DsbA and DsbC affect extracellular enzyme formation in Pseudomonas aeruginosa

DsbA and DsbC proteins involved in the periplasmic formation of disulfide bonds in Pseudomonas aeruginosa were identified and shown to play an important role for the formation of extracellular enzymes. Mutants deficient in either dsbA or dsbC or both genes were constructed, and extracellular elastase, alkaline phosphatase, and lipase activities were determined. The dsbA mutant no longer produced these enzymes, whereas the lipase activity was doubled in the dsbC mutant. Also, extracellar lipase production was severely reduced in a P. aeruginosa dsbA mutant in which an inactive DsbA variant carrying the mutation C34S was expressed. Even when the lipase gene lipA was constitutively expressed in trans in a lipA dsbA double mutant, lipase activity in cell extracts and culture supernatants was still reduced to about 25%. Interestingly, the presence of dithiothreitol in the growth medium completely inhibited the formation of extracellular lipase whereas the addition of dithiothreitol to a cell-free culture supernatant did not affect lipase activity. We conclude that the correct formation of the disulfide bond catalyzed in vivo by DsbA is necessary to stabilize periplasmic lipase. Such a stabilization is the prerequisite for efficient secretion using the type II pathway.     

Heterospecific cloning of Arabidopsis thaliana cDNAs by direct complementation of pyrimidine auxotrophic mutants of Saccharomyces cerevisiae. I. Cloning and sequence analysis of two cDNAs catalysing the second,fifth and sixth steps of the de novo pyrimidine biosynthesis pathway

A one-step mutant of Escherichia coli K-12 lacking both glucose-1-phosphatase (Agp) and pH 2.5 acid phosphatase (AppA) activities in the periplasmic space was isolated. The mutation which mapped close to ch1B, at 87 min on the E. coli linkage map, also caused the loss of alkaline phosphatase (PhoA) activity, even when this activity was expressed from TnphoA fusions to genes encoding periplasmic or membrane proteins. A DNA fragment that complements the mutation was cloned and shown to carry the dsbA gene, which encodes a periplasmic disulphide bond-forming factor. The mutant had an ochre triplet in dsbA, truncating the protein at amino acid 70. Introduction of TnphoA fusions into a plasmid-borne dsbA gene resulted in DsbA-PhoA hybrid proteins that were all exported to the periplasmic space in both dsbA ⁺ and dsbA strains. They belong to three different classes, depending on the length of the DsbA fragment fused to PhoA. When PhoA was fused to an amino-terminal DsbA heptapeptide, the protein was only seen in the periplasm of a dsbA ⁺ strain, as in the case of wild-type PhoA. Hybrid proteins missing up to 29 amino acids at the carboxy-terminus of DsbA were stable and retained both the DsbA and PhoA activities. Those with shorter DsbA fragments that still carried the -Cys-ProHis-Cys-motif were rapidly degraded (no DsbA activity). The presence is discussed of a structural domain lying around amino acid 170 of DsbA and which is probably essential for its folding into a proteolytic-resistant and enzymatically active form.     

DsbB catalyzes disulfide bond formation de novo

DsbA and DsbB are responsible for disulfide bond formation. DsbA is the direct donor of disulfides, and DsbB oxidizes DsbA. DsbB has the unique ability to generate disulfides by quinone reduction. It is thought that DsbB oxidizes DsbA via thiol disulfide exchange. In this mechanism, a disulfide is formed across the N-terminal pair of cysteines (Cys-41/Cys-44) in DsbB by quinone reduction. This disulfide is then transferred on to the second pair of cysteine residues in DsbB (Cys-104/Cys-130) and then finally transferred to DsbA. We have shown here the redox potential of the two disulfides in DsbB are -271 and -284 mV, respectively, and considerably less oxidizing than the disulfide of DsbA at -120 mV. In addition, we have found the Cys-104/Cys-130 disulfide of DsbB to actually be a substrate for DsbA in vitro. These findings indicate that the disulfides in DsbB are unsuitable to function as the oxidant of DsbA. Furthermore, we have shown that mutants in DsbB that lack either pair or all of its cysteines are also capable of oxidizing DsbA. These unexpected findings raise the possibility that the oxidation of DsbA by DsbB does not occur via thiol disulfide exchange as is widely assumed but rather, directly via quinone reduction.     

