming the Shell of Primitive Bacterial Organelles. Science 2005, 309 (5736), 936– 938, DOI: 10.1126/science.11133975https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXmvFSks7w%253D&md5=9001e7177689f87280fe8c491ea46f49Protein Structures Forming the Shell of Primitive Bacterial OrganellesKerfeld, Cheryl A.; Sawaya, Michael R.; Tanaka, Shiho; Nguyen, Chau V.; Phillips, Martin; Beeby, Morgan; Yeates, Todd O.Science (Washington, DC, United States) (2005), 309 (5736), 936-938CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Bacterial microcompartments are primitive organelles composed entirely of protein subunits. Genomic sequence databases reveal the widespread occurrence of microcompartments across diverse microbes. The prototypical bacterial microcompartment is the carboxysome, a protein shell for sequestering carbon fixation reactions. The authors report three-dimensional crystal structures of multiple carboxysome shell proteins, revealing a hexameric unit as the basic microcompartment building block and showing how these hexamers assemble to form flat facets of the polyhedral shell. The structures suggest how mol. transport across the shell may be controlled and how structural variations might govern the assembly and architecture of these subcellular compartments.
- 6Klein, 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 (2), 319– 333, DOI: 10.1016/j.jmb.2009.03.0566https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVKkt77O&md5=5555ea1b2add3c076176135c9c3b0c69Identification and Structural Analysis of a Novel Carboxysome Shell Protein with Implications for Metabolite TransportKlein, 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.
- 7Ferlez, B. H.; Kirst, H.; Greber, B. J.; Nogales, E.; Sutter, M.; Kerfeld, C. A. Heterologous Assembly of Pleomorphic Bacterial Microcompartment Shell Architectures Spanning the Nano- to Microscale. Adv. Mater. 2023, 35 (23), e2212065 DOI: 10.1002/adma.202212065There is no corresponding record for this reference.
- 8Tan, Y. Q.; Ali, S.; Xue, B.; Teo, W. Z.; Ling, L. H.; Go, M. K.; Lv, H.; Robinson, R. C.; Narita, A.; Yew, W. S. Structure of a Minimal Α-Carboxysome-Derived Shell and Its Utility in Enzyme Stabilization. Biomacromolecules 2021, 22 (10), 4095– 4109, DOI: 10.1021/acs.biomac.1c005338https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhslGrs7nP&md5=658a0654c1640d34ebd1f66251d15fb5Structure of a Minimal α-Carboxysome-Derived Shell and Its Utility in Enzyme StabilizationTan, Yong Quan; Ali, Samson; Xue, Bo; Teo, Wei Zhe; Ling, Lay Hiang; Go, Maybelle Kho; Lv, Hong; Robinson, Robert C.; Narita, Akihiro; Yew, Wen ShanBiomacromolecules (2021), 22 (10), 4095-4109CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)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 at.-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 enzymic activities against heat shock, presence of methanol co-solvent, consecutive freeze-thawing, and alk. 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.
- 9Cesle, E. E.; Filimonenko, A.; Tars, K.; Kalnins, G. Variety of Size and Form of GRM2 Bacterial Microcompartment Particles. Protein Sci. 2021, 30 (5), 1035– 1043, DOI: 10.1002/pro.40699https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXnslGnu7Y%253D&md5=2932e8028787a2d625c5fc54fcd10618Variety of size and form of GRM2 bacterial microcompartment particlesCesle, Eva Emilija; Filimonenko, Anatolij; Tars, Kaspars; Kalnins, GintsProtein Science (2021), 30 (5), 1035-1043CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)Bacterial microcompartments (BMCs) are bacterial organelles involved in enzymic processes, such as carbon fixation, choline, ethanolamine and propanediol degrdn., and others. Formed of a semi-permeable protein shell and an enzymic core, they can enhance enzyme performance and protect the cell from harmful intermediates. With the ability to encapsulate non-native enzymes, BMCs show high potential for applied use. For this goal, a detailed look into shell form variability is significant to predict shell adaptability. Here we present four novel 3D cryo-EM maps of recombinant Klebsiella pneumoniae GRM2 BMC shell particles with the resoln. in range of 9 to 22 Å and nine novel 2D classes corresponding to discrete BMC shell forms. These structures reveal icosahedral, elongated, oblate, multi-layered and polyhedral traits of BMCs, indicating considerable variation in size and form as well as adaptability during shell formation processes.
- 10Sutter, M.; Laughlin, T. G.; Sloan, N. B.; Serwas, D.; Davies, K. M.; Kerfeld, C. A. Structure of a Synthetic β-Carboxysome Shell. Plant Physiol. 2019, 181 (3), 1050– 1058, DOI: 10.1104/pp.19.0088510https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitl2kt73N&md5=fb5f3642ac5a252a91af2096cb82027eStructure of a synthetic β-carboxysome shellSutter, Markus; Laughlin, Thomas G.; Sloan, Nancy B.; Serwas, Daniel; Davies, Karen M.; Kerfeld, Cheryl A.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.
- 11Evans, S. L.; Al-Hazeem, M. M. J.; Mann, D.; Smetacek, N.; Beavil, A. J.; Sun, Y.; Chen, T.; Dykes, G. F.; Liu, L.-N.; Bergeron, J. R. C. Single-Particle Cryo-EM Analysis of the Shell Architecture and Internal Organization of an Intact α-Carboxysome. Structure 2023, 31 (6), 677– 688, DOI: 10.1016/j.str.2023.03.008There is no corresponding record for this reference.
- 12Greber, B. J.; Sutter, M.; Kerfeld, C. A. The Plasticity of Molecular Interactions Governs Bacterial Microcompartment Shell Assembly. Structure 2019, 27 (5), 749– 763, DOI: 10.1016/j.str.2019.01.01712https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXktFSjtr0%253D&md5=3b6a4b08167dd834a8d553d05f382eb4The Plasticity of Molecular Interactions Governs Bacterial Microcompartment Shell AssemblyGreber, Basil J.; Sutter, Markus; Kerfeld, Cheryl A.Structure (Oxford, United Kingdom) (2019), 27 (5), 749-763.e4CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)Bacterial microcompartments (BMCs) are composed of an enzymic core encapsulated by a selectively permeable protein shell that enhances catalytic efficiency. Many pathogenic bacteria derive competitive advantages from their BMC-based catabolism, implicating BMCs as drug targets. BMC shells are of interest for bioengineering due to their diverse and selective permeability properties and because they self-assemble. A complete understanding of shell compn. and organization is a prerequisite for biotechnol. applications. Here, we report the cryoelectron microscopy structure of a BMC shell at 3.0-Å resoln., using an image-processing strategy that allowed us to det. the previously uncharacterized structural details of the interactions formed by the BMC-TS and BMC-TD shell subunits in the context of the assembled shell. We found unexpected structural plasticity among these interactions, resulting in distinct shell populations assembled from varying nos. of the BMC-TS and BMC-TD subunits. We discuss the implications of these findings on shell assembly and function.
- 13Zhou, R.-Q.; Jiang, Y.-L.; Li, H.; Hou, P.; Kong, W.-W.; Deng, J.-X.; Chen, Y.; Zhou, C.-Z.; Zeng, Q. Structure and Assembly of the α-Carboxysome in the Marine Cyanobacterium Prochlorococcus. Nat. Plants 2024, 10, 661– 672, DOI: 10.1038/s41477-024-01660-9There is no corresponding record for this reference.
