Disulfide bond formation in prokaryotes: History, diversity and design (original) (raw)
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From Biology to Biotechnology: Disulfide Bond Formation in Escherichia coli
Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications, 2017
Disulfide bonds formed between a pair of oxidized cysteines are important to the structural integrity and proper folding of many proteins. Accordingly, Nature has evolved several systems for the genesis and maintenance of such bonds. Beginning with the discovery of protein disulfide isomerase, which provided the first evidence for enzyme-catalyzed disulfide-bond formation, many years of research have resulted in the explication of the complex network of electron transport pathways needed for this process. Herein, we take a historical approach in describing the elucidation of disulfide-bond formation in E. coli. We frame this topic in the context of genome sequencing eras. The first section describes the discovery of eukaryotic protein disulfide isomerase and the subsequent research that followed from the early 1960s to the early 1990s, a time period we have named the pre-genomic sequencing era. The second section details the renaissance in research on disulfide-bond formation in the periplasm of prokaryotes, fueled by bacterial genetic screens and the development of genomic sequencing technology. Accordingly, we have named this section the genomic sequencing era, which ranges from the early 1990s to approximately 2010. The final section outlines the use of bacterial genetic screens to select for new oxidoreductase enzymes and their potential uses in biotechnological and pharmaceutical applications. This era we have dubbed the post-genomic sequencing era, and we envision it to represent the future of research on oxidative folding.
From Biology to Biotechnology: Disulfide Bond Formation in <i>Escherichia coli</i>
InTech eBooks, 2017
Disulfide bonds formed between a pair of oxidized cysteines are important to the structural integrity and proper folding of many proteins. Accordingly, Nature has evolved several systems for the genesis and maintenance of such bonds. Beginning with the discovery of protein disulfide isomerase, which provided the first evidence for enzyme-catalyzed disulfide-bond formation, many years of research have resulted in the explication of the complex network of electron transport pathways needed for this process. Herein, we take a historical approach in describing the elucidation of disulfide-bond formation in E. coli. We frame this topic in the context of genome sequencing eras. The first section describes the discovery of eukaryotic protein disulfide isomerase and the subsequent research that followed from the early 1960s to the early 1990s, a time period we have named the pre-genomic sequencing era. The second section details the renaissance in research on disulfide-bond formation in the periplasm of prokaryotes, fueled by bacterial genetic screens and the development of genomic sequencing technology. Accordingly, we have named this section the genomic sequencing era, which ranges from the early 1990s to approximately 2010. The final section outlines the use of bacterial genetic screens to select for new oxidoreductase enzymes and their potential uses in biotechnological and pharmaceutical applications. This era we have dubbed the post-genomic sequencing era, and we envision it to represent the future of research on oxidative folding.
A pathway for disulfide bond formation in vivo
Proceedings of the National Academy of Sciences, 1993
Protein disulfide bond formation in Escherichia coli requires the periplasmic protein DsbA. We describe here mutations in the gene for a second protein, DsbB, which is also necessary for disulfide bond formation. Evidence suggests that DsbB may act by reoxidizing DsbA, thereby regenerating its ability to donate its disulde bond to target proteins. We propose that DsbB, an integral membrane protein, may be involved in transducing redox potential across the cytoplasmic membrane.
An in vivo Pathway for Disulfide Bond Isomerization in Escherichia coli
Proceedings of The National Academy of Sciences, 1996
Biochemical studies have shown that the periplasmic protein disulfide oxidoreductase DsbC can isomerize aberrant disulfide bonds. Here we present the first evidence for an in vivo role of DsbC in disulfide bond isomerization. Furthermore, our data suggest that the enzymes DsbA and DsbC play distinct roles in the cell in disulfide bond formation and isomerization, respectively. We have shown that mutants in dsbC display a defect in disulfide bond formation specific for proteins with multiple disulfide bonds. The defect can be complemented by the addition of reduced dithiothreitol to the medium, suggesting that absence of DsbC results in accumulation of misoxidized proteins. Mutations in the dipZ and trxA genes have similar phenotypes. We propose that DipZ, a cytoplasmic membrane protein with a thioredoxin-like domain, and thioredoxin, the product of the trxA gene, are components of a pathway for maintaining DsbC active as a protein disulfide bond isomerase.
