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Jon Beckwith - One of the best experts on this subject based on the ideXlab platform.
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identification of the thioredoxin partner of vitamin k epoxide reductase in mycobacterial Disulfide Bond formation
Journal of Bacteriology, 2018Co-Authors: Cristina Landeta, Dana Boyd, Xiaoyun Wang, Markus Eser, Jon BeckwithAbstract:ABSTRACT Disulfide Bonds influence the stability and activity of many proteins. In Escherichia coli, the DsbA and DsbB enzymes promote Disulfide Bond formation. Other bacteria, including the Actinobacteria, use instead of DsbB the enzyme vitamin K epoxide reductase (VKOR), whose gene is found either fused to or in the same operon as a dsbA-like gene. Mycobacterium tuberculosis and other Gram-positive actinobacteria secrete many proteins with even numbers of cysteines to the cell envelope. These organisms have predicted oxidoreductases and VKOR orthologs. These findings indicate that such bacteria likely form Disulfide Bonds in the cell envelope. The M. tuberculosisvkor gene complements an E. colidsbB deletion strain, restoring the oxidation of E. coli DsbA. While we have suggested that the dsbA gene linked to the vkor gene may express VKOR9s partner in mycobacteria, others have suggested that two other extracytoplasmic oxidoreductases (DsbE or DsbF) may be catalysts of protein Disulfide Bond formation. However, there is no direct evidence for interactions of VKOR with either DsbA, DsbE, or DsbF. To identify the actual substrate of VKOR, we identified two additional predicted extracytoplasmic DsbA-like proteins using bioinformatics analysis of the M. tuberculosis genome. Using the five potential DsbAs, we attempted to reconstitute Disulfide Bond pathways in E. coli and in Mycobacterium smegmatis, a close relative of M. tuberculosis. Our results show that only M. tuberculosis DsbA is oxidized by VKOR. Comparison of the properties of dsbA- and vkor-null mutants in M. smegmatis shows parallels to the properties of dsb mutations in E. coli. IMPORTANCE Disulfide Bond formation has a great impact on bacterial pathogenicity. Thus, Disulfide-Bond-forming proteins represent new targets for the development of antibacterials, since the inhibition of Disulfide Bond formation would result in the simultaneous loss of the activity of several classes of virulence factors. Here, we identified five candidate proteins encoded by the M. tuberculosis genome as possible substrates of the M. tuberculosis VKOR protein involved in Disulfide Bond formation. We then reconstituted the mycobacterial Disulfide Bond formation pathway in E. coli and showed that of the five candidates, only M. tuberculosis DsbA is efficiently oxidized by VKOR in E. coli. We also present evidence for the involvement of VKOR in DsbA oxidation in M. smegmatis.
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Disulfide Bond formation in prokaryotes
Nature Microbiology, 2018Co-Authors: Cristina Landeta, Dana Boyd, Jon BeckwithAbstract:Interest in protein Disulfide Bond formation has recently increased because of the prominent role of Disulfide Bonds in bacterial virulence and survival. The first discovered pathway that introduces Disulfide Bonds into cell envelope proteins consists of Escherichia coli enzymes DsbA and DsbB. Since its discovery, variations on the DsbAB pathway have been found in bacteria and archaea, probably reflecting specific requirements for survival in their ecological niches. One variation found amongst Actinobacteria and Cyanobacteria is the replacement of DsbB by a homologue of human vitamin K epoxide reductase. Many Gram-positive bacteria express enzymes involved in Disulfide Bond formation that are similar, but non-homologous, to DsbAB. While bacterial pathways promote Disulfide Bond formation in the bacterial cell envelope, some archaeal extremophiles express proteins with Disulfide Bonds both in the cytoplasm and in the extra-cytoplasmic space, possibly to stabilize proteins in the face of extreme conditions, such as growth at high temperatures. Here, we summarize the diversity of Disulfide-Bond-catalysing systems across prokaryotic lineages, discuss examples for understanding the biological basis of such systems, and present perspectives on how such systems are enabling advances in biomedical engineering and drug development. A review of the diversity of Disulfide-Bond-catalysing systems across prokaryotes.