- 14Ni, T.; Jiang, Q.; Ng, P. C.; Shen, J.; Dou, H.; Zhu, Y.; Radecke, J.; Dykes, G. F.; Huang, F.; Liu, L.-N.; Zhang, P. Intrinsically Disordered CsoS2 Acts as a General Molecular Thread for α-Carboxysome Shell Assembly. Nat. Commun. 2023, 14 (1), 5512, DOI: 10.1038/s41467-023-41211-yThere is no corresponding record for this reference.
- 15Tanaka, S.; Kerfeld, C. A.; Sawaya, M. R.; Cai, F.; Heinhorst, S.; Cannon, G. C.; Yeates, T. O. Atomic-Level Models of the Bacterial Carboxysome Shell. Science 2008, 319 (5866), 1083– 1086, DOI: 10.1126/science.115145815https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXit1yhsbw%253D&md5=229ca40f815313eb56f84253e2f1a828Atomic-Level Models of the Bacterial Carboxysome ShellTanaka, Shiho; Kerfeld, Cheryl A.; Sawaya, Michael R.; Cai, Fei; Heinhorst, Sabine; Cannon, Gordon C.; Yeates, Todd O.Science (Washington, DC, United States) (2008), 319 (5866), 1083-1086CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)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.
- 16Kerfeld, C. A.; Erbilgin, O. Bacterial Microcompartments and the Modular Construction of Microbial Metabolism. Trends Microbiol. 2015, 23 (1), 22– 34, DOI: 10.1016/j.tim.2014.10.00316https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsl2ltb7O&md5=96858d72be4d76b3821cb00da17ba412Bacterial microcompartments and the modular construction of microbial metabolismKerfeld, Cheryl A.; Erbilgin, OnurTrends in Microbiology (2015), 23 (1), 22-34CODEN: TRMIEA; ISSN:0966-842X. (Elsevier Ltd.)A review. Bacterial microcompartments (BMCs) are protein-bound organelles predicted to be present across 23 bacterial phyla. BMCs facilitate carbon fixation as well as the aerobic and anaerobic catabolism of a variety of org. compds. These functions have been linked to ecol. nutrient cycling, symbiosis, pathogenesis, and cardiovascular disease. Within bacterial cells, BMCs are metabolic modules that can be further dissocd. into their constituent structural and functional protein domains. Viewing BMCs as genetic, structural, functional, and evolutionary modules provides a framework for understanding both BMC-mediated metab. and for adapting their architectures for applications in synthetic biol.
- 17Liu, L.-N. Advances in the Bacterial Organelles for CO2 Fixation. Trends Microbiol. 2022, 30 (6), 567– 580, DOI: 10.1016/j.tim.2021.10.004There is no corresponding record for this reference.
- 18Turmo, A.; Gonzalez-Esquer, C. R.; Kerfeld, C. A. Carboxysomes: Metabolic Modules for CO2 Fixation. FEMS Microbiol. Lett. 2017, 364 (18), 1000118, DOI: 10.1093/femsle/fnx176There is no corresponding record for this reference.
- 19Faulkner, 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 (1), 17501, DOI: 10.1038/s41598-020-74536-5There is no corresponding record for this reference.
- 20Tsai, 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 (6), e144 DOI: 10.1371/journal.pbio.0050144There is no corresponding record for this reference.
- 21Mahinthichaichan, 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 (39), 9110– 9118, DOI: 10.1021/acs.jpcb.8b0682221https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhs1ygtrjO&md5=fa585e72f333795d11f776b0953f9092Selective Permeability of Carboxysome Shell Pores to Anionic MoleculesMahinthichaichan, Paween; Morris, Dylan M.; Wang, Yi; Jensen, Grant J.; Tajkhorshid, EmadJournal 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.
- 22Trettel, D. S.; Neale, C.; Zhao, M.; Gnanakaran, S.; Gonzalez-Esquer, C. R. Monatomic Ions Influence Substrate Permeation across Bacterial Microcompartment Shells. Sci. Rep. 2023, 13 (1), 15738, DOI: 10.1038/s41598-023-42688-9There is no corresponding record for this reference.
- 23Park, 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 (34), 8149– 8154, DOI: 10.1021/acs.jpcb.7b0723223https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlKisb7L&md5=265fa3d2313ef00e57959feb755c2f14Molecular Dynamics Simulations of Selective Metabolite Transport across the Propanediol Bacterial Microcompartment ShellPark, 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.
- 24Sarkar, D.; Maffeo, C.; Sutter, M.; Aksimentiev, A.; Kerfeld, C. A.; Vermaas, J. V. Atomic View of Photosynthetic Metabolite Permeability Pathways and Confinement in Synthetic Carboxysome Shells. Proc. Natl. Acad. Sci. U. S. A. 2024, 121 (45), e2402277121 DOI: 10.1073/pnas.2402277121There is no corresponding record for this reference.
- 25Penrod, J. T.; Roth, J. R. Conserving a Volatile Metabolite: A Role for Carboxysome-Like Organelles in Salmonella Enterica. J. Bacteriol. 2006, 188 (8), 2865– 2874, DOI: 10.1128/JB.188.8.2865-2874.200625https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xjslejt78%253D&md5=67c1e4f99f8ce19b258541f34b92f98bConserving a volatile metabolite: a role for carboxysome-like organelles in Salmonella entericaPenrod, Joseph T.; Roth, John R.Journal of Bacteriology (2006), 188 (8), 2865-2874CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)Salmonellae can use ethanolamine (EA) as a sole source of carbon and nitrogen. This ability is encoded by an operon (eut) contg. 17 genes, only 6 of which are required under std. conditions (37°C; pH 7.0). Five of the extra genes (eutM, -N, -L, -K, and -G) become necessary under conditions that favor loss of the volatile intermediate, acetaldehyde, which escapes as a gas during growth on EA and is lost at a higher rate from these mutants. The eutM, -N, -L, and -K genes encode homologs of shell proteins of the carboxysome, an organelle shown (in other organisms) to conc. CO2. We propose that carboxysome-like organelles help bacteria conserve certain volatile metabolites-CO2 or acetaldehyde-perhaps by providing a low-pH compartment. The EutG enzyme converts acetaldehyde to ethanol, which may improve carbon retention by forming acetals; alternatively, EutG may recycle NADH within the carboxysome.