Engineering antibody fragments to fold in the absence of disulfide bonds

Disulfide bonds play a critical role in the stabilization of the immunoglobulin β-sandwich sandwich. Under reducing conditions, such as those that prevail in the cytoplasm, disulfide bonds do not normally form and as a result most antibodies expressed in that compartment (intrabodies) accumulate in a misfolded and inactive state. We have developed a simple method for the quantitative isolation of antibody fragments that retain full activity under reducing conditions from large mutant libraries. In E. coli, inactivation of the cysteine oxidoreductase DsbA abolishes protein oxidation in the periplasm, which leads to the accumulation of scFvs and other disulfide-containing proteins in a reduced form. Libraries of mutant scFvs were tethered onto the inner membrane of dsbA cells and mutants that could bind fluorescently labeled antigen in the reducing periplasm were screened by Anchored Periplasmic Expression (APEx; Harvey et al., Proc Natl Acad Sci USA 2004;101:9193–9198.). Using this approach, we isolated scFv antibody variants that are fully active when expressed in the cytoplasm or when the four Cys residues that normally form disulfides are substituted by Ser residues.     

霍乱弧菌ToxS蛋白间的互作增强ToxR蛋白诱导毒力基因的表达

【目的】阐明霍乱弧菌ToxR蛋白功能调控的分子机制。【方法】利用巯基捕获(thiol-trapping)的方法分析DsbA蛋白对ToxR周质空间结构域半胱氨酸残基的氧化作用;采用定点突变的方法构建ToxR半胱氨酸突变株(ToxR_(C236/293S));利用荧光素酶基因作为报告基因分析ToxR野生型(ToxR_(wt))和半胱氨酸突变体(ToxR_(C236/293S))诱导下游基因表达的活性;通过细菌双杂交系统分析ToxR_(wt)和ToxR_(C236/293S)蛋白之间、ToxR与ToxS之间以及ToxS之间的相互作用。【结果】ToxR周质空间结构域半胱氨酸残基确实可以被DsbA蛋白氧化,且当ToxR与ToxS共表达时,ToxR诱导ctxAB转录表达的活性显著增强,且在dsbA基因缺失突变株中ToxR诱导ctxAB转录表达的活性更高;成功构建株霍乱弧菌ToxR半胱氨酸突变株(ToxR_(C236/293S)),在没有ToxS存在的条件下,ToxR_(C236/293S)诱导毒力基因表达的活性与ToxRwt相当;细菌双杂交系统分析发现当ToxR与ToxS共转录表达时,ToxS极大增强ToxR蛋白之间的互作;在dsbA基因缺失突变株中,ToxS之间的相互作用显著增强。【结论】ToxR蛋白本身的氧还状态对其诱导毒力基因表达的活性没有影响;ToxS通过增强ToxR形成二聚体的能力从而增强其诱导毒力基因的表达,而DsbA对ToxS蛋白之间的相互作用具有抑制作用,DsbA通过影响ToxS的蛋白互作从而影响ToxR蛋白的功能。本文为进一步阐明霍乱弧菌毒力基因表达调控的分子机制提供重要的理论依据。     

Differential effect of dsbA and dsbC mutations on extracellular enzyme secretion in Erwinia chrysanthemi

An Erwinia chrysanthemi gene able to complement an Escherichia coli dsbA mutation has been cloned and sequenced. This gene codes for a periplasmic protein with disulphide isomerase activity that has 69% identity and 94% similarity with the E. coli DsbA protein. An E. chrysanthemi dsbA-uidA fusion mutant has been constructed. dsbA expression seems to be constitutive. This mutant has multiple phenotypes resulting from the absence of disulphide bond formation in periplasmic and secreted proteins. Pectate lyases and the cellulase EGZ are rapidly degraded in the periplasm of the dsbA mutant. E. chrysanthemi synthesizes another periplasmic protein with disulphide isomerase activity, namely DsbC. The dsbC gene introduced on a multicopy plasmid in a dsbA mutant was only partially able to restore EGZ secretion, indicating that even if DsbA and DsbC possess disulphide oxydoreductase activity, they are not completely interchangeable. Moreover, pectate lyases expressed in an E. coli dsbA mutant were very instable but their stability was unaffected in a dsbC mutant. These results indicate that DsbA and DsbC could have different substrate specificities.     