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Disulfide Bond formation in prokaryotes
Nature microbiology, 2018Co-Authors: Cristina Landeta, Dana Boyd, Jon BeckwithAbstract:Interest in protein Disulfide Bond formation has recently increased because of the prominent role of Disulfide Bonds in bacterial virulence and survival. The first discovered pathway that introduces Disulfide Bonds into cell envelope proteins consists of Escherichia coli enzymes DsbA and DsbB. Since its discovery, variations on the DsbAB pathway have been found in bacteria and archaea, probably reflecting specific requirements for survival in their ecological niches. One variation found amongst Actinobacteria and Cyanobacteria is the replacement of DsbB by a homologue of human vitamin K epoxide reductase. Many Gram-positive bacteria express enzymes involved in Disulfide Bond formation that are similar, but non-homologous, to DsbAB. While bacterial pathways promote Disulfide Bond formation in the bacterial cell envelope, some archaeal extremophiles express proteins with Disulfide Bonds both in the cytoplasm and in the extra-cytoplasmic space, possibly to stabilize proteins in the face of extreme conditions, such as growth at high temperatures. Here, we summarize the diversity of Disulfide-Bond-catalysing systems across prokaryotic lineages, discuss examples for understanding the biological basis of such systems, and present perspectives on how such systems are enabling advances in biomedical engineering and drug development.
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bacterial species exhibit diversity in their mechanisms and capacity for protein Disulfide Bond formation
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Rachel J Dutton, Dana Boyd, Mehmet Berkmen, Jon BeckwithAbstract:Protein Disulfide Bond formation contributes to the folding and activity of many exported proteins in bacteria. However, information about Disulfide Bond formation is limited to only a few bacterial species. We used a multifaceted bioinformatic approach to assess the capacity for Disulfide Bond formation across this biologically diverse group of organisms. We combined data from a cysteine counting method, in which a significant bias for even numbers of cysteine in proteins is taken as an indicator of Disulfide Bond formation, with data on the presence of homologs of known Disulfide Bond formation enzymes. These combined data enabled us to make predictions about Disulfide Bond formation in the cell envelope across bacterial species. Our bioinformatic and experimental results suggest that many bacteria may not generally oxidatively fold proteins, and implicate the bacterial homolog of the enzyme vitamin K epoxide reductase, a protein required for blood clotting in humans, as part of a Disulfide Bond formation pathway present in several major bacterial phyla.
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Disulfide Bond formation in periplasm of Escherichia coli.
Methods in enzymology, 2002Co-Authors: Federico Katzen, Jon BeckwithAbstract:Publisher Summary This chapter outlines the process of Disulfide Bond formation in periplasm of Escherichia coli . The formation of stable Disulfide Bonds in proteins takes place in specific compartments in the cell. The periplasm of Escherichia coli contains a mechanism devoted to the correct and rapid formation of Disulfide Bonds. This machinery comprises two main thiol/Disulfide exchange processes: (1) the enzymatic oxidation of free protein thiols into Disulfide Bonds and (2) the Disulfide Bond isomerization or reshuffling of incorrect Disulfide Bonds. This chapter summarizes the genetic approaches that have been used to identify the components of this machinery. In addition, it describes protocols for detecting and quantitating these two E. coli periplasmic thiol/Disulfide activities in vivo. Finally, the chapter describes a technique for detecting mixed Disulfide intermediates formed in vivo by the rapid reactions carried out by thiol/Disulfide oxidoreductases. This technique allows the definition of aspects of the reaction mechanism and the identification of novel substrates of these enzymes.
James C.a. Bardwell - One of the best experts on this subject based on the ideXlab platform.
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Converting a Sulfenic Acid Reductase into a Disulfide Bond Isomerase
Antioxidants & redox signaling, 2015Co-Authors: Claire Chatelle, Dana Boyd, Guoping Ren, Stéphanie Kraemer, Hannah Chmura, Nils Marechal, Caroline Roggemans, Paul Riggs, James C.a. BardwellAbstract:Abstract Aims: Posttranslational formation of Disulfide Bonds is essential for the folding of many secreted proteins. Formation of Disulfide Bonds in a protein with more than two cysteines is inherently fraught with error and can result in incorrect Disulfide Bond pairing and, consequently, misfolded protein. Protein Disulfide Bond isomerases, such as DsbC of Escherichia coli, can recognize mis-oxidized proteins and shuffle the Disulfide Bonds of the substrate protein into their native folded state. Results: We have developed a simple blue/white screen that can detect Disulfide Bond isomerization in vivo, using a mutant alkaline phosphatase (PhoA*) in E. coli. We utilized this screen to isolate mutants of the sulfenic acid reductase (DsbG) that allowed this protein to act as a Disulfide Bond isomerase. Characterization of the isolated mutants in vivo and in vitro allowed us to identify key amino acid residues responsible for oxidoreductase properties of thioredoxin-like proteins such as DsbC or DsbG. Inno...
