Distinct enzymatic strategies for de novo generation of disulfide bonds in membranes.

Disulfide bond formation is a catalyzed reaction essential for the folding and stability of proteins in the secretory pathway. In prokaryotes, disulfide bonds are generated by DsbB or VKOR homologs that couple the oxidation of a cysteine pair to quinone reduction. Vertebrate VKOR and VKOR-like enzymes have gained the epoxide reductase activity to support blood coagulation. The core structures of DsbB and VKOR variants share the architecture of a four-transmembrane-helix bundle that supports the coupled redox reaction and a flexible region containing another cysteine pair for electron transfer. Despite considerable similarities, recent high-resolution crystal structures of DsbB and VKOR variants reveal significant differences. DsbB activates the cysteine thiolate by a catalytic triad of polar residues, a reminiscent of classical cysteine/serine proteases. In contrast, bacterial VKOR homologs create a hydrophobic pocket to activate the cysteine thiolate. Vertebrate VKOR and VKOR-like maintain this hydrophobic pocket and further evolved two strong hydrogen bonds to stabilize the reaction intermediates and increase the quinone redox potential. These hydrogen bonds are critical to overcome the higher energy barrier required for epoxide reduction. The electron transfer process of DsbB and VKOR variants uses slow and fast pathways, but their relative contribution may be different in prokaryotic and eukaryotic cells. The quinone is a tightly bound cofactor in DsbB and bacterial VKOR homologs, whereas vertebrate VKOR variants use transient substrate binding to trigger the electron transfer in the slow pathway. Overall, the catalytic mechanisms of DsbB and VKOR variants have fundamental differences.

Warfarin analogs target disulfide bond-forming enzymes and suggest a residue important for quinone and coumarin binding.

Disulfide bond formation has a central role in protein folding of both eukaryotes and prokaryotes. In bacteria, disulfide bonds are catalyzed by DsbA and DsbB/VKOR enzymes. First, DsbA, a periplasmic disulfide oxidoreductase, introduces disulfide bonds into substrate proteins. Then, the membrane enzyme, either DsbB or VKOR, regenerate DsbA's activity by the formation of de novo disulfide bonds which reduce quinone. We have previously performed a high-throughput chemical screen and identified a family of warfarin analogs that target either bacterial DsbB or VKOR. In this work, we expressed functional human VKORc1 in Escherichia coli and performed a structure-activity-relationship analysis to study drug selectivity between bacterial and mammalian enzymes. We found that human VKORc1 can function in E. coli by removing two positive residues, allowing the search for novel anticoagulants using bacteria. We also found one warfarin analog capable of inhibiting both bacterial DsbB and VKOR and a second one antagonized only the mammalian enzymes when expressed in E. coli. The difference in the warfarin structure suggests that substituents at positions three and six in the coumarin ring can provide selectivity between the bacterial and mammalian enzymes. Finally, we identified the two amino acid residues responsible for drug binding. One of these is also essential for de novo disulfide bond formation in both DsbB and VKOR enzymes. Our studies highlight a conserved role of this residue in de novo disulfide-generating enzymes and enable the design of novel anticoagulants or antibacterials using coumarin as a scaffold.

Development of a sensor for disulfide bond formation in diverse bacteria.

In bacteria, disulfide bonds contribute to the folding and stability of proteins important for processes in the cellular envelope. In Escherichia coli, disulfide bond formation is catalyzed by DsbA and DsbB enzymes. DsbA is a periplasmic protein that catalyzes disulfide bond formation in substrate proteins, while DsbB is an inner membrane protein that transfers electrons from DsbA to quinones, thereby regenerating the DsbA active state. Actinobacteria including mycobacteria use an alternative enzyme named VKOR, which performs the same function as DsbB. Disulfide bond formation enzymes, DsbA and DsbB/VKOR, represent novel drug targets because their inhibition could simultaneously affect the folding of several cell envelope proteins including virulence factors, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. We have previously developed a cell-based and target-based assay to identify molecules that inhibit the DsbB and VKOR in pathogenic bacteria, using E. coli cells expressing a periplasmic ß-Galactosidase sensor (ß-Galdbs), which is only active when disulfide bond formation is inhibited. Here, we report the construction of plasmids that allows fine-tuning of the expression of the ß-Galdbs sensor and can be mobilized into other gram-negative organisms. As an example, when expressed in Pseudomonas aeruginosa UCBPP-PA14, which harbors two DsbB homologs, ß-Galdbs behaves similarly as in E. coli, and the biosensor responds to the inhibition of the two DsbB proteins. Thus, these ß-Galdbs reporter plasmids provide a basis to identify novel inhibitors of DsbA and DsbB/VKOR in multidrug-resistant gram-negative pathogens and to further study oxidative protein folding in diverse gram-negative bacteria. IMPORTANCE: Disulfide bonds contribute to the folding and stability of proteins in the bacterial cell envelope. Disulfide bond-forming enzymes represent new drug targets against multidrug-resistant bacteria because inactivation of this process would simultaneously affect several proteins in the cell envelope, including virulence factors, toxins, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. Identifying the enzymes involved in disulfide bond formation in gram-negative pathogens as well as their inhibitors can contribute to the much-needed antibacterial innovation. In this work, we developed sensors of disulfide bond formation for gram-negative bacteria. These tools will enable the study of disulfide bond formation and the identification of inhibitors for this crucial process in diverse gram-negative pathogens.