Biochemical and Structural Study of the Homologues of the Thiol-Disulfide Oxidoreductase DsbA in Neisseria meningitidis

Bacterial virulence depends on the correct folding of surface-exposed proteins, a process catalyzed by the thiol-disulfide oxidoreductase DsbA, which facilitates the synthesis of disulfide bonds in Gram-negative bacteria. The Neisseria meningitidis genome possesses three genes encoding active DsbAs: DsbA1, DsbA2 and DsbA3. DsbA1 and DsbA2 have been characterized as lipoproteins involved in natural competence and in host interactive biology, while the function of DsbA3 remains unknown.This work reports the biochemical characterization of the three neisserial enzymes and the crystal structures of DsbA1 and DsbA3. As predicted by sequence homology, both enzymes adopt the classic Escherichia coli DsbA fold. The most striking feature shared by all three proteins is their exceptional oxidizing power. With a redox potential of − 80 mV, the neisserial DsbAs are the most oxidizing thioredoxin-like enzymes known to date. Consistent with these findings, thermal studies indicate that their reduced form is also extremely stable. For each of these enzymes, this study shows that a threonine residue found within the active-site region plays a key role in dictating this extraordinary oxidizing power. This result highlights how residues located outside the CXXC motif may influence the redox potential of members of the thioredoxin family.     

Randomization of the entire active-site helix alpha 1 of the thiol-disulfide oxidoreductase DsbA from Escherichia coli

DsbA from Escherichia coli is the most oxidizing member of the thiol-disulfide oxidoreductase family (E(o)' = -122 mV) and is required for efficient disulfide bond formation in the periplasm. The reactivity of the catalytic disulfide bond (Cys(30)-Pro(31)-His(32)-Cys(33)) is primarily due to an extremely low pK(a) value (3.4) of Cys(30), which is stabilized by the partial positive dipole charge of the active-site helix alpha1 (residues 30-37). We have randomized all non-cysteine residues of helix alpha1 (residues 31, 32, and 34-37) and found that two-thirds of the resulting variants complement DsbA deficiency in a dsbA deletion strain. Sequencing of 98 variants revealed a large number of non-conservative replacements in active variants, even at well conserved positions. This indicates that tertiary structure context strongly determines alpha-helical secondary structure formation of the randomized sequence. A subset of active and inactive variants was further characterized. All these variants were more reducing than wild type DsbA, but the redox potentials of active variants did not drop below -210 mV. All inactive variants had redox potentials lower than -210 mV, although some of the inactive proteins were still re-oxidized by DsbB. This demonstrates that efficient oxidation of substrate polypeptides is the crucial property of DsbA in vivo.     

The oxidase DsbA folds a protein with a nonconsecutive disulfide

One of the last unsolved problems of molecular biology is how the sequential amino acid information leads to a functional protein. Correct disulfide formation within a protein is hereby essential. We present periplasmic ribonuclease I (RNase I) from Escherichia coli as a new endogenous substrate for the study of oxidative protein folding. One of its four disulfides is between nonconsecutive cysteines. In general view, the folding of proteins with nonconsecutive disulfides requires the protein disulfide isomerase DsbC. In contrast, our study with RNase I shows that DsbA is a sufficient catalyst for correct disulfide formation in vivo and in vitro. DsbA is therefore more specific than generally assumed. Further, we show that the redox potential of the periplasm depends on the presence of glutathione and the Dsb proteins to maintain it at-165 mV. We determined the influence of this redox potential on the folding of RNase I. Under the more oxidizing conditions of dsb(-) strains, DsbC becomes necessary to correct non-native disulfides, but it cannot substitute for DsbA. Altogether, DsbA folds a protein with a nonconsecutive disulfide as long as no incorrect disulfides are formed.     

Spanin function requires subunit homodimerization through intermolecular disulfide bonds

The λ Rz and Rz1 proteins are the subunits of the spanin complex, required for the disruption of the outer membrane during host lysis. Rz, the inner membrane or i‐spanin, has a largely alpha‐helical periplasmic domain, whereas Rz1, the outer membrane or o‐spanin, has a 25% proline content with no predicted secondary structure. We report that both Rz and Rz1 accumulate as homodimers covalently linked by intermolecular disulfide bonds involving all three Cys residues, two in Rz and one in Rz1. Moreover, of these three intermolecular disulfides, spanin function requires the presence of at least one of the two linkages nearest the Rz–Rz1 C‐terminal interaction domains; i.e. either the Rz1–Rz1 disulfide or the distal Rz–Rz disulfide link. In a dsbC host, but not in dsbA or dsbA dsbC hosts, formation of the covalent homodimers of Rz is severely reduced and outer membrane disruption is significantly delayed, suggesting that the spanin pathway normally proceeds through DsbA‐mediated formation of an intramolecular disulfide in Rz. In contrast, efficient formation of the Rz1–Rz1 disulfide requires DsbA. Finally, Dsb‐independent formation of the covalent homodimer of either subunit requires the presence of the other, presumably as a template for close apposition of the thiols.     

