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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.

Vitamin K epoxide reductase (VKOR) generates vitamin K hydroquinone to sustain γ-carboxylation of many blood coagulation factors. Here, we report the 3.6 Å crystal structure of a bacterial homologue of VKOR from Synechococcus sp. The structure shows VKOR in complex with its naturally fused redox partner, a thioredoxin-like domain, and corresponds to an arrested state of electron transfer. The catalytic core of VKOR is a four transmembrane helix bundle that surrounds a quinone, connected through an additional transmembrane segment with the periplasmic thioredoxin-like domain. We propose a pathway for how VKOR uses electrons from cysteines of newly synthesized proteins to reduce a quinone, a mechanism confirmed by in vitro reconstitution of vitamin K-dependent disulphide bridge formation. Our results have implications for the mechanism of the mammalian VKOR and explain how mutations can cause resistance to the VKOR inhibitor warfarin, the most commonly used oral anticoagulant. Structure of a warfarin target Mammalian vitamin K epoxide reductase (VKOR) catalyses the generation of vitamin K hydroquinone, a decisive step in the vitamin K cycle that is required to sustain blood coagulation. The X-ray crystal structure of a bacterial homologue of VKOR has now been determined. It shows VKOR in a complex with its redox partner, a thioredoxin-like domain, and corresponds to an arrested state of electron transfer. This points to a possible mechanism by which VKOR uses electrons from newly synthesized proteins to reduce the quinone. This work may help explain how mutations to VKOR cause resistance to warfarin, the ubiquitous anticoagulant that acts by inhibiting VKOR. The γ-carboxylation of many blood coagulation factors relies on the generation of vitamin K hydroquinone by the enzyme vitamin K epoxide reductase (VKOR), of which the anticoagulant warfarin is an inhibitor. Here, the X-ray crystal structure of a bacterial homologue of VKOR is presented; the results have implications for the mechanism of action of mammalian VKOR and explain how mutations can cause warfarin resistance.

Abstract Disulfide bonds formed between pairs of cysteines are important features of the structure of many proteins. Elaborate electron transfer pathways have evolved Escherichia coli to promote the formation of these covalent bonds and to ensure that the correct pairs of cysteines are joined in the final folded protein. These transfers of electrons consist, in the main, of cascades of disulfide bond formation or reduction steps between a series of proteins (DsbA, DsbB, DsbC, and DsbD). A surprising variety of mechanisms and protein structures are involved in carrying out these steps.

Disulfide bonds in proteins play various important roles. They are either formed as structural features to stabilize the protein or are found only transiently as part of a catalytic or regulatory cycle. In vivo, the formation and reduction of disulfide bonds is catalyzed by specialized thiol-disulfide exchanging enzymes that contain an active site with the sequence motif Cys-X-X-Cys. These proteins have structurally evolved to catalyze predominantly either oxidative reactions or reductive steps. There is mounting evidence that, in addition to the thiol redox potential, the spatial distribution within different cell compartments and the overall redox state of the cell are equally important. In the cytoplasm, multiple pathways play overlapping roles in the reduction of disulfide bonds and additionally, the expression of several components of thiol-redox pathways was shown to respond to the changes in the cellular thiol-redox equilibrium. In the periplasm, two systems coexist, one catalyzing thiol oxidation and the other disulfide reduction. Recent results suggest that two different mechanisms are used to translocate reducing power from the cytoplasm or to dissipate the electrons after oxidation.

β-lactam antibiotics inhibit penicillin binding proteins (PBPs) involved in peptidoglycan synthesis. Although inhibition of peptidoglycan biosynthesis is generally thought to induce cell lysis, the pattern and mechanism of cell lysis can vary substantially. β-lactams that inhibit FtsI, the only division specific PBP, block cell division and result in growth as filaments. These filaments ultimately lyse through a poorly understood mechanism. Here we find that one such β-lactam, cephalexin, can, under certain conditions, lead instead to rapid lysis at nascent division sites through a process that requires the complete and ordered assembly of the divisome, the essential machinery involved in cell division. We propose that this assembly process (in which the localization of cell wall hydrolases depends on properly targeted FtsN, which in turn depends on the presence of FtsI) ensures that the biosynthetic machinery to form new septa is in place before the machinery to degrade septated daughter cells is enabled. β-lactams that target FtsI subvert this mechanism by inhibiting FtsI without perturbing the normal assembly of the cell division machinery and the consequent activation of cell wall hydrolases. One seemingly paradoxical implication of our results is that β-lactam therapy may be improved by promoting active cell division.