- 26Havemann, G. D.; Sampson, E. M.; Bobik, T. A. PduA Is a Shell Protein of Polyhedral Organelles Involved in Coenzyme B 12 -Dependent Degradation of 1,2-Propanediol in Salmonella Enterica Serovar Typhimurium LT2. J. Bacteriol. 2002, 184 (5), 1253– 1261, DOI: 10.1128/JB.184.5.1253-1261.200226https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XhtlKnsbo%253D&md5=f830432faeeed6d38718dfb5df33a7abPduA is a shell protein of polyhedral organelles involved in coenzyme B12-dependent degradation of 1,2-propanediol in Salmonella enterica serovar typhimurium LT2Havemann, Gregory D.; Sampson, Edith M.; Bobik, Thomas A.Journal of Bacteriology (2002), 184 (5), 1253-1261CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)Salmonella enterica forms polyhedral organelles involved in coenzyme B12-dependent 1,2-propanediol degrdn. These organelles are thought to consist of a proteinaceous shell that encases coenzyme B12-dependent diol dehydratase and perhaps other enzymes involved in 1,2-propanediol degrdn. The function of these organelles is unknown, and no detailed studies of their structure have been reported. Genes needed for organelle formation and for 1,2-propanediol degrdn. are located at the 1,2-propanediol utilization (pdu) locus, but the specific genes involved in organelle formation have not been identified. Here, we show that the pduA gene encodes a shell protein required for the formation of polyhedral organelles involved in coenzyme B12-dependent 1,2-propanediol degrdn. A His6-PduA fusion protein was purified from a recombinant Escherichia coli strain and used for the prepn. of polyclonal antibodies. The anti-PduA antibodies obtained were partially purified by a subtraction procedure and used to demonstrate that the PduA protein localized to the shell of the polyhedral organelles. In addn., electron microscopy studies established that strains with nonpolar pduA mutations were unable to form organelles. These results show that the pduA gene is essential for organelle formation and indicate that the PduA protein is a structural component of the shell of these organelles. Physiol. studies of nonpolar pduA mutants were also conducted. Such mutants grew similarly to the wild-type strain at low concns. of 1,2-propanediol but exhibited a period of interrupted growth in the presence of higher concns. of this growth substrate. Growth tests also showed that a nonpolar pduA deletion mutant grew faster than the wild-type strain at low vitamin B12 concns. These results suggest that the polyhedral organelles formed by S. enterica during growth on 1,2-propanediol are not involved in the concn. of 1,2-propanediol or coenzyme B12, but are consistent with the hypothesis that these organelles moderate aldehyde prodn. to minimize toxicity.
- 27Chowdhury, C.; Chun, S.; Pang, A.; Sawaya, M. R.; Sinha, S.; Yeates, T. O.; Bobik, T. A. Selective Molecular Transport through the Protein Shell of a Bacterial Microcompartment Organelle. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (10), 2990– 2995, DOI: 10.1073/pnas.142367211227https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjtFemtbk%253D&md5=ba014ac2a2ef0b2e41aeaf9f7f4af6b6Selective molecular transport through the protein shell of a bacterial microcompartment organelleChowdhury, Chiranjit; Chun, Sunny; Pang, Allan; Sawaya, Michael R.; Sinha, Sharmistha; Yeates, Todd O.; Bobik, Thomas A.Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (10), 2990-2995CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Bacterial microcompartments are widespread prokaryotic organelles that have important and diverse roles ranging from carbon fixation to enteric pathogenesis. Current models for microcompartment function propose that their outer protein shell is selectively permeable to small mols., but whether a protein shell can mediate selective permeability and how this occurs are unresolved questions. Here, biochem. and physiol. studies of structure-guided mutants are used to show that the hexameric PduA shell protein of the 1,2-propanediol utilization (Pdu) microcompartment forms a selectively permeable pore tailored for the influx of 1,2-propanediol (the substrate of the Pdu microcompartment) while restricting the efflux of propionaldehyde, a toxic intermediate of 1,2-propanediol catabolism. Crystal structures of various PduA mutants provide a foundation for interpreting the obsd. biochem. and phenotypic data in terms of mol. diffusion across the shell. Overall, these studies provide a basis for understanding a class of selectively permeable channels formed by nonmembrane proteins.
- 28Chowdhury, C.; Chun, S.; Sawaya, M. R.; Yeates, T. O.; Bobik, T. A. The Function of the PduJ Microcompartment Shell Protein Is Determined by the Genomic Position of Its Encoding Gene. Mol. Microbiol. 2016, 101 (5), 770– 783, DOI: 10.1111/mmi.13423There is no corresponding record for this reference.
- 29Chowdhury, C.; Bobik, T. A. Engineering the PduT Shell Protein to Modify the Permeability of the 1,2-Propanediol Microcompartment of Salmonella. Microbiology 2019, 165 (12), 1355– 1364, DOI: 10.1099/mic.0.000872There is no corresponding record for this reference.
- 30Cai, F.; Menon, B. B.; Cannon, G. C.; Curry, K. J.; Shively, J. M.; Heinhorst, S. The Pentameric Vertex Proteins Are Necessary for the Icosahedral Carboxysome Shell to Function as a CO2 Leakage Barrier. PLoS One 2009, 4 (10), e7521 DOI: 10.1371/journal.pone.0007521There is no corresponding record for this reference.
- 31Lee, M. F. S.; Jakobson, C. M.; Tullman-Ercek, D. Evidence for Improved Encapsulated Pathway Behavior in a Bacterial Microcompartment through Shell Protein Engineering. ACS Synth. Biol. 2017, 6 (10), 1880– 1891, DOI: 10.1021/acssynbio.7b00042There is no corresponding record for this reference.
- 32Huang, J.; Jiang, Q.; Yang, M.; Dykes, G. F.; Weetman, S. L.; Xin, W.; He, H.-L.; Liu, L.-N. Probing the Internal PH and Permeability of a Carboxysome Shell. Biomacromolecules 2022, 23 (10), 4339– 4348, DOI: 10.1021/acs.biomac.2c00781There is no corresponding record for this reference.
- 33Rae, B. D.; Long, B. M.; Badger, M. R.; Price, G. D. Structural Determinants of the Outer Shell of β-Carboxysomes in Synechococcus Elongatus PCC 7942: Roles for CcmK2, K3-K4, CcmO, and CcmL. PLoS One 2012, 7 (8), e43871 DOI: 10.1371/journal.pone.0043871There is no corresponding record for this reference.
- 34Cai, F.; Sutter, M.; Bernstein, S. L.; Kinney, J. N.; Kerfeld, C. A. Engineering Bacterial Microcompartment Shells: Chimeric Shell Proteins and Chimeric Carboxysome Shells. ACS Synth. Biol. 2015, 4 (4), 444– 453, DOI: 10.1021/sb500226j34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlalu7bL&md5=b8df8cf38136dbffd8a57cace90fcb85Engineering Bacterial Microcompartment Shells: Chimeric Shell Proteins and Chimeric Carboxysome ShellsCai, Fei; Sutter, Markus; Bernstein, Susan L.; Kinney, James N.; Kerfeld, Cheryl A.ACS Synthetic Biology (2015), 4 (4), 444-453CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Bacterial microcompartments (BMCs) are self-assembling organelles composed entirely of protein. Depending on the enzymes they encapsulate, BMCs function in either inorg. carbon fixation (carboxysomes) or org. carbon use (metabolosomes). The hallmark feature of all BMCs is a selectively permeable shell formed by multiple paralogous proteins, each proposed to confer specific flux characteristics. Gene clusters encoding diverse BMCs are distributed broadly across bacterial phyla, providing a rich variety of building blocks with a predicted range of permeability properties. In theory, shell permeability can be engineered by modifying residues flanking the pores (symmetry axes) of hexameric shell proteins or by combining shell proteins from different types of BMCs into chimeric shells. The authors undertook both approaches to altering shell properties using the carboxysome as a model system. There are two types of carboxysomes, α and β. In both, the predominant shell protein(s) contain a single copy of the BMC domain (pfam00936), but they are significantly different in primary structure. Indeed, phylogenetic anal. shows that the two types of carboxysome shell proteins are more similar to their counterparts in metabolosomes than to each other. The authors solved high resoln. crystal structures of the major shell proteins, CsoS1 and CcmK2, and the presumed minor shell protein CcmK4, representing both types of cyanobacterial carboxysomes and then tested the interchangeability. The in vivo study presented here confirms that both engineering pores to mimic those of other shell proteins and the construction of chimeric shells is feasible.