Light-induced disulfide dimerization of recoverin under ex vivo and in vivo conditions

Despite vast knowledge of the molecular mechanisms underlying photochemical damage of photoreceptors, linked to progression of age-related macular degeneration, information on specific protein targets of the light-induced oxidative stress is scarce. Here, we demonstrate that prolonged intense illumination (halogen bulb, 1500 lx, 1–5 h) of mammalian eyes under ex vivo (cow) or in vivo (rabbit) conditions induces disulfide dimerization of recoverin, a Ca²⁺-dependent inhibitor of rhodopsin kinase. Western blotting and mass spectrometry analysis of retinal extracts reveals illumination time-dependent accumulation of disulfide homodimers of recoverin and its higher order disulfide cross-linked species, including a minor fraction of mixed disulfides with intracellular proteins (tubulins, etc.). Meanwhile, monomeric bovine recoverin remains mostly reduced. These effects are accompanied by accumulation of disulfide homodimers of visual arrestin. Histological studies demonstrate that the light-induced oxidation of recoverin and arrestin occurs in intact retina (illumination for 2 h), while illumination for 5 h is associated with damage of the photoreceptor layer. A comparison of ex vivo levels of disulfide homodimers of bovine recoverin with redox dependence of its in vitro thiol–disulfide equilibrium (glutathione redox pair) gives the lowest estimate of redox potential in rod outer segments under illumination from −160 to −155  mV. Chemical crosslinking and dynamic light scattering data demonstrate an increased propensity of disulfide dimer of bovine recoverin to multimerization/aggregation. Overall, the oxidative stress caused by the prolonged intense illumination of retina might affect rhodopsin desensitization via concerted disulfide dimerization of recoverin and arrestin. The developed herein models of eye illumination are useful for studies of the light-induced thiol oxidation of visual proteins.     

Characterization of Escherichia coli thioredoxin variants mimicking the active-sites of other thiol/disulfide oxidoreductases.

Thiol/disulfide oxidoreductases like thioredoxin, glutaredoxin, DsbA, or protein disulfide isomerase (PDI) share the thioredoxin fold and a catalytic disulfide bond with the sequence Cys-Xaa-Xaa-Cys (Xaa corresponds to any amino acid). Despite their structural similarities, the enzymes have very different redox properties, which is reflected by a 100,000-fold difference in the equilibrium constant (K(eq)) with glutathione between the most oxidizing member, DsbA, and the most reducing member, thioredoxin. Here we present a systematic study on a series of variants of thioredoxin from Escherichia coli, in which the Xaa-Xaa dipeptide was exchanged by that of glutaredoxin, PDI, and DsbA. Like the corresponding natural enzymes, all thioredoxin variants proved to be stronger oxidants than the wild-type, with the order wild-type < PDI-type < DsbA-type < glutaredoxin-type. The most oxidizing, glutaredoxin-like variant has a 420-fold decreased value of K(eq), corresponding to an increase in redox potential by 75 mV. While oxidized wild-type thioredoxin is more stable than the reduced form (delta deltaG(ox/red) = 16.9 kJ/mol), both redox forms have almost the same stability in the variants. The pH-dependence of the reactivity with the alkylating agent iodoacetamide proved to be the best method to determine the pKa value of thioredoxin's nucleophilic active-site thiol (Cys32). A pKa of 7.1 was measured for Cys32 in the reduced wild-type. All variants showed a lowered pKa of Cys32, with the lowest value of 5.9 for the glutaredoxin-like variant. A correlation of redox potential and the Cys32 pKa value could be established on a quantitative level. However, the predicted correlation between the measured delta deltaG(ox/red) values and Cys32 pKa values was only qualitative.     