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Engineered Pathways for Correct Disulfide Bond Oxidation
Antioxidants & redox signaling, 2011Co-Authors: Guoping Ren, James C.a. BardwellAbstract:Correct formation of Disulfide Bonds is critical for protein folding. We find that cells lacking protein Disulfide isomerases (PDIs) can use alternative mechanisms for correct Disulfide Bond formation. By linking correct Disulfide Bond formation to antibiotic resistance, we selected mutants that catalyze correct Disulfide formation in the absence of DsbC, Escherichia coli's PDI. Most of our mutants massively overproduce the Disulfide oxidase DsbA and change its redox status. They enhance DsbA's ability to directly form the correct Disulfides by increasing the level of mixed Disulfides between DsbA and substrate proteins. One mutant operates via a different mechanism; it contains mutations in DsbB and CpxR that alter the redox environment of the periplasm and increases the level of the chaperone/protease DegP, allowing DsbA to gain Disulfide isomerase ability in vivo. Thus, given the proper expression level, redox status, and chaperone assistance, the oxidase DsbA can readily function in vivo to catalyze the folding of proteins with complex Disulfide Bond connectivities. Our selection reveals versatile strategies for correct Disulfide formation in vivo. Remarkably, our evolution of new pathways for correct Disulfide Bond formation in E. coli mimics eukaryotic PDI, a highly abundant partially reduced protein with chaperone activity. Antioxid. Redox Signal. 14, 2399–2412.
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Disulfide Bond Formation in Prokaryotes and Eukaryotes
Protein Targeting Transport and Translocation, 2008Co-Authors: James Regeimbal, James C.a. BardwellAbstract:Publisher Summary This chapter reviews Disulfide Bond formation in prokaryotes and eukaryote. The formation of a Disulfide Bond in vivo is a controlled and catalyzed process. The catalysis of Disulfide Bond formation occurs via a thiol–Disulfide exchange reaction between a Disulfide donor and a target protein. Disulfide Bond formation and isomerization are catalyzed processes in both prokaryotes and eukaryotes, and are achieved via thiol–Disulfide exchange reactions with specific Disulfide donors and isomerases. A mechanism for DsbB's oxidation of reduced DsbA has been proposed, and it involves a thiol–Disulfide exchange cascade between DsbA and the two presumed Disulfides of DsbB. Like DsbA, DsbC has a very reactive CXXC, which is only slightly less oxidizing than the CXXC of DsbA. Most of the proteins that are secreted from a eukaryotic cell contain Disulfide Bonds. In prokaryotes there is a clear separation between the oxidative and isomerase pathways. In eukaryotes a large number of PDI homologs with both PDI and oxidase activities cooperate to catalyze proper Disulfide Bond formation. Finally, this chapter concludes with a summary note on Disulfide Bond formation.
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Disulfide Bond Formation Enzymes
Molecular Machines Involved in Protein Transport across Cellular Membranes, 2007Co-Authors: James C.a. BardwellAbstract:Publisher Summary This chapter discusses the Disulfide Bond (Dsb) formation and isomerization in the periplasm of the model organism E.coli and focuses on the enzymatic properties of Disulfide catalysts and isomerases. Disulfide Bond formation and isomerization are catalyzed processes in both prokaryotes and eukaryotic organisms, and the enzymes responsible are called the “Disulfide Bond (Dsb) enzymes” for their ability to affect the formation and isomerization of Disulfide Bonds. DsbA and DsbB are responsible for thiol oxidation and DsbC, DsbG, and DsbD are responsible for Disulfide isomerization. DsbA is a strong oxidant and is capable of very rapid thiol–Disulfide exchange reactions, while the membrane-bound protein DsbB performs the reoxidation of DsbA. DsbC can rearrange Disulfides in vivo and in vitro , and DsbD is an integral membrane protein whose function is to keep DsbC in a reduced state. However, DsbB is a central player in Disulfide Bond formation in prokaryotes as it links Disulfide Bond formation to electron transport. The structural characterization of the full-length protein and/or intermediates of electron transfer should give further insight into the detailed molecular mechanism.