- 35Cameron, J. C.; Wilson, S. C.; Bernstein, S. L.; Kerfeld, C. A. Biogenesis of a Bacterial Organelle: The Carboxysome Assembly Pathway. Cell 2013, 155 (5), 1131– 1140, DOI: 10.1016/j.cell.2013.10.04435https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFWjtbjI&md5=04e529a8a8a3a5ad3af30d9d7c887b9bBiogenesis of a bacterial organelle: The carboxysome assembly pathwayCameron, Jeffrey C.; Wilson, Steven C.; Bernstein, Susan L.; Kerfeld, Cheryl A.Cell (Cambridge, MA, United States) (2013), 155 (5), 1131-1140CODEN: CELLB5; ISSN:0092-8674. (Cell Press)The carboxysome is a protein-based organelle for carbon fixation in cyanobacteria, keystone organisms in the global carbon cycle. It is composed of thousands of subunits including hexameric and pentameric proteins that form a shell to encapsulate the enzymes ribulose 1,5-bisphosphate carboxylase/oxygenase and carbonic anhydrase. Here, the authors describe the stages of carboxysome assembly and the requisite gene products necessary for progression through each. Unlike membrane-bound organelles of eukaryotes, in carboxysomes the interior of the compartment forms first, at a distinct site within the cell. Subsequently, shell proteins encapsulate this procarboxysome, inducing budding and distribution of functional organelles within the cell. The authors propose that the principles of carboxysome assembly that we have uncovered extend to diverse bacterial microcompartments.
- 36Yang, M.; Wenner, N.; Dykes, G. F.; Li, Y.; Zhu, X.; Sun, Y.; Huang, F.; Hinton, J. C. D.; Liu, L.-N. Biogenesis of a Bacterial Metabolosome for Propanediol Utilization. Nat. Commun. 2022, 13 (1), 2920, DOI: 10.1038/s41467-022-30608-wThere is no corresponding record for this reference.
- 37Huseby, D. L.; Roth, J. R. Evidence That a Metabolic Microcompartment Contains and Recycles Private Cofactor Pools. J. Bacteriol. 2013, 195 (12), 2864– 2879, DOI: 10.1128/JB.02179-1237https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXpsVartbc%253D&md5=c859df086c8e90e4f6664e2d49bec440Evidence that a metabolic microcompartment contains and recycles private cofactor poolsHuseby, Douglas L.; Roth, John R.Journal of Bacteriology (2013), 195 (12), 2864-2879CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)Microcompartments are loose protein cages that encapsulate enzymes for particular bacterial metabolic pathways. These structures are thought to retain and perhaps conc. pools of small, uncharged intermediates that would otherwise diffuse from the cell. In Salmonella enterica, a microcompartment encloses enzymes for ethanolamine catabolism. The cage has been thought to retain the volatile intermediate acetaldehyde but allow diffusion of the much larger cofactors NAD and CoA (CoA). Genetic tests support an alternative idea that the microcompartment contains and recycles private pools of the large cofactors NAD and CoA. Two central enzymes convert ethanolamine to acetaldehyde (EutBC) and then to acetyl-CoA (EutE). Two seemingly peripheral redundant enzymes encoded by the eut operon proved to be essential for ethanolamine utilization, when subjected to sufficiently stringent tests. These are EutD (acetyl-CoA to acetyl phosphate) and EutG (acetaldehyde to ethanol). Obligatory recycling of cofactors couples the three reactions and drives acetaldehyde consumption. Loss and toxic effects of acetaldehyde are minimized by accelerating its consumption. In a eutD mutant, acetyl-CoA cannot escape the compartment but is released by mutations that disrupt the structure. The model predicts that EutBC (ethanolamine-ammonia lyase) lies outside the compartment, using external coenzyme B12 and injecting its product, acetaldehyde, into the lumen, where it is degraded by the EutE, EutD, and EutG enzymes using private pools of CoA and NAD. The compartment appears to allow free diffusion of the intermediates ethanol and acetyl-PO4 but (to our great surprise) restricts diffusion of acetaldehyde.
- 38Sommer, M.; Sutter, M.; Gupta, S.; Kirst, H.; Turmo, A.; Lechno-Yossef, S.; Burton, R. L.; Saechao, C.; Sloan, N. B.; Cheng, X.; Chan, L.-J. G.; Petzold, C. J.; Fuentes-Cabrera, M.; Ralston, C. Y.; Kerfeld, C. A. Heterohexamers Formed by CcmK3 and CcmK4 Increase the Complexity of Beta Carboxysome Shells. Plant Physiol. 2019, 179 (1), 156– 167, DOI: 10.1104/pp.18.0119038https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXotFeru70%253D&md5=879e497ec69aa657f0c5395107665404Heterohexamers formed by CcmK3 and CcmK4 increase the complexity of beta carboxysome shellsSommer, Manuel; Sutter, Markus; Gupta, Sayan; Kirst, Henning; Turmo, Aiko; Lechno-Yossef, Sigal; Burton, Rodney L.; Saechao, Christine; Sloan, Nancy B.; Cheng, Xiaolin; Chan, Leanne-Jade G.; Petzold, Christopher J.; Fuentes-Cabrera, Miguel; Ralston, Corie Y.; Kerfeld, Cheryl A.Plant Physiology (2019), 179 (1), 156-167CODEN: PLPHAY; ISSN:1532-2548. (American Society of Plant Biologists)Bacterial microcompartments (BMCs) encapsulate enzymes within a selectively permeable, proteinaceous shell. Carboxysomes are BMCs contg. ribulose-1,5-bisphosphate carboxylase oxygenase and carbonic anhydrase that enhance carbon dioxide fixation. The carboxysome shell consists of three structurally characterized protein types, each named after the oligomer they form: BMC-H (hexamer), BMC-P (pentamer), and BMC-T (trimer). These three protein types form cyclic homooligomers with pores at the center of symmetry that enable metabolite transport across the shell. Carboxysome shells contain multiple BMC-H paralogs, each with distinctly conserved residues surrounding the pore, which are assumed to be assocd. with specific metabolites. We studied the regulation of β-carboxysome shell compn. by investigating the BMC-H genes ccmK3 and ccmK4 situated in a locus remote from other carboxysome genes. We made single and double deletion mutants of ccmK3 and ccmK4 in Synechococcus elongatus PCC7942 and show that, unlike CcmK3, CcmK4 is necessary for optimal growth. In contrast to other CcmK proteins, CcmK3 does not form homohexamers; instead CcmK3 forms heterohexamers with CcmK4 with a 1:2 stoichiometry. The CcmK3-CcmK4 heterohexamers form stacked dodecamers in a pH-dependent manner.
- 39Garcia-Alles, L. F.; Root, K.; Maveyraud, L.; Aubry, N.; Lesniewska, E.; Mourey, L.; Zenobi, R.; Truan, G. Occurrence and Stability of Hetero-Hexamer Associations Formed by β-Carboxysome CcmK Shell Components. PLoS One 2019, 14 (10), e0223877 DOI: 10.1371/journal.pone.0223877There is no corresponding record for this reference.
- 40Sun, Y.; Wollman, A. J. M.; Huang, F.; Leake, M. C.; Liu, L.-N. Single-Organelle Quantification Reveals Stoichiometric and Structural Variability of Carboxysomes Dependent on the Environment. Plant Cell 2019, 31 (7), 1648– 1664, DOI: 10.1105/tpc.18.00787There is no corresponding record for this reference.