Revisiting long-chain fatty acid metabolism in Escherichia coli: integration with stress responses.

Long-chain fatty acids (LCFAs) are a tremendous source of metabolic energy, an essential component of membranes, and important effector molecules that regulate a myriad of cellular processes. As an energy-rich nutrient source, the role of LCFAs in promoting bacterial survival and infectivity is well appreciated. LCFA degradation generates a large number of reduced cofactors that may confer redox stress; therefore, it is imperative to understand how bacteria deal with this paradoxical situation. Although the LCFA utilization pathway has been studied in great detail, especially in Escherichia coli, where the earliest studies date back to the 1960s, the interconnection of LCFA degradation with bacterial stress responses remained largely unexplored. Recent work in E. coli shows that LCFA degradation induces oxidative stress and also impedes oxidative protein folding. Importantly, both issues arise due to the insufficiency of ubiquinone, a lipid-soluble electron carrier in the electron transport chain. However, to maintain redox homeostasis, bacteria induce sophisticated cellular responses. Here, we review these findings in light of our current knowledge of the LCFA metabolic pathway, metabolism-induced oxidative stress, the process of oxidative protein folding, and stress combat mechanisms. We discuss probable mechanisms for the activation of defense players during LCFA metabolism and the likely feedback imparted by them. We suggest that besides defending against intrinsic stresses, LCFA-mediated upregulation of stress response pathways primes bacteria to adapt to harsh external environments. Collectively, the interplay between LCFA metabolism and stress responses is likely an important factor that underlies the success of LCFA-utilizing bacteria in the host.

Bacterial Periplasmic Oxidoreductases Control the Activity of Oxidized Human Antimicrobial ß-Defensin 1.

The antimicrobial peptide human ß-defensin 1 (hBD1) is continuously produced by epithelial cells in many tissues. Compared to other defensins, hBD1 has only minor antibiotic activity in its native state. After reduction of its disulfide bridges, however, it becomes a potent antimicrobial agent against bacteria, while the oxidized native form (hBD1ox) shows specific activity against Gram-negative bacteria. We show that the killing mechanism of hBD1ox depends on aerobic growth conditions and bacterial enzymes. We analyzed the different activities of hBD1 using mutants of Escherichia coli lacking one or more specific proteins of their outer membrane, cytosol, or redox systems. We discovered that DsbA and DsbB are essential for the antimicrobial activity of hBD1ox but not for that of reduced hBD1 (hBD1red). Furthermore, our results strongly suggest that hBD1ox uses outer membrane protein FepA to penetrate the bacterial periplasm space. In contrast, other bacterial proteins in the outer membrane and cytosol did not modify the antimicrobial activity. Using immunogold labeling, we identified the localization of hBD1ox in the periplasmic space and partly in the outer membrane of E. coli However, in resistant mutants lacking DsbA and DsbB, hBD1ox was detected mainly in the bacterial cytosol. In summary, we discovered that hBD1ox could use FepA to enter the periplasmic space, where its activity depends on presence of DsbA and DsbB. HBD1ox concentrates in the periplasm in Gram-negative bacteria, which finally leads to bleb formation and death of the bacteria. Thus, the bacterial redox system plays an essential role in mechanisms of resistance against host-derived peptides such as hBD1.

The Disulfide Bond Formation Pathway Is Essential for Anaerobic Growth of Escherichia coli.

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.

Targeting Bacterial Dsb Proteins for the Development of Anti-Virulence Agents.