A screen for compounds that inhibit disulfide bond formation in β-galactosidase in Escherichia coli found inhibitors of the membrane enzyme DsbB. Given the importance of DsbB in bacterial virulence, the inhibitors are potentially useful as antibacterials. In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond. These proteins include numerous bacterial virulence factors, and thus bacterial enzymes that promote disulfide bond formation represent targets for compounds inhibiting bacterial virulence. Here, we describe a new target- and cell-based screening methodology for identifying compounds that inhibit the disulfide bond–forming enzymes Escherichia coli DsbB ( Ec DsbB) or Mycobacterium tuberculosis VKOR ( Mtb VKOR), which can replace Ec DsbB, although the two are not homologs. Initial screening of 51,487 compounds yielded six specifically inhibiting Ec DsbB. These compounds share a structural motif and do not inhibit Mtb VKOR. A medicinal chemistry approach led us to select related compounds, some of which are much more effective DsbB inhibitors than those found in the screen. These compounds inhibit purified DsbB and prevent anaerobic growth of E. coli . Furthermore, these compounds inhibit all but one of the DsbBs of nine other Gram-negative pathogenic bacteria tested.

Numerous aerobic bacteria depend on glutathione but are lacking the first enzyme in its biosynthetic pathway. An evolutionary experiment identifies a likely natural route to compensate for this loss through mutations in two enzymes in proline biosynthesis. The first and highly conserved step in glutathione (GSH) biosynthesis is formation of γ-glutamyl cysteine by the enzyme glutamate-cysteine ligase (GshA). However, bioinformatic analysis revealed that many prokaryotic species that encode GSH-dependent proteins lack the gene for this enzyme. To understand how bacteria cope without gshA , we isolated Escherichia coli Δ gshA multigenic suppressors that accumulated physiological levels of GSH. Mutations in both proB and proA , the first two genes in L -proline biosynthesis, provided a new pathway for γ-glutamyl cysteine formation via the selective interception of ProB-bound γ-glutamyl phosphate by amino acid thiols, likely through an S-to-N acyl shift mechanism. Bioinformatic analysis suggested that the L -proline biosynthetic pathway may have a second role in γ-glutamyl cysteine formation in prokaryotes. Also, we showed that this mechanism could be exploited to generate cytoplasmic redox buffers bioorthogonal to GSH.

DsbA, a thioredoxin superfamily member, introduces disulfide bonds into newly translocated proteins. This process is thought to occur via formation of mixed disulfide complexes between DsbA and its substrates. However, these complexes are difficult to detect, probably because of their short-lived nature. Here we show that it is possible to detect such covalent intermediates in vivo by a mutation in DsbA that alters cis proline-151. Further, this mutant allowed us to identify substrates of DsbA. Alteration of the cis proline, highly conserved among thioredoxin superfamily members, may be useful for the detection of substrates and intermediate complexes in other systems.

The Escherichia coli transcription factor OxyR is activated by the formation of an intramolecular disulfide bond and subsequently is deactivated by enzymatic reduction of the disulfide bond. Here we show that OxyR can be activated by two possible pathways. In mutants defective in the cellular disulfide-reducing systems, OxyR is constitutively activated by a change in the thiol--disulfide redox status in the absence of added oxidants. In wild-type cells, OxyR is activated by hydrogen peroxide. By monitoring the presence of the OxyR disulfide bond after exposure to hydrogen peroxide in vivo and in vitro, we also show that the kinetics of OxyR oxidation by low concentrations of hydrogen peroxide is significantly faster than the kinetics of OxyR reduction, allowing for transient activation in an overall reducing environment. We propose that the activity of OxyR in vivo is determined by the balance between hydrogen peroxide levels and the cellular redox environment.

Blood coagulation in humans requires the activity of vitamin K epoxide reductase (VKOR), the target of the anticoagulant warfarin (Coumadin). Bacterial homologs of VKOR were recently found to participate in a pathway leading to disulfide bond formation in secreted proteins of many bacteria. Here we show that the VKOR homolog from the bacterium Mycobacterium tuberculosis, the causative agent of human tuberculosis, is inhibited by warfarin and that warfarin-resistant mutations of mycobacterial VKOR appear in similar locations to mutations found in human patients who require higher doses of warfarin. Deletion of VKOR results in a severe growth defect in mycobacteria, and the growth of M. tuberculosis is inhibited by warfarin. The bacterial VKOR homolog may represent a target for antibiotics and a model for genetic studies of human VKOR. We present a simple assay in Escherichia coli, based on a disulfide-sensitive ß-galactosidase, which can be used to screen for stronger inhibitors of the M. tuberculosis VKOR homolog.