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Catalysis of Disulfide Bond formation and isomerization in Escherichia coli.
Advances in protein chemistry, 2001Co-Authors: Martin W. Bader, James C.a. BardwellAbstract:Publisher Summary This chapter deals with folding catalysts, in particular with catalysts that are essential for Disulfide Bond formation in Escherichia coli. Nevertheless, some of these catalysts contain chaperone activity, demonstrating that these two activities are sometimes found within the same protein molecule. The folding of proteins into their three-dimensional structure is essential for their biological function. For proteins that contain Disulfide Bonds, formation of these Bonds is often an important step in the folding reaction. DsbA is identified by the use of a Disulfide indicator protein, MalF-fi-galactosidase. This fusion protein lacks fl-galactosidase activity when present in a wild-type E. coli background that is competent in forming Disulfides. Despite their common structures, thioredoxin and DsbA fulfill different functions and exist in different cellular compartments. While thioredoxin acts as a reductant of Disulfide Bonds in the cytosol introduces Disulfide Bonds into newly synthesized proteins during their translocation to the periplasm. The small equilibrium constant of DsbA with glutathione demonstrates that the Disulfide Bond formed by DsbA is highly unstable. The stability of a particular Disulfide Bond corresponds to the extent to which a protein is stabilized by this Bond. In other words, the more stable the Disulfide Bond, the more stable the protein conformation.
Hiram F. Gilbert - One of the best experts on this subject based on the ideXlab platform.
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Disulfide Bond Formation
Encyclopedia of Biological Chemistry, 2013Co-Authors: Hiram F. GilbertAbstract:Disulfide Bond formation is a posttranslational protein modification that introduces a covalent cross-link between the sulfhydryl groups of specific cysteine residues. Biologically, Disulfide Bonds are most often used to increase the conformational stability of extracellular proteins, but Disulfides may also serve catalytic or regulatory roles through their effects on protein structure. Disulfide Bonds are formed as the protein folds into its correct biological structure. Elaborate quality control systems in the bacterial periplasm and eukaryotic endoplasmic reticulum assure that correct Disulfides are formed as the proper conformation is achieved.
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Thiol/Disulfide exchange equilibria and Disulfide Bond stability
Methods in enzymology, 1995Co-Authors: Hiram F. GilbertAbstract:Publisher Summary Disulfide-Bond formation is a versatile oxidation used biologically in diverse processes, such as enzyme catalysis, protection against oxidative damage, stabilization of extracellular proteins, and regulation of biological activity. Because Disulfide formation is a reversible process, Disulfide-Bond stability often plays an important role in the biological utility of Disulfide Bonds. In turn, the ability to form and break a specific Disulfide Bond under appropriate biological conditions depends on the nature of the oxidant or reductant, the Disulfide stability, the kinetics of the forward and reverse reactions, and the nature and redox state of the environment in which the reaction occurs. The stability of Disulfide Bonds in small molecules and proteins spans an enormous range, a factor of approximately 1011, corresponding to a free-energy difference of about 15 kcal/mol or a redox potential difference of 0.33 V. This chapter outlines the importance of reversible thiol/Disulfide exchange and discusses some practical considerations in measuring Disulfide Bond stability.
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thiol Disulfide exchange equilibria and Disulfide Bond stability
Methods in Enzymology, 1995Co-Authors: Hiram F. GilbertAbstract:Publisher Summary Disulfide-Bond formation is a versatile oxidation used biologically in diverse processes, such as enzyme catalysis, protection against oxidative damage, stabilization of extracellular proteins, and regulation of biological activity. Because Disulfide formation is a reversible process, Disulfide-Bond stability often plays an important role in the biological utility of Disulfide Bonds. In turn, the ability to form and break a specific Disulfide Bond under appropriate biological conditions depends on the nature of the oxidant or reductant, the Disulfide stability, the kinetics of the forward and reverse reactions, and the nature and redox state of the environment in which the reaction occurs. The stability of Disulfide Bonds in small molecules and proteins spans an enormous range, a factor of approximately 1011, corresponding to a free-energy difference of about 15 kcal/mol or a redox potential difference of 0.33 V. This chapter outlines the importance of reversible thiol/Disulfide exchange and discusses some practical considerations in measuring Disulfide Bond stability.