- 41Sommer, M.; Cai, F.; Melnicki, M.; Kerfeld, C. A. β-Carboxysome Bioinformatics: Identification and Evolution of New Bacterial Microcompartment Protein Gene Classes and Core Locus Constraints. J. Exp. Bot. 2017, 68 (14), 3841– 3855, DOI: 10.1093/jxb/erx115There is no corresponding record for this reference.
- 42Hagen, A.; Sutter, M.; Sloan, N.; Kerfeld, C. A. Programmed Loading and Rapid Purification of Engineered Bacterial Microcompartment Shells. Nat. Commun. 2018, 9 (1), 2881, DOI: 10.1038/s41467-018-05162-z42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3c7htF2nug%253D%253D&md5=1486d359a87968f84608690e5ec53d7dProgrammed loading and rapid purification of engineered bacterial microcompartment shellsHagen Andrew; Sutter Markus; Sloan Nancy; Kerfeld Cheryl A; Sutter Markus; Kerfeld Cheryl A; Kerfeld Cheryl ANature 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.
- 43Trettel, D. S.; Resager, W.; Ueberheide, B. M.; Jenkins, C. C.; Winkler, W. C. Chemical Probing Provides Insight into the Native Assembly State of a Bacterial Microcompartment. Structure 2022, 30 (4), 537– 550, DOI: 10.1016/j.str.2022.02.002There is no corresponding record for this reference.
- 44Jiang, Q.; Li, T.; Yang, J.; Aitchison, C. M.; Huang, J.; Chen, Y.; Huang, F.; Wang, Q.; Cooper, A. I.; Liu, L.-N. Synthetic Engineering of a New Biocatalyst Encapsulating [NiFe]-Hydrogenases for Enhanced Hydrogen Production. J. Mater. Chem. B 2023, 11 (12), 2684– 2692, DOI: 10.1039/D2TB02781JThere is no corresponding record for this reference.
- 45Nguyen, N. D.; Pulsford, S. B.; Hee, W. Y.; Rae, B. D.; Rourke, L. M.; Price, G. D.; Long, B. M. Towards Engineering a Hybrid Carboxysome. Photosynth. Res. 2023, 156 (2), 265– 277, DOI: 10.1007/s11120-023-01009-xThere is no corresponding record for this reference.
- 46Kirst, H.; Ferlez, B. H.; Lindner, S. N.; Cotton, C. A. R.; Bar-Even, A.; Kerfeld, C. A. Toward a Glycyl Radical Enzyme Containing Synthetic Bacterial Microcompartment to Produce Pyruvate from Formate and Acetate. Proc. Natl. Acad. Sci. U. S. A. 2022, 119 (8), e2116871119 DOI: 10.1073/pnas.2116871119There is no corresponding record for this reference.
- 47Li, 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 (1), 5448, DOI: 10.1038/s41467-020-19280-047https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit1Ojur%252FK&md5=f9a4fcfafcb7204877e067c4fbbb1152Reprogramming bacterial protein organelles as a nanoreactor for hydrogen productionLi, 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-NingNature 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.
- 48Wagner, H. J.; Capitain, C. C.; Richter, K.; Nessling, M.; Mampel, J. Engineering Bacterial Microcompartments with Heterologous Enzyme Cargos. Eng. Life Sci. 2017, 17 (1), 36– 46, DOI: 10.1002/elsc.201600107There is no corresponding record for this reference.
- 49Lawrence, 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 (7), 454– 465, DOI: 10.1021/sb400111849https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVOjurk%253D&md5=f82c98604ef70551c623a4cfb008b0ccSolution Structure of a Bacterial Microcompartment Targeting Peptide and Its Application in the Construction of an Ethanol BioreactorLawrence, 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.
- 50Choudhary, S.; Quin, M. B.; Sanders, M. A.; Johnson, E. T.; Schmidt-Dannert, C. Engineered Protein Nano-Compartments for Targeted Enzyme Localization. PLoS One 2012, 7 (3), e33342 DOI: 10.1371/journal.pone.003334250https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XksVGgsLs%253D&md5=0b56b21e0cbad66c0be1d4ecbbc53988Engineered protein nano-compartments for targeted enzyme localizationChoudhary, Swati; Quin, Maureen B.; Sanders, Mark A.; Johnson, Ethan T.; Schmidt-Dannert, ClaudiaPLoS One (2012), 7 (3), e33342CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Compartmentalized co-localization of enzymes and their substrates represents an attractive approach for multi-enzymic synthesis in engineered cells and biocatalysis. Sequestration of enzymes and substrates would greatly increase reaction efficiency while also protecting engineered host cells from potentially toxic reaction intermediates. Several bacteria form protein-based polyhedral microcompartments which sequester functionally related enzymes and regulate their access to substrates and other small metabolites. Such bacterial microcompartments may be engineered into protein-based nano-bioreactors, provided that they can be assembled in a non-native host cell, and that heterologous enzymes and substrates can be targeted into the engineered compartments. Here, we report that recombinant expression of Salmonella enterica ethanolamine utilization (eut) bacterial microcompartment shell proteins in E. coli results in the formation of polyhedral protein shells. Purified recombinant shells are morphol. similar to the native Eut microcompartments purified from S. enterica. Surprisingly, recombinant expression of only one of the shell proteins (EutS) is sufficient and necessary for creating properly delimited compartments. Co-expression with EutS also facilitates the encapsulation of EGFP fused with a putative Eut shell-targeting signal sequence. We also demonstrate the functional localization of a heterologous enzyme (β-galactosidase) targeted to the recombinant shells. Together our results provide proof-of-concept for the engineering of protein nano-compartments for biosynthesis and biocatalysis.
- 51Li, T.; Chang, P.; Chen, W.; Shi, Z.; Xue, C.; Dykes, G. F.; Huang, F.; Wang, Q.; Liu, L.-N. Nanoengineering Carboxysome Shells for Protein Cages with Programmable Cargo Targeting. ACS Nano 2024, 18, 7473, DOI: 10.1021/acsnano.3c11559There is no corresponding record for this reference.
- 52Lassila, 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 (11), 2217– 2228, DOI: 10.1016/j.jmb.2014.02.02552https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXkslGgu7o%253D&md5=0dfa6cfb708e477d860aa712e53ab221Assembly of Robust Bacterial Microcompartment Shells Using Building Blocks from an Organelle of Unknown FunctionLassila, 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.
- 53Ferlez, 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.2019.04.01153https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXps1Gltbc%253D&md5=a952be90a5194ff9fdd5003de0968f8aA designed bacterial microcompartment shell with tunable composition and precision cargo loadingFerlez, 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.
- 54Dale, R.; Ohmuro-Matsuyama, Y.; Ueda, H.; Kato, N. Mathematical Model of the Firefly Luciferase Complementation Assay Reveals a Non-Linear Relationship between the Detected Luminescence and the Affinity of the Protein Pair Being Analyzed. PLoS One 2016, 11 (2), e0148256 DOI: 10.1371/journal.pone.0148256There is no corresponding record for this reference.
- 55Dale, R.; Ohmuro-Matsuyama, Y.; Ueda, H.; Kato, N. Non-Steady State Analysis of Enzyme Kinetics in Real Time Elucidates Substrate Association and Dissociation Rates: Demonstration with Analysis of Firefly Luciferase Mutants. Biochemistry 2019, 58 (23), 2695– 2702, DOI: 10.1021/acs.biochem.9b00272There is no corresponding record for this reference.