Catalysis of disulfide bond formation and isomerization in the Escherichia coli periplasm
Biochimica Et Biophysica Acta-molecular Cell Research, 2004
Disulfide bond formation is a catalyzed process in vivo. In prokaryotes, the oxidation of cysteine pairs is achieved by the transfer of disulfides from the highly oxidizing DsbA/DsbB catalytic machinery to substrate proteins. The oxidizing power utilized by this system comes from the membrane-embedded electron transport system, which utilizes molecular oxygen as a final oxidant. Proofreading of disulfide bond formation is performed by the DsbC/DsbD system, which has the ability to rearrange non-native disulfides to their native configuration. These disulfide isomerization reactions are sustained by a constant supply of reducing power provided by the cytoplasmic thioredoxin system, utilizing NADPH as the ultimate electron source. D
2011
Background: Disulfide bonds are one of the most common post-translational modifications found in proteins. The production of proteins that contain native disulfide bonds is challenging, especially on a large scale. Either the protein needs to be targeted to the endoplasmic reticulum in eukaryotes or to the prokaryotic periplasm. These compartments that are specialised for disulfide bond formation have an active catalyst for their formation, along with catalysts for isomerization to the native state. We have recently shown that it is possible to produce large amounts of prokaryotic disulfide bond containing proteins in the cytoplasm of wild-type bacteria such as E. coli by the introduction of catalysts for both of these processes.
Tuned Escherichia coli as a host for the expression of disulfide-rich proteins
Biotechnology Journal, 2011
Disulfide-bond formation is a major post-translational modification and is essential for protein folding, stability, and function. This is especially true for secreted proteins, many of which possess great potential for biotechnological applications. Focusing on the use of Escherichia coli for the production of this class of proteins, we describe the mechanisms that maintain redox compartmentalization in the cell, with an emphasis on those that promote the formation and isomerization of disulfide bonds in the bacterial periplasm, while presenting parallel pathways in the eukaryotic endoplasmic reticulum. Based on these concepts, we review the use of E. coli as a cell factory for the production of heterologous disulfide-containing proteins using either peri-or cytoplasmic expression and, in particular, how these compartments can be tuned to improve the yield of correctly folded recombinant proteins. Finally, we describe a few examples of the production of small disulfide-rich proteins (protease inhibitors) to illustrate how soluble, active, and fully oxidized recombinants may be successfully obtained upon peri-or cytoplasmic expression in E. coli.
1998
Cytoplasmic proteins do not generally contain structural disulfide bonds, although certain cytoplasmic enzymes form such bonds as part of their catalytic cycles. The disulfide bonds in these latter enzymes are reduced in Escherichia coli by two systems; the thioredoxin pathway and the glutathione/glutaredoxin pathway. However, structural disulfide bonds can form in proteins in the cytoplasm when the gene (trxB) for the enzyme thioredoxin reductase is inactivated by mutation. This disulfide bond formation can be detected by assessing the state of the normally periplasmic enzyme alkaline phosphatase (AP) when it is localized to the cytoplasm. Here we show that the formation of disulfide bonds in cytoplasmic AP in the trxB mutant is dependent on the presence of two thioredoxins in the cell, thioredoxins 1 and 2, the products of the genes trxA and trxC, respectively. Our evidence supports a model in which the oxidized forms of these thioredoxins directly catalyze disulfide bond formation in cytoplasmic AP, a reversal of their normal role. In addition, we show that the recently discovered thioredoxin 2 can perform many of the roles of thioredoxin 1 in vivo, and thus is able to reduce certain essential cytoplasmic enzymes. Our results suggest that the three most effective cytoplasmic disulfide-reducing proteins are thioredoxin 1, thioredoxin 2 and glutaredoxin 1; expression of any one of these is sufficient to support aerobic growth. Our results help to explain how the reducing environment in the cytoplasm is maintained so that disulfide bonds do not normally occur.
Production of disulfide-bonded proteins in Escherichia coli
Protein Expression and Purification, 2012
Disulfide bonds are covalent bonds formed post-translationally by the oxidation of a pair of cysteines. A disulfide bond can serve structural, catalytic, and signaling roles. However, there is an inherent problem to the process of disulfide bond formation: mis-pairing of cysteines can cause misfolding, aggregation and ultimately result in low yields during protein production. Recent developments in the understanding of the mechanisms involved in the formation of disulfide bonds have allowed the research community to engineer and develop methods to produce multi-disulfide-bonded proteins to high yields. This review attempts to highlight the mechanisms responsible for disulfide bond formation in Escherichia coli, both in its native periplasmic compartment in wild-type strains and in the genetically modified cytoplasm of engineered strains. The purpose of this review is to familiarize the researcher with the biological principles involved in the formation of disulfide-bonded proteins with the hope of guiding the scientist in choosing the optimum expression system.
Disulfide Bond Formation in the Periplasm of Escherichia coli
Ecosal plus, 2019
The formation of disulfide bonds is critical to the folding of many extracytoplasmic proteins in all domains of life. With the discovery in the early 1990s that disulfide bond formation is catalyzed by enzymes, the field of oxidative folding of proteins was born. Escherichia coli played a central role as a model organism for the elucidation of the disulfide bond-forming machinery. Since then, many of the enzymatic players and their mechanisms of forming, breaking, and shuffling disulfide bonds have become understood in greater detail. This article summarizes the discoveries of the past 3 decades, focusing on disulfide bond formation in the periplasm of the model prokaryotic host E. coli.