Heather Desaire - One of the best experts on this subject based on the ideXlab platform.
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Recent mass spectrometry-based techniques and considerations for Disulfide Bond characterization in proteins
Analytical and Bioanalytical Chemistry, 2018Co-Authors: Jude Lakbub, Joshua T. Shipman, Heather DesaireAbstract:Disulfide Bonds are important structural moieties of proteins: they ensure proper folding, provide stability, and ensure proper function. With the increasing use of proteins for biotherapeutics, particularly monoclonal antibodies, which are highly Disulfide Bonded, it is now important to confirm the correct Disulfide Bond connectivity and to verify the presence, or absence, of Disulfide Bond variants in the protein therapeutics. These studies help to ensure safety and efficacy. Hence, Disulfide Bonds are among the critical quality attributes of proteins that have to be monitored closely during the development of biotherapeutics. However, Disulfide Bond analysis is challenging because of the complexity of the biomolecules. Mass spectrometry (MS) has been the go-to analytical tool for the characterization of such complex biomolecules, and several methods have been reported to meet the challenging task of mapping Disulfide Bonds in proteins. In this review, we describe the relevant, recent MS-based techniques and provide important considerations needed for efficient Disulfide Bond analysis in proteins. The review focuses on methods for proper sample preparation, fragmentation techniques for Disulfide Bond analysis, recent Disulfide Bond mapping methods based on the fragmentation techniques, and automated algorithms designed for rapid analysis of Disulfide Bonds from liquid chromatography–MS/MS data. Researchers involved in method development for protein characterization can use the information herein to facilitate development of new MS-based methods for protein Disulfide Bond analysis. In addition, individuals characterizing biotherapeutics, especially by Disulfide Bond mapping in antibodies, can use this review to choose the best strategies for Disulfide Bond assignment of their biologic products. Graphical Abstract This review, describing characterization methods for Disulfide Bonds in proteins, focuses on three critical components: sample preparation, mass spectrometry data, and software tools
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Recent mass spectrometry-based techniques and considerations for Disulfide Bond characterization in proteins.
Analytical and bioanalytical chemistry, 2017Co-Authors: Jude Lakbub, Joshua T. Shipman, Heather DesaireAbstract:Disulfide Bonds are important structural moieties of proteins: they ensure proper folding, provide stability, and ensure proper function. With the increasing use of proteins for biotherapeutics, particularly monoclonal antibodies, which are highly Disulfide Bonded, it is now important to confirm the correct Disulfide Bond connectivity and to verify the presence, or absence, of Disulfide Bond variants in the protein therapeutics. These studies help to ensure safety and efficacy. Hence, Disulfide Bonds are among the critical quality attributes of proteins that have to be monitored closely during the development of biotherapeutics. However, Disulfide Bond analysis is challenging because of the complexity of the biomolecules. Mass spectrometry (MS) has been the go-to analytical tool for the characterization of such complex biomolecules, and several methods have been reported to meet the challenging task of mapping Disulfide Bonds in proteins. In this review, we describe the relevant, recent MS-based techniques and provide important considerations needed for efficient Disulfide Bond analysis in proteins. The review focuses on methods for proper sample preparation, fragmentation techniques for Disulfide Bond analysis, recent Disulfide Bond mapping methods based on the fragmentation techniques, and automated algorithms designed for rapid analysis of Disulfide Bonds from liquid chromatography–MS/MS data. Researchers involved in method development for protein characterization can use the information herein to facilitate development of new MS-based methods for protein Disulfide Bond analysis. In addition, individuals characterizing biotherapeutics, especially by Disulfide Bond mapping in antibodies, can use this review to choose the best strategies for Disulfide Bond assignment of their biologic products.
Cristina Landeta - One of the best experts on this subject based on the ideXlab platform.