Recent years have witnessed a dramatic increase in bacterial antimicrobial resistance and a decline in the development of novel antibiotics. New therapeutic strategies are urgently needed to combat the growing threat posed by multidrug resistant bacterial infections. The Dsb disulfide bond forming pathways are potential targets for the development of antimicrobial agents because they play a central role in bacterial pathogenesis. In particular, the DsbA/DsbB system catalyses disulfide bond formation in a wide array of virulence factors, which are essential for many pathogens to establish infections and cause disease. These redox enzymes are well placed as antimicrobial targets because they are taxonomically widespread, share low sequence identity with human proteins, and many years of basic research have provided a deep molecular understanding of these systems in bacteria. In this review, we discuss disulfide bond catalytic pathways in bacteria and their significance in pathogenesis. We also review the use of different approaches to develop inhibitors against Dsb proteins as potential anti-virulence agents, including fragment-based drug discovery, high-throughput screening and other structure-based drug discovery methods.

Disulfide bond formation in prokaryotes: history, diversity and design.

The formation of structural disulfide bonds is essential for the function and stability of a great number of proteins, particularly those that are secreted. There exists a variety of dedicated cellular catalysts and pathways from archaea to humans that ensure the formation of native disulfide bonds. In this review we describe the initial discoveries of these pathways and report progress in recent years in our understanding of the diversity of these pathways in prokaryotes, including those newly discovered in some archaea. We will also discuss the various successful efforts to achieve laboratory-based evolution and design of synthetic disulfide bond formation machineries in the bacterium Escherichia coli. These latter studies have also led to new more general insights into the redox environment of the cytoplasm and bacterial cell envelope. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.

Rv2969c, essential for optimal growth in Mycobacterium tuberculosis, is a DsbA-like enzyme that interacts with VKOR-derived peptides and has atypical features of DsbA-like disulfide oxidases.

The bacterial disulfide machinery is an attractive molecular target for developing new antibacterials because it is required for the production of multiple virulence factors. The archetypal disulfide oxidase proteins in Escherichia coli (Ec) are DsbA and DsbB, which together form a functional unit: DsbA introduces disulfides into folding proteins and DsbB reoxidizes DsbA to maintain it in the active form. In Mycobacterium tuberculosis (Mtb), no DsbB homologue is encoded but a functionally similar but structurally divergent protein, MtbVKOR, has been identified. Here, the Mtb protein Rv2969c is investigated and it is shown that it is the DsbA-like partner protein of MtbVKOR. It is found that it has the characteristic redox features of a DsbA-like protein: a highly acidic catalytic cysteine, a highly oxidizing potential and a destabilizing active-site disulfide bond. Rv2969c also has peptide-oxidizing activity and recognizes peptide segments derived from the periplasmic loops of MtbVKOR. Unlike the archetypal EcDsbA enzyme, Rv2969c has little or no activity in disulfide-reducing and disulfide-isomerase assays. The crystal structure of Rv2969c reveals a canonical DsbA fold comprising a thioredoxin domain with an embedded helical domain. However, Rv2969c diverges considerably from other DsbAs, including having an additional C-terminal helix (H8) that may restrain the mobility of the catalytic helix H1. The enzyme is also characterized by a very shallow hydrophobic binding surface and a negative electrostatic surface potential surrounding the catalytic cysteine. The structure of Rv2969c was also used to model the structure of a paralogous DsbA-like domain of the Ser/Thr protein kinase PknE. Together, these results show that Rv2969c is a DsbA-like protein with unique properties and a limited substrate-binding specificity.

Protein Disulfide Exchange by the Intramembrane Enzymes DsbB, DsbD, and CcdA.

The formation of disulfide bonds in proteins is an essential process in both prokaryotes and eukaryotes. In gram-negative bacteria including Escherichia coli, the proteins DsbA and DsbB mediate the formation of disulfide bonds in the periplasm. DsbA acts as the periplasmic oxidant of periplasmic substrate proteins. DsbA is reoxidized by transfer of reducing equivalents to the 4 TM helix membrane protein DsbB, which transfers reducing equivalents to ubiquinone or menaquinone. Multiple structural studies of DsbB have provided detailed structural information on intermediates in the process of DsbB catalyzed oxidation of DsbA. These structures and the insights gained are described. In proteins with more than one pair of Cys residues, there is the potential for formation of non-native disulfide bonds, making it necessary for the cell to have a mechanism for the isomerization of such non-native disulfide bonds. In E. coli, this is mediated by the proteins DsbC and DsbD. DsbC reduces mis-formed disulfide bonds. The eight-TM-helix protein DsbD reduces DsbC and is itself reduced by cytoplasmic thioredoxin. DsbD also contributes reducing equivalents for the reduction of cytochrome c to facilitate heme attachment. The DsbD functional homolog CcdA is a six-TM-helix membrane protein that provides reducing equivalents for the reduction of cytochrome c. A recent structure determination of CcdA has provided critical insights into how reducing equivalents are transferred across the membrane that likely also provides understanding how this is achieved by DsbD as well. This structure and the insights gained are described.