- 56Zakeri, B.; Fierer, J. O.; Celik, E.; Chittock, E. C.; Schwarz-Linek, U.; Moy, V. T.; Howarth, M. Peptide Tag Forming a Rapid Covalent Bond to a Protein, through Engineering a Bacterial Adhesin. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (12), E690– E697, DOI: 10.1073/pnas.111548510956https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XkvFGhtr4%253D&md5=eef3f7f046b9c6595b75d460294ba2c5Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesinZakeri, Bijan; Fierer, Jacob O.; Celik, Emrah; Chittock, Emily C.; Schwarz-Linek, Ulrich; Moy, Vincent T.; Howarth, MarkProceedings of the National Academy of Sciences of the United States of America (2012), 109 (12), E690-E697, SE690/1-SE690/20CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Protein interactions with peptides generally have low thermodn. and mech. stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (Spy-Catcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temp., and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-mol. dynamic force spectroscopy showed that SpyTag did not sep. from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures.
- 57Ribeiro, C.; Esteves da Silva, J. C. G. Kinetics of Inhibition of Firefly Luciferase by Oxyluciferin and Dehydroluciferyl-Adenylate. Photochem. Photobiol. Sci. 2008, 7 (9), 1085– 1090, DOI: 10.1039/b809935aThere is no corresponding record for this reference.
- 58Yang, N. J.; Hinner, M. J. Site-Specific Protein Labeling, Methods and Protocols. Methods Mol. Biol. 2015, 1266, 29– 53, DOI: 10.1007/978-1-4939-2272-7_358https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xls1Siur4%253D&md5=4002e2e8ab2fcc86d9ea400e1aa2a29bGetting Across the Cell Membrane: An Overview for Small Molecules, Peptides, and ProteinsYang, Nicole J.; Hinner, Marlon J.Methods in Molecular Biology (New York, NY, United States) (2015), 1266 (Site-Specific Protein Labeling), 29-53CODEN: MMBIED; ISSN:1940-6029. (Springer)The ability to efficiently access cytosolic proteins is desired in both biol. research and medicine. However, targeting intracellular proteins is often challenging, because to reach the cytosol, exogenous mols. must first traverse the cell membrane. This review provides a broad overview of how certain mols. are thought to cross this barrier, and what kinds of approaches are being made to enhance the intracellular delivery of those that are impermeable. We first discuss rules that govern the passive permeability of small mols. across the lipid membrane, and mechanisms of membrane transport that have evolved in nature for certain metabolites, peptides, and proteins. Then, we introduce design strategies that have emerged in the development of small mols. and peptides with improved permeability. Finally, intracellular delivery systems that have been engineered for protein payloads are surveyed. Viewpoints from varying disciplines have been brought together to provide a cohesive overview of how the membrane barrier is being overcome.
- 59Jakobson, C. M.; Tullman-Ercek, D.; Slininger, M. F.; Mangan, N. M. A Systems-Level Model Reveals That 1,2-Propanediol Utilization Microcompartments Enhance Pathway Flux through Intermediate Sequestration. PLoS Comput. Biol. 2017, 13 (5), e1005525 DOI: 10.1371/journal.pcbi.100552559https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1CitrnN&md5=c502a788a472a729bfad09ba0d5138aaA systems-level model reveals that 1,2-propanediol utilization microcompartments enhance pathway flux through intermediate sequestrationJakobson, Christopher M.; Tullman-Ercek, Danielle; Slininger, Marilyn F.; Mangan, Niall M.PLoS Computational Biology (2017), 13 (5), e1005525/1-e1005525/24CODEN: PCBLBG; ISSN:1553-7358. (Public Library of Science)The spatial organization of metab. is common to all domains of life. Enteric and other bacteria use subcellular organelles known as bacterial microcompartments to spatially organize the metab. of pathogenicity-relevant carbon sources, such as 1,2-propanediol. The organelles are thought to sequester a private cofactor pool, minimize the effects of toxic intermediates, and enhance flux through the encapsulated metabolic pathways. We develop a math. model of the function of the 1,2-propanediol utilization microcompartment of Salmonella enterica and use it to analyze the function of the microcompartment organelles in detail. Our model makes accurate ests. of doubling times based on an optimized compartment shell permeability detd. by maximizing metabolic flux in the model. The compartments function primarily to decouple cytosolic intermediate concns. from the concns. in the microcompartment, allowing significant enhancement in pathway flux by the generation of large concn. gradients across the microcompartment shell. We find that selective permeability of the microcompartment shell is not absolutely necessary, but is often beneficial in establishing this intermediate-trapping function. Our findings also implicate active transport of the 1,2-propanediol substrate under conditions of low external substrate concn., and we present a math. bound, in terms of external 1,2-propanediol substrate concn. and diffusive rates, on when active transport of the substrate is advantageous. By allowing us to predict exptl. inaccessible aspects of microcompartment function, such as intra-microcompartment metabolite concns., our model presents avenues for future research and underscores the importance of carefully considering changes in external metabolite concns. and other conditions during batch cultures. Our results also suggest that the encapsulation of heterologous pathways in bacterial microcompartments might yield significant benefits for pathway flux, as well as for toxicity mitigation.
- 60Long, B. M.; Förster, B.; Pulsford, S. B.; Price, G. D.; Badger, M. R. Rubisco Proton Production Can Drive the Elevation of CO2 within Condensates and Carboxysomes. Proc. Natl. Acad. Sci. U. S. A. 2021, 118 (18), e2014406118 DOI: 10.1073/pnas.2014406118There is no corresponding record for this reference.
- 61Mangan, N. M.; Flamholz, A.; Hood, R. D.; Milo, R.; Savage, D. F. PH Determines the Energetic Efficiency of the Cyanobacterial CO2 Concentrating Mechanism. Proc. Natl. Acad. Sci. U. S. A. 2016, 113 (36), E5354– E5362, DOI: 10.1073/pnas.152514511361https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhtl2mtL3I&md5=c99a9f8f5381655436f418c0e99132bcpH Determines the energetic efficiency of the cyanobacterial CO2 concentrating mechanismMangan, Niall M.; Flamholz, Avi; Hood, Rachel D.; Milo, Ron; Savage, David F.Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (36), E5354-E5362CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Many carbon-fixing bacteria rely on a CO2 concg. mechanism (CCM) to elevate the CO2 concn. around the carboxylating enzyme ribulose bisphosphate carboxylase/oxygenase (RuBisCO). The CCM is postulated to simultaneously enhance the rate of carboxylation and minimize oxygenation, a competitive reaction with O2 also catalyzed by RuBisCO. To achieve this effect, the CCM combines 2 features: active transport of inorg. carbon into the cell and colocalization of carbonic anhydrase and RuBisCO inside proteinaceous microcompartments called carboxysomes. Understanding the significance of the various CCM components requires reconciling biochem. intuition with a quant. description of the system. To this end, we have developed a math. model of the CCM to analyze its energetic costs and the inherent intertwining of physiol. and pH. We find that intracellular pH greatly affects the cost of inorg. carbon accumulation. At low pH the inorg. carbon pool contains more of the highly cell-permeable H2CO3, necessitating a substantial expenditure of energy on transport to maintain internal inorg. carbon levels. An intracellular pH ≈8 reduces leakage, making the CCM significantly more energetically efficient. This pH prediction coincides well with our measurement of intracellular pH in a model cyanobacterium. We also demonstrate that CO2 retention in the carboxysome is necessary, whereas selective uptake of HCO3- into the carboxysome would not appreciably enhance energetic efficiency. Altogether, integration of pH produces a model that is quant. consistent with cyanobacterial physiol., emphasizing that pH cannot be neglected when describing biol. systems interacting with inorg. carbon pools.
