Differential in Vivo Roles Played by DsbA and DsbC in the Formation of Protein Disulfide Bonds

Sone, Michio; Akiyama, Yoshinori; Ito, Koreaki

doi:10.1074/jbc.272.16.10349

Cited by 94 publications

(70 citation statements)

References 33 publications

(35 reference statements)

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“…There is some evidence supporting this model: when CcrA, a metallo-␤-lactamase from Bacteroides fragilis that normally contains no disulfides, is expressed in the E. coli periplasm, DsbA introduces a non-native disulfide bond (29). Additionally, DsbA introduces a nonnative disulfide bond into a mutant version of alkaline phosphatase that is missing its first cysteine (30). Here we show that although both copper and DsbA catalyze disulfide bond formation in vivo, DsbA appears to form correct disulfide bonds more frequently than copper does.…”

Section: Figure 2 Dsbc Isomerizes Copper-catalyzed Non-native Disulfmentioning

confidence: 96%

Copper Stress Causes an in Vivo Requirement for the Escherichia coli Disulfide Isomerase DsbC

Hiniker

Collet

Bardwell

2005

Journal of Biological Chemistry

151

173

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In Escherichia coli, the periplasmic disulfide oxidoreductase DsbA is thought to be a powerful but nonspecific oxidant, joining cysteines together the moment they enter the periplasm. DsbC, the primary disulfide isomerase, likely resolves incorrect disulfides. Given the reliance of protein function on correct disulfide bonds, it is surprising that no phenotype has been established for null mutations in dsbC. Here we demonstrate that mutations in the entire DsbC disulfide isomerization pathway cause an increased sensitivity to the redox-active metal copper. We find that copper catalyzes periplasmic disulfide bond formation under aerobic conditions and that copper catalyzes the formation of disulfide-bonded oligomers in vitro, which DsbC can resolve. Our data suggest that the copper sensitivity of dsbC ؊ strains arises from the inability of the cell to rearrange copper-catalyzed non-native disulfides in the absence of functional DsbC. Absence of functional DsbA augments the deleterious effects of copper on a dsbC ؊ strain, even though the dsbA ؊ single mutant is unaffected by copper. This may indicate that DsbA successfully competes with copper and forms disulfide bonds more accurately than copper does. These findings lead us to a model in which DsbA may be significantly more accurate in disulfide oxidation than previously thought, and in which the primary role of DsbC may be to rearrange incorrect disulfide bonds that are formed during certain oxidative stresses.Most periplasmic Escherichia coli proteins contain at least two cysteine residues and many are stable and active only when these cysteines form their native disulfide bond pairings (1). In E. coli, a family of thioldisulfide oxidoreductases ensures that periplasmic and secreted proteins form correct disulfide bonds. DsbA is the primary disulfide oxidant in the periplasm. It rapidly donates its disulfide directly to substrate proteins and oxidizes them (2). DsbA is believed to act as a relatively nonspecific oxidant, joining any two cysteines that approach each other (3). A dsbA Ϫ strain shows several in vivo phenotypes, including attenuated virulence and loss of motility, because of the absence of disulfide bonds in proteins involved in these pathways (4, 5). DsbC, a second periplasmic thiol-disulfide oxidoreductase, appears to function as a disulfide isomerase both in vitro and in vivo. In vitro, DsbC has been shown to rearrange non-native disulfides in well studied isomerization substrates such as BPTI and RNase A (6, 7). In vivo, DsbC is required for full activity of a handful of proteins containing at least one non-consecutive disulfide bond (1). We have found that the periplasmic proteins RNase I (four disulfides, one non-consecutive) and MepA (three disulfides, two non-consecutive) require DsbC for their stability and, in the case of RNase I, in vivo activity (1). Berkmen et al. (3) recently showed that the folding of Agp (three consecutive disulfides) becomes DsbC-dependent with the introduction of a non-consecutive disulfide bond. These results s...