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inhibition of pseudomonas aeruginosa and mycobacterium tuberculosis Disulfide Bond forming enzymes
Molecular Microbiology, 2019Co-Authors: Cristina Landeta, Brian M. Meehan, Laura Mcpartland, Ngoc Q Tran, Yifan Zhang, Zaidi Tanweer, Shoko Wakabayashi, Jeremy M Rock, Taehyun Kim, Deepak BalasubramanianAbstract:In bacteria, Disulfide Bonds confer stability on many proteins exported to the cell envelope or beyond, including bacterial virulence factors. Thus, proteins involved in Disulfide Bond formation represent good targets for the development of inhibitors that can act as antibiotics or anti-virulence agents, resulting in the simultaneous inactivation of several types of virulence factors. Here, we present evidence that the Disulfide Bond forming enzymes, DsbB and VKOR, are required for Pseudomonas aeruginosa pathogenicity and Mycobacterium tuberculosis survival respectively. We also report the results of a HTS of 216,767 compounds tested against P. aeruginosa DsbB1 and M. tuberculosis VKOR using Escherichia coli cells. Since both P. aeruginosa DsbB1 and M. tuberculosis VKOR complement an E. coli dsbB knockout, we screened simultaneously for inhibitors of each complemented E. coli strain expressing a Disulfide-Bond sensitive β-galactosidase reported previously. The properties of several inhibitors obtained from these screens suggest they are a starting point for chemical modifications with potential for future antibacterial development.
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identification of the thioredoxin partner of vitamin k epoxide reductase in mycobacterial Disulfide Bond formation
Journal of Bacteriology, 2018Co-Authors: Cristina Landeta, Dana Boyd, Xiaoyun Wang, Markus Eser, Jon BeckwithAbstract:ABSTRACT Disulfide Bonds influence the stability and activity of many proteins. In Escherichia coli, the DsbA and DsbB enzymes promote Disulfide Bond formation. Other bacteria, including the Actinobacteria, use instead of DsbB the enzyme vitamin K epoxide reductase (VKOR), whose gene is found either fused to or in the same operon as a dsbA-like gene. Mycobacterium tuberculosis and other Gram-positive actinobacteria secrete many proteins with even numbers of cysteines to the cell envelope. These organisms have predicted oxidoreductases and VKOR orthologs. These findings indicate that such bacteria likely form Disulfide Bonds in the cell envelope. The M. tuberculosisvkor gene complements an E. colidsbB deletion strain, restoring the oxidation of E. coli DsbA. While we have suggested that the dsbA gene linked to the vkor gene may express VKOR9s partner in mycobacteria, others have suggested that two other extracytoplasmic oxidoreductases (DsbE or DsbF) may be catalysts of protein Disulfide Bond formation. However, there is no direct evidence for interactions of VKOR with either DsbA, DsbE, or DsbF. To identify the actual substrate of VKOR, we identified two additional predicted extracytoplasmic DsbA-like proteins using bioinformatics analysis of the M. tuberculosis genome. Using the five potential DsbAs, we attempted to reconstitute Disulfide Bond pathways in E. coli and in Mycobacterium smegmatis, a close relative of M. tuberculosis. Our results show that only M. tuberculosis DsbA is oxidized by VKOR. Comparison of the properties of dsbA- and vkor-null mutants in M. smegmatis shows parallels to the properties of dsb mutations in E. coli. IMPORTANCE Disulfide Bond formation has a great impact on bacterial pathogenicity. Thus, Disulfide-Bond-forming proteins represent new targets for the development of antibacterials, since the inhibition of Disulfide Bond formation would result in the simultaneous loss of the activity of several classes of virulence factors. Here, we identified five candidate proteins encoded by the M. tuberculosis genome as possible substrates of the M. tuberculosis VKOR protein involved in Disulfide Bond formation. We then reconstituted the mycobacterial Disulfide Bond formation pathway in E. coli and showed that of the five candidates, only M. tuberculosis DsbA is efficiently oxidized by VKOR in E. coli. We also present evidence for the involvement of VKOR in DsbA oxidation in M. smegmatis.