- 62Vermaas, J. V.; Dixon, R. A.; Chen, F.; Mansfield, S. D.; Boerjan, W.; Ralph, J.; Crowley, M. F.; Beckham, G. T. Passive Membrane Transport of Lignin-Related Compounds. Proc. Natl. Acad. Sci. U. S. A. 2019, 116 (46), 23117– 23123, DOI: 10.1073/pnas.190464311662https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitFGqsrnM&md5=bc9ec48cc3af2ae7f17ebeb7d23687a9Passive membrane transport of lignin-related compoundsVermaas, Josh V.; Dixon, Richard A.; Chen, Fang; Mansfield, Shawn D.; Boerjan, Wout; Ralph, John; Crowley, Michael F.; Beckham, Gregg T.Proceedings of the National Academy of Sciences of the United States of America (2019), 116 (46), 23117-23123CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Lignin is an abundant arom. polymer found in plant secondary cell walls. In recent years, lignin has attracted renewed interest as a feedstock for bio-based chems. via catalytic and biol. approaches and has emerged as a target for genetic engineering to improve lignocellulose digestibility by altering its compn. In lignin biosynthesis and microbial conversion, small phenolic lignin precursors or degrdn. products cross membrane bilayers through an unidentified translocation mechanism prior to incorporation into lignin polymers (synthesis) or catabolism (bioconversion), with both passive and transporter-assisted mechanisms postulated. To test the passive permeation potential of these phenolics, we performed mol. dynamics simulations for 69 monomeric and dimeric lignin-related phenolics with 3 model membranes to det. the membrane partitioning and permeability coeffs. for each compd. The results support an accessible passive permeation mechanism for most compds., including monolignols, dimeric phenolics, and the flavonoid, tricin. Computed lignin partition coeffs. are consistent with concn. enrichment near lipid carbonyl groups, and permeability coeffs. are sufficient to keep pace with cellular metab. Interactions between methoxy and hydroxy groups are found to reduce membrane partitioning and improve permeability. Only carboxylate-modified or glycosylated lignin phenolics are predicted to require transporters for membrane translocation. Overall, the results suggest that most lignin-related compds. can passively traverse plant and microbial membranes on timescales commensurate with required biol. activities, with any potential transport regulation mechanism in lignin synthesis, catabolism, or bioconversion requiring compd. functionalization.
- 63Branchini, B. R.; Magyar, R. A.; Murtiashaw, M. H.; Anderson, S. M.; Zimmer, M. Site-Directed Mutagenesis of Histidine 245 in Firefly Luciferase: A Proposed Model of the Active Site †. Biochemistry 1998, 37 (44), 15311– 15319, DOI: 10.1021/bi981150d63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXmsFChsr8%253D&md5=0cc5cae21ff14d0777b453df4c9ac4aaSite-directed mutagenesis of histidine 245 in firefly luciferase: A proposed model of the active siteBranchini, Bruce R.; Magyar, Rachelle A.; Murtiashaw, Martha H.; Anderson, Shannon M.; Zimmer, MarcBiochemistry (1998), 37 (44), 15311-15319CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Firefly luciferase (I) catalyzes the highly efficient emission of yellow-green light from the substrate, luciferin, by a sequence of reactions that require Mg-ATP and O2. The authors previously reported that 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a firefly luciferin analog, was a potent photoinactivation reagent for I. A I tetrapeptide [244HHGF247] was identified, the degrdn. of which was directly correlated to the photooxidn. process. Here, the authors report the construction and purifn. of wild-type (WT) I and mutants H244F, H245F, H245A, and H245D. The results of photoinactivation and kinetic and bioluminescence studies with these proteins were consistent with His-245 being the primary functional target of BPTC-catalyzed enzyme inactivation. The possibility that His-245 is oxidized to Asp during the photooxidn. reaction was supported by the extremely low specific activity (∼300-fold lower than WT I) of the H245D mutant. Using the previously reported crystal structures of I without substrates and the functionally related phenylalanine-activating subunit of gramicidin synthetase 1 as a starting point, the authors performed mol. modeling studies and propose here a model for the I active site with substrates, luciferin and Mg-ATP, bound. This model was used to provide a structure-based interpretation of the role of peptide 244HHGF247 in firefly bioluminescence.
- 64Schenkmayerova, A.; Pinto, G. P.; Toul, M.; Marek, M.; Hernychova, L.; Planas-Iglesias, J.; Liskova, V. D.; Pluskal, D.; Vasina, M.; Emond, S.; Dörr, M.; Chaloupkova, R.; Bednar, D.; Prokop, Z.; Hollfelder, F.; Bornscheuer, U. T.; Damborsky, J. Engineering the Protein Dynamics of an Ancestral Luciferase. Nat. Commun. 2021, 12 (1), 3616, DOI: 10.1038/s41467-021-23450-z64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsVOqtLjK&md5=9ee528ee46ef28cdd9fbd0c6cef11e72Engineering the protein dynamics of an ancestral luciferaseSchenkmayerova, Andrea; Pinto, Gaspar P.; Toul, Martin; Marek, Martin; Hernychova, Lenka; Planas-Iglesias, Joan; Daniel Liskova, Veronika; Pluskal, Daniel; Vasina, Michal; Emond, Stephane; Dorr, Mark; Chaloupkova, Radka; Bednar, David; Prokop, Zbynek; Hollfelder, Florian; Bornscheuer, Uwe T.; Damborsky, JiriNature Communications (2021), 12 (1), 3616CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Abstr.: Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein AncHLD-RLuc which catalyzes both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of AncHLD-RLuc challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallog. B-factors, hydrogen exchange, and mol. dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins.
- 65Melnicki, M. R.; Sutter, M.; Kerfeld, C. A. Evolutionary Relationships among Shell Proteins of Carboxysomes and Metabolosomes. Curr. Opin. Microbiol. 2021, 63, 1– 9, DOI: 10.1016/j.mib.2021.05.011There is no corresponding record for this reference.
- 66Du, P.; Xu, S.; Xu, Z.; Wang, Z. Bioinspired Self-Assembling Materials for Modulating Enzyme Functions. Adv. Funct. Mater. 2021, 31 (38), 2104819, DOI: 10.1002/adfm.202104819There is no corresponding record for this reference.
- 67Küchler, A.; Yoshimoto, M.; Luginbühl, S.; Mavelli, F.; Walde, P. Enzymatic Reactions in Confined Environments. Nat. Nanotechnol. 2016, 11 (5), 409– 420, DOI: 10.1038/nnano.2016.5467https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XnsVWruro%253D&md5=f476bcda9da7f4fccdb11cb2cd11ccd2Enzymatic reactions in confined environmentsKuchler, Andreas; Yoshimoto, Makoto; Luginbuhl, Sandra; Mavelli, Fabio; Walde, PeterNature 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.
- 68Caparco, A. A.; Dautel, D. R.; Champion, J. A. Protein Mediated Enzyme Immobilization. Small 2022, 18 (19), e2106425 DOI: 10.1002/smll.202106425There is no corresponding record for this reference.
