The Escherichia coli dsbC (xprA) gene encodes a periplasmic protein involved in disulfide bond formation.
We have identified and functionally characterized a new Escherichia coli gene, dsbC, whose product is involved in disulfide bond formation in the periplasmic space. It corresponds to a previously sequenced open reading frame mapping upstream of recJ with no previously assigned function. Null mutations in dsbC were obtained using a screen for dithiothreitol (DTT)-sensitive mutants and were shown to result in the accumulation of reduced forms of a variety of disulfide bond-containing periplasmic proteins. This defect could be rescued by the addition of either oxidized DTT or cystine or by multicopy expression of dsbA, a known periplasmic disulfide oxidase. The DsbC protein is synthesized as a precursor form of 25.5 kDa which is processed to a 23.3 kDa mature species located in the periplasmic space. The DsbC protein was overexpressed, purified to homogeneity and shown to catalyse the reduction of insulin in a DTT-dependent manner at levels comparable with those of purified DsbA. The replacement of either cysteine residue of the predicted active site, F-(X4)-C-G-Y-C, completely inactivates DsbC protein function. We have further shown that in vivo overexpression of DsbC can functionally substitute for a loss of DsbA function. Taken together, all of our results demonstrate that DsbC acts in vivo as a disulfide oxidase.
Missiakas
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Département de Biochimie Médicale, Centre Médical Universitaire, Genève, Switzerland.
Georgopoulos
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Raina
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004220:Disulfides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