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Disulfide Bond formation in prokaryotes
Nature Microbiology, 2018Co-Authors: Cristina Landeta, Dana Boyd, Jon BeckwithAbstract:Interest in protein Disulfide Bond formation has recently increased because of the prominent role of Disulfide Bonds in bacterial virulence and survival. The first discovered pathway that introduces Disulfide Bonds into cell envelope proteins consists of Escherichia coli enzymes DsbA and DsbB. Since its discovery, variations on the DsbAB pathway have been found in bacteria and archaea, probably reflecting specific requirements for survival in their ecological niches. One variation found amongst Actinobacteria and Cyanobacteria is the replacement of DsbB by a homologue of human vitamin K epoxide reductase. Many Gram-positive bacteria express enzymes involved in Disulfide Bond formation that are similar, but non-homologous, to DsbAB. While bacterial pathways promote Disulfide Bond formation in the bacterial cell envelope, some archaeal extremophiles express proteins with Disulfide Bonds both in the cytoplasm and in the extra-cytoplasmic space, possibly to stabilize proteins in the face of extreme conditions, such as growth at high temperatures. Here, we summarize the diversity of Disulfide-Bond-catalysing systems across prokaryotic lineages, discuss examples for understanding the biological basis of such systems, and present perspectives on how such systems are enabling advances in biomedical engineering and drug development. A review of the diversity of Disulfide-Bond-catalysing systems across prokaryotes.
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Disulfide Bond formation in prokaryotes
Nature microbiology, 2018Co-Authors: Cristina Landeta, Dana Boyd, Jon BeckwithAbstract:Interest in protein Disulfide Bond formation has recently increased because of the prominent role of Disulfide Bonds in bacterial virulence and survival. The first discovered pathway that introduces Disulfide Bonds into cell envelope proteins consists of Escherichia coli enzymes DsbA and DsbB. Since its discovery, variations on the DsbAB pathway have been found in bacteria and archaea, probably reflecting specific requirements for survival in their ecological niches. One variation found amongst Actinobacteria and Cyanobacteria is the replacement of DsbB by a homologue of human vitamin K epoxide reductase. Many Gram-positive bacteria express enzymes involved in Disulfide Bond formation that are similar, but non-homologous, to DsbAB. While bacterial pathways promote Disulfide Bond formation in the bacterial cell envelope, some archaeal extremophiles express proteins with Disulfide Bonds both in the cytoplasm and in the extra-cytoplasmic space, possibly to stabilize proteins in the face of extreme conditions, such as growth at high temperatures. Here, we summarize the diversity of Disulfide-Bond-catalysing systems across prokaryotic lineages, discuss examples for understanding the biological basis of such systems, and present perspectives on how such systems are enabling advances in biomedical engineering and drug development.
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The Disulfide Bond Formation Pathway Is Essential for Anaerobic Growth of Escherichia coli.
Journal of bacteriology, 2017Co-Authors: Brian M. Meehan, Cristina Landeta, Dana Boyd, Jonathan BeckwithAbstract:Disulfide Bonds are critical to the stability and function of many bacterial proteins. In the periplasm of Escherichia coli, intramolecular Disulfide Bond formation is catalyzed by the two-component Disulfide Bond forming (DSB) system. Inactivation of the DSB pathway has been shown to lead to a number of pleotropic effects, although cells remain viable under standard laboratory conditions. However, we show here that dsb strains of E. coli reversibly filament under aerobic conditions and fail to grow anaerobically unless a strong oxidant is provided in the growth medium. These findings demonstrate that the background Disulfide Bond formation necessary to maintain the viability of dsb strains is oxygen dependent. LptD, a key component of the lipopolysaccharide transport system, fails to fold properly in dsb strains exposed to anaerobic conditions, suggesting that these mutants may have defects in outer membrane assembly. We also show that anaerobic growth of dsb mutants can be restored by suppressor mutations in the Disulfide Bond isomerization system. Overall, our results underscore the importance of proper Disulfide Bond formation to pathways critical to E. coli viability under conditions where oxygen is limited.IMPORTANCE While the Disulfide Bond formation (DSB) system of E. coli has been studied for decades and has been shown to play an important role in the proper folding of many proteins, including some associated with virulence, it was considered dispensable for growth under most laboratory conditions. This work represents the first attempt to study the effects of the DSB system under strictly anaerobic conditions, simulating the environment encountered by pathogenic E. coli strains in the human intestinal tract. By demonstrating that the DSB system is essential for growth under such conditions, this work suggests that compounds inhibiting Dsb enzymes might act not only as antivirulents but also as true antibiotics.