- 69Schindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.; Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.; Schmid, B.; Tinevez, J.-Y.; White, D. J.; Hartenstein, V.; Eliceiri, K.; Tomancak, P.; Cardona, A. Fiji: An Open-Source Platform for Biological-Image Analysis. Nat. Methods 2012, 9 (7), 676– 682, DOI: 10.1038/nmeth.201969https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtVKnurbJ&md5=ad150521a33367d37a800bee853dd9dbFiji: an open-source platform for biological-image analysisSchindelin, Johannes; Arganda-Carreras, Ignacio; Frise, Erwin; Kaynig, Verena; Longair, Mark; Pietzsch, Tobias; Preibisch, Stephan; Rueden, Curtis; Saalfeld, Stephan; Schmid, Benjamin; Tinevez, Jean-Yves; White, Daniel James; Hartenstein, Volker; Eliceiri, Kevin; Tomancak, Pavel; Cardona, AlbertNature Methods (2012), 9 (7_part1), 676-682CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Fiji is a distribution of the popular open-source software ImageJ focused on biol.-image anal. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biol. research communities.
- 70Mirdita, M.; Schütze, K.; Moriwaki, Y.; Heo, L.; Ovchinnikov, S.; Steinegger, M. ColabFold: Making Protein Folding Accessible to All. Nat. Methods 2022, 19 (6), 679– 682, DOI: 10.1038/s41592-022-01488-170https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsVWjsLfL&md5=3b7437a490bd18770450c773d23bd6f6ColabFold: making protein folding accessible to allMirdita, Milot; Schuetze, Konstantin; Moriwaki, Yoshitaka; Heo, Lim; Ovchinnikov, Sergey; Steinegger, MartinNature Methods (2022), 19 (6), 679-682CODEN: NMAEA3; ISSN:1548-7091. (Nature Portfolio)ColabFold offers accelerated prediction of protein structures and complexes by combining the fast homol. search of MMseqs2 with AlphaFold2 or RoseTTAFold. ColabFold's 40-60-fold faster search and optimized model utilization enables prediction of close to 1,000 structures per day on a server with one graphics processing unit. Coupled with Google Colab., ColabFold becomes a free and accessible platform for protein folding. ColabFold is open-source software.
- 71Virtanen, P.; Gommers, R.; Oliphant, T. E.; Haberland, M.; Reddy, T.; Cournapeau, D.; Burovski, E.; Peterson, P.; Weckesser, W.; Bright, J.; van der Walt, S. J.; Brett, M.; Wilson, J.; Millman, K. J.; Mayorov, N.; Nelson, A. R. J.; Jones, E.; Kern, R.; Larson, E.; Carey, C. J.; Polat, İ.; Feng, Y.; Moore, E. W.; VanderPlas, J.; Laxalde, D.; Perktold, J.; Cimrman, R.; Henriksen, I.; Quintero, E. A.; Harris, C. R.; Archibald, A. M.; Ribeiro, A. H.; Pedregosa, F.; van Mulbregt, P.; Contributors, S.; Vijaykumar, A.; Bardelli, A. P.; Rothberg, A.; Hilboll, A.; Kloeckner, A.; Scopatz, A.; Lee, A.; Rokem, A.; Woods, C. N.; Fulton, C.; Masson, C.; Häggström, C.; Fitzgerald, C.; Nicholson, D. A.; Hagen, D. R.; Pasechnik, D. V.; Olivetti, E.; Martin, E.; Wieser, E.; Silva, F.; Lenders, F.; Wilhelm, F.; Young, G.; Price, G. A.; Ingold, G.-L.; Allen, G. E.; Lee, G. R.; Audren, H.; Probst, I.; Dietrich, J. P.; Silterra, J.; Webber, J. T.; Slavič, J.; Nothman, J.; Buchner, J.; Kulick, J.; Schönberger, J. L.; Cardoso, J. V. de M.; Reimer, J.; Harrington, J.; Rodríguez, J. L. C.; Nunez-Iglesias, J.; Kuczynski, J.; Tritz, K.; Thoma, M.; Newville, M.; Kümmerer, M.; Bolingbroke, M.; Tartre, M.; Pak, M.; Smith, N. J.; Nowaczyk, N.; Shebanov, N.; Pavlyk, O.; Brodtkorb, P. A.; Lee, P.; McGibbon, R. T.; Feldbauer, R.; Lewis, S.; Tygier, S.; Sievert, S.; Vigna, S.; Peterson, S.; More, S.; Pudlik, T.; Oshima, T.; Pingel, T. J.; Robitaille, T. P.; Spura, T.; Jones, T. R.; Cera, T.; Leslie, T.; Zito, T.; Krauss, T.; Upadhyay, U.; Halchenko, Y. O.; Vázquez-Baeza, Y. SciPy 1.0: Fundamental Algorithms for Scientific Computing in Python. Nat. Methods 2020, 17 (3), 261– 272, DOI: 10.1038/s41592-019-0686-271https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXislCjuro%253D&md5=f007632188adeb57a43469157898e0a8SciPy 1.0: fundamental algorithms for scientific computing in PythonVirtanen, Pauli; Gommers, Ralf; Oliphant, Travis E.; Haberland, Matt; Reddy, Tyler; Cournapeau, David; Burovski, Evgeni; Peterson, Pearu; Weckesser, Warren; Bright, Jonathan; van der Walt, Stefan J.; Brett, Matthew; Wilson, Joshua; Millman, K. Jarrod; Mayorov, Nikolay; Nelson, Andrew R. J.; Jones, Eric; Kern, Robert; Larson, Eric; Carey, C. J.; Polat, Ilhan; Feng, Yu; Moore, Eric W.; Vander Plas, Jake; Laxalde, Denis; Perktold, Josef; Cimrman, Robert; Henriksen, Ian; Quintero, E. A.; Harris, Charles R.; Archibald, Anne M.; Ribeiro, Antonio H.; Pedregosa, Fabian; van Mulbregt, PaulNature Methods (2020), 17 (3), 261-272CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)Abstr.: SciPy is an open-source scientific computing library for the Python programming language. Since its initial release in 2001, SciPy has become a de facto std. for leveraging scientific algorithms in Python, with over 600 unique code contributors, thousands of dependent packages, over 100,000 dependent repositories and millions of downloads per yr. In this work, we provide an overview of the capabilities and development practices of SciPy 1.0 and highlight some recent tech. developments.
- 72Marquardt, D. W. An Algorithm for Least-Squares Estimation of Nonlinear Parameters. J. Soc. Ind. Appl. Math. 1963, 11 (2), 431– 441, DOI: 10.1137/0111030There is no corresponding record for this reference.
Supporting Information
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.4c00290.
Purification of in vivo covalently loaded SpyCatcher-LucZ-uncapped BMC shells; purification of in vivo covalently loaded LucZ-SpyCatcher-uncapped BMC shells; luminescence activity comparison between LucZ and SpyCatcher fusion orientation; activity assay of His-LucZ-SpyCatcher-loaded uncapped BMC shells; capping pentamer protein purification, and workflow for comparing shell permeability in uncapped versus capped configuration; LucZ-SpyCatcher Shells after incubation in substrate buffer; stopped-flow spectrophotometry of uncapped and capped BMC shells; stopped-flow spectrophotometry of uncapped and capped shells in 10 and 20% PEG6000 buffer; stopped-flow spectrophotometry of MonoQ-purified shells after capping; calculation of kM of uncapped and capped LucZ-SpyCatcher BMC shells; protein expression vectors; coding sequences of expression vectors; differential equations for luciferase activity without shells; and addition of permeability into the model (PDF)
The code for the fitting, and reading in the experimental data, is provided on github (DOI: 10.5281/zenodo.10963263).
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