Novel Sequence Features of DNA Repair Genes/Proteins from Deinococcus Species Implicated in Protection from Oxidatively Generated Damage.

Deinococcus species display a high degree of resistance to radiation and desiccation due to their ability to protect critical proteome from oxidatively generated damage; however, the underlying mechanisms are not understood. Comparative analysis of DNA repair proteins reported here has identified 22 conserved signature indels (CSIs) in the proteins UvrA1, UvrC, UvrD, UvsE, MutY, MutM, Nth, RecA, RecD, RecG, RecQ, RecR, RuvC, RadA, PolA, DnaE, LigA, GyrA and GyrB, that are uniquely shared by all/most Deinococcus homologs. Of these CSIs, a 30 amino acid surface-exposed insert in the Deinococcus UvrA1, which distinguishes it from all other UvrA homologs, is of much interest. The uvrA1 gene in Deinococcus also exhibits specific genetic linkage (predicted operonic arrangement) to genes for three other proteins including a novel Deinococcus-specific transmembrane protein (designated dCSP-1) and the proteins DsbA and DsbB, playing central roles in protein disulfide bond formation by oxidation-reduction of CXXC (C represents cysteine, X any other amino acid) motifs. The CXXC motifs provide important targets for oxidation damage and they are present in many DNA repair proteins including five in UvrA, which are part of Zinc-finger elements. A conserved insert specific for Deinococcus is also present in the DsbA protein. Additionally, the uvsE gene in Deinococcus also shows specific linkage to the gene for a membrane-associated protein. To account for these novel observations, a model is proposed where specific interaction of the Deinococcus UvrA1 protein with membrane-bound dCSP-1 enables the UvrA1 to receive electrons from DsbA-DsbB oxido-reductase machinery to ameliorate oxidation damage in the UvrA1 protein.

Phylogeny of the Vitamin K 2,3-Epoxide Reductase (VKOR) Family and Evolutionary Relationship to the Disulfide Bond Formation Protein B (DsbB) Family.

In humans and other vertebrate animals, vitamin K 2,3-epoxide reductase (VKOR) family enzymes are the gatekeepers between nutritionally acquired K vitamins and the vitamin K cycle responsible for posttranslational modifications that confer biological activity upon vitamin K-dependent proteins with crucial roles in hemostasis, bone development and homeostasis, hormonal carbohydrate regulation and fertility. We report a phylogenetic analysis of the VKOR family that identifies five major clades. Combined phylogenetic and site-specific conservation analyses point to clade-specific similarities and differences in structure and function. We discovered a single-site determinant uniquely identifying VKOR homologs belonging to human pathogenic, obligate intracellular prokaryotes and protists. Building on previous work by Sevier et al. (Protein Science 14:1630), we analyzed structural data from both VKOR and prokaryotic disulfide bond formation protein B (DsbB) families and hypothesize an ancient evolutionary relationship between the two families where one family arose from the other through a gene duplication/deletion event. This has resulted in circular permutation of primary sequence threading through the four-helical bundle protein folds of both families. This is the first report of circular permutation relating distant a-helical membrane protein sequences and folds. In conclusion, we suggest a chronology for the evolution of the five extant VKOR clades.

Topological plasticity of enzymes involved in disulfide bond formation allows catalysis in either the periplasm or the cytoplasm.

The transmembrane enzymes disulfide bond forming enzyme B (DsbB) and vitamin K epoxide reductase (VKOR) are central to oxidative protein folding in the periplasm of prokaryotes. Catalyzed formation of structural disulfide bonds in proteins also occurs in the cytoplasm of some hyperthermophilic prokaryotes through currently, poorly defined mechanisms. We aimed to determine whether DsbB and VKOR can be inverted in the membrane with retention of activity. By rational design of inversion of membrane topology, we engineered DsbB mutants that catalyze disulfide bond formation in the cytoplasm of Escherichia coli. This represents the first engineered inversion of a transmembrane protein with demonstrated conservation of activity and substrate specificity. This successful designed engineering led us to identify two naturally occurring and oppositely oriented VKOR homologues from the hyperthermophile Aeropyrum pernix that promote oxidative protein folding in the periplasm or cytoplasm, respectively, and hence defines the probable route for disulfide bond formation in the cytoplasm of hyperthermophiles. Our findings demonstrate how knowledge on the determinants of membrane protein topology can be used to de novo engineer a metabolic pathway and to unravel an intriguingly simple evolutionary scenario where a new "adaptive" cellular process is constructed by means of membrane protein topology inversion.