Going through the Barrier: COUPLED DISULFIDE EXCHANGE REACTIONS PROMOTE EFFICIENT CATALYSIS IN QUIESCIN SULFHYDRYL OXIDASE*

The quiescin sulfhydryl oxidase (QSOX) family of enzymes generates disulfide bonds in peptides and proteins with the reduction of oxygen to hydrogen peroxide. Determination of the potentials of the redox centers in Trypanosoma brucei QSOX provides a context for understanding catalysis by this facile oxidant of protein thiols. The CXXC motif of the thioredoxin domain is comparatively oxidizing (E′₀ of −144 mV), consistent with an ability to transfer disulfide bonds to a broad range of thiol substrates. In contrast, the proximal CXXC disulfide in the ERV (essential for respiration and vegetative growth) domain of TbQSOX is strongly reducing (E′₀ of −273 mV), representing a major apparent thermodynamic barrier to overall catalysis. Reduction of the oxidizing FAD cofactor (E′₀ of −153 mV) is followed by the strongly favorable reduction of molecular oxygen. The role of a mixed disulfide intermediate between thioredoxin and ERV domains was highlighted by rapid reaction studies in which the wild-type CGAC motif in the thioredoxin domain of TbQSOX was replaced by the more oxidizing CPHC or more reducing CGPC sequence. Mixed disulfide bond formation is accompanied by the generation of a charge transfer complex with the flavin cofactor. This provides thermodynamic coupling among the three redox centers of QSOX and avoids the strongly uphill mismatch between the formal potentials of the thioredoxin and ERV disulfides. This work identifies intriguing mechanistic parallels between the eukaryotic QSOX enzymes and the DsbA/B system catalyzing disulfide bond generation in the bacterial periplasm and suggests that the strategy of linked disulfide exchanges may be exploited in other catalysts of oxidative protein folding.     

Redox properties of protein disulfide isomerase (DsbA) from Escherichia coli.

The redox properties of periplasmic protein disulfide isomerase (DsbA) from Escherichia coli were analyzed by measuring the equilibrium constant of the oxidation of reduced DsbA by oxidized glutathione. The experiments are based on the finding that the intrinsic tryptophan fluorescence of DsbA increases about threefold upon reduction of the enzyme, which can be explained by the catalytic disulfide bridge quenching the fluorescence of a neighboring tryptophan residue. From the specific fluorescence of DsbA equilibrated in the presence of different ratios of reduced and oxidized glutathione at pH 7, an equilibrium constant of 1.2 x 10(-4) M was determined, corresponding to a standard redox potential (E'0) of DsbA of -0.089 V. Thus, DsbA is a significantly stronger oxidant than cytoplasmic thioredoxins and its redox properties are similar to those of eukaryotic protein disulfide isomerase. The equilibrium constants for the DsbA/glutathione equilibrium were found to be strongly dependent on pH and varied from 2.5 x 10(-3) M to 3.9 x 10(-5) M between pH 4 and 8.5. The redox state-dependent fluorescence properties of DsbA should allow detailed physicochemical studies of the enzyme as well as the quantitative determination of the oxidized protein by fluorescence titration with dithiothreitol and open the possibility to observe bacterial protein disulfide isomerase "at work" during catalysis of oxidative protein folding.     

Glutamine and KGF each regulate extracellular thiol/disulfide redox and enhance proliferation in Caco-2 cells

Glutamine (Gln) and keratinocyte growth factor (KGF) each stimulate intestinal epithelial cell growth, but regulatory mechanisms are not well understood. We determined whether Gln and KGF alter intra- and extracellular thiol/disulfide redox pools in Caco-2 cells cultured in oxidizing or reducing cell medium and whether such redox variations are a determinant of proliferative responses to these agents. Cells were cultured over a physiological range of oxidizing to reducing extracellular thiol/disulfide redox (Eh) conditions, obtained by varying cysteine (Cys) and cystine (CySS) concentrations in cell medium. Cell proliferation was determined by 5-bromo-2-deoxyuridine (BrdU) incorporation. Gln (10 mmol/l) or KGF (10 microg/l) did not alter BrdU incorporation at reducing Eh (-131 to -150 mV), but significantly increased incorporation at more oxidizing Eh (Gln at 0 to -109 mV; KGF at -46 to -80 mV). Cellular glutathione/glutathione disulfide (GSH/GSSG) Eh was unaffected by Gln, KGF, or variations in extracellular Cys/CySS Eh. Control cells largely maintained extracellular Eh at initial values after 24 h (-36 to -136 mV). However, extracellular Eh shifted toward a narrow physiological range with Gln and KGF treatment (Gln -56 to -88 mV and KGF -76 to -92 mV, respectively; P < 0.05 vs. control). The results indicate that thiol/disulfide redox state in the extracellular milieu is an important determinant of Caco-2 cell proliferation induced by Gln and KGF, that this control is independent of intracellular GSH redox status, and that both Gln and KGF enhance the capability of Caco-2 cells to modulate extremes of extracellular redox.     