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Section: Figure 2 Dsbc Isomerizes Copper-catalyzed Non-native Disulfmentioning

confidence: 96%

Copper Stress Causes an in Vivo Requirement for the Escherichia coli Disulfide Isomerase DsbC

Hiniker

Collet

Bardwell

2005

Journal of Biological Chemistry

151

173

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“…Motility of various strains of B. cepacia and E. coli was slightly rescued by dsbA on pMA507. The level of alkaline phosphatase of an E. coli dsbA mutant is low (Table 3) because the formation of two intramolecular disulfide bonds and the correct folding of the enzyme in the periplasmic space is impaired (34). The enzyme activity was fully restored by the introduction of either E. coli dsbA or B. cepacia dsbA.…”

Section: Sequencing the Dsba Genementioning

confidence: 97%

The DsbA‐DsbB Disulfide Bond Formation System of Burkholderia cepacia Is Involved in the Production of Protease and Alkaline Phosphatase, Motility, Metal Resistance, and Multi‐Drug Resistance

Hayashi

Abe

Kimoto

et al. 2000

Microbiology and Immunology

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Abstract:In Once considered as a phytopathogen, Burkholderia cepacia, a multi-drug-resistant bacteria, is now recognized as an important pathogen in the lung of patients with cystic fibrosis (CF) and can lead to severe pneumonia and death (15). B. cepacia strain KF1 isolated from a non-CF patient with pneumonia, produces extracellular metalloprotease in large quantities (37), and causes lung infections in mice on intratracheal inoculation (42). We have been analyzing the mechanism of protease production to explore its involvement in the pathogenesis.In a previous study, we showed that the protease-negative, lipase-positive mutant KFT1007, a Tn5-Tp insertion mutant of B. cepacia KF1, was impaired in the dsbB gene which encodes a membranebound disulfide bond oxidoreductase, DsbB, resulting in secretion of a premature and catalytically inactive form of protease (1). In Escherichia coli, DsbB couples with DsbA, a periplasmic disulfide bond oxidoreductase that directly makes S-S bonds on target molecules (26). Thus, the reduced-DsbA is reoxidized by oxidized-DsbB that is recycled with the aid of a respiratory electron transfer chain (14). The DsbA-DsbB circuit in E. coli is involved in flagellar basal body assembly (9), type IV pilin biogenesis (44), and the maturation of heat-stable enterotoxin (22) and alkaline phosphatase (10). In some Gram-negative bacteria, homologs of E. coli DsbA and their target proteins are found in Vibrio cholerae TcpG for enterotoxin (23)

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“…The oxidation of protein cysteines by DsbA is very rapid but often results in the formation of incorrect disulfide bonds. The rearrangement of nonnative disulfides is catalyzed primarily by the dimeric periplasmic enzyme DsbC (8,14,15). For DsbC to be able to catalyze disulfide bond isomerization, its active site Cys-X-XCys sequence must be present in the dithiol form.…”

mentioning

confidence: 99%

In Vivo and in Vitro Function of theEscherichia coli Periplasmic Cysteine Oxidoreductase DsbG

Bessette

Cotto

Gilbert

et al. 1999

Journal of Biological Chemistry

162

180

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We have characterized in vivo and in vitro the recently identified DsbG from Escherichia coli. In addition to sharing sequence homology with the thiol disulfide exchange protein DsbC, DsbG likewise was shown to form a stable periplasmic dimer, and it displays an equilibrium constant with glutathione comparable with DsbA and DsbC. DsbG was found to be expressed at approximately 25% the level of DsbC. In contrast to earlier results (Andersen, C. L., Matthey-Dupraz, A., Missiakas, D., and Raina, S. (1997) Mol. Microbiol. 26, 121-132), we showed that dsbG is not essential for growth and that dsbG null mutants display no defect in folding of multiple disulfide-containing heterologous proteins. Overexpression of DsbG, however, was able to restore the ability of dsbC mutants to express heterologous multidisulfide proteins, namely bovine pancreatic trypsin inhibitor, a protein with three disulfides, and to a lesser extent, mouse urokinase (12 disulfides). As in DsbC, the putative active site thiols in DsbG are completely reduced in vivo in a dsbD-dependent fashion, as would be expected if DsbG is acting as a disulfide isomerase or reductase. However, the latter is not likely because DsbG could not catalyze insulin reduction in vitro. Overall, our results indicate that DsbG functions primarily as a periplasmic disulfide isomerase with a narrower substrate specificity than DsbC.

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Differential in Vivo Roles Played by DsbA and DsbC in the Formation of Protein Disulfide Bonds

Cited by 94 publications

References 33 publications

Copper Stress Causes an in Vivo Requirement for the Escherichia coli Disulfide Isomerase DsbC

Copper Stress Causes an in Vivo Requirement for the Escherichia coli Disulfide Isomerase DsbC

The DsbA‐DsbB Disulfide Bond Formation System of Burkholderia cepacia Is Involved in the Production of Protease and Alkaline Phosphatase, Motility, Metal Resistance, and Multi‐Drug Resistance

In Vivo and in Vitro Function of theEscherichia coli Periplasmic Cysteine Oxidoreductase DsbG

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