Identification of a protein required for disulfide bond formation in vivo

We describe a mutation (dsbA) that renders Escherichia coli severely defective in disulfide bond formation. In dsbA mutant cells, pulse-labeled beta-lactamase, alkaline phosphatase, and OmpA are secreted but largely lack disulfide bonds. These disulfideless proteins may represent in vivo folding intermediates, since they are protease sensitive and chase slowly into stable oxidized forms. The dsbA gene codes for a 21,000 Mr periplasmic protein containing the sequence cys-pro-his-cys, which resembles the active sites of certain disulfide oxidoreductases. The purified DsbA protein is capable of reducing the disulfide bonds of insulin, an activity that it shares with these disulfide oxidoreductases. Our results suggest that disulfide bond formation is facilitated by DsbA in vivo.     

FrnE,a Cadmium-Inducible Protein in Deinococcus radiodurans,Is Characterized as a Disulfide Isomerase Chaperone In Vitro and for Its Role in Oxidative Stress Tolerance In Vivo

Deinococcus radiodurans R1 exposed to a lethal dose of cadmium shows differential expression of a large number of genes, including frnE (drfrnE) and some of those involved in DNA repair and oxidative stress tolerance. The drfrnE::nptII mutant of D. radiodurans showed growth similar to that of the wild type, but its tolerance to 10 mM cadmium and 10 mM diamide decreased by ∼15- and ∼3-fold, respectively. These cells also showed nearly 6 times less resistance to gamma radiation at 12 kGy and ∼2-fold-higher sensitivity to 40 mM hydrogen peroxide than the wild type. In trans expression of drFrnE increased cytotoxicity of dithiothreitol (DTT) in the dsbA mutant of Escherichia coli. Recombinant drFrnE showed disulfide isomerase activity and could maintain insulin in its reduced form in the presence of DTT. While an equimolar ratio of wild-type protein could protect malate dehydrogenase completely from thermal denaturation at 42°C, the C22S mutant of drFrnE provided reduced protection to malate dehydrogenase from thermal inactivation. These results suggested that drFrnE is a protein disulfide isomerase in vitro and has a role in oxidative stress tolerance of D. radiodurans possibly by protecting the damaged cellular proteins from inactivation.     

Legionella pneumophila utilizes a single‐player disulfide‐bond oxidoreductase system to manage disulfide bond formation and isomerization

Legionella pneumophila uses a single homodimeric disulfide bond (DSB) oxidoreductase DsbA2 to catalyze extracytoplasmic protein folding and to correct DSB errors through protein‐disulfide isomerase (PDI) activity. In Escherichia coli, these functions are separated to avoid futile cycling. In L. pneumophila, DsbA2 is maintained as a mixture of disulfides (S‐S) and free thiols (SH), but when expressed in E. coli, only the SH form is observed. We provide evidence to suggest that structural differences in DsbB oxidases (LpDsbB1 and LpDsbB2) and DsbD reductases (LpDsbD1 and LpDsbD2) (compared with E. coli) permit bifunctional activities without creating a futile cycle. LpdsbB1 and LpdsbB2 partially complemented an EcdsbB mutant while neither LpdsbD1 nor LpdsbD2 complemented an EcdsbD mutant unless DsbA2 was also expressed. When the dsb genes of E. coli were replaced with those of L. pneumophila, motility was restored and DsbA2 was present as a mixture of redox forms. A dominant‐negative approach to interfere with DsbA2 function in L. pneumophila determined that DSB oxidase activity was necessary for intracellular multiplication and assembly/function of the Dot/Icm Type IVb secretion system. Our studies show that a single‐player system may escape the futile cycle trap by limiting transfer of reducing equivalents from LpDsbDs to DsbA2.     

The name's bond......disulfide bond

A repeating theme in the structural biology of disulfide oxidants and isomerases is the extraordinary architectural similarity between functionally related proteins from prokaryotes and eukaryotes. The recently determined structure of full-length yeast protein disulfide isomerase (PDI) reveals a U-shaped molecule with two redox-active sites. It bears a remarkable resemblance to the V-shaped, but dimeric, bacterial disulfide isomerases DsbC and DsbG. Similarly, the much-anticipated structure of the bacterial membrane protein DsbB, the redox partner of DsbA, comprises a flexible redox loop embedded in an antiparallel four-helix bundle. This architecture is similar to that of soluble eukaryotic Ero1p and Erv2p proteins, the redox partners of PDI. Importantly, the DsbB crystal structure is a complex with DsbA, providing our first view of the molecular interactions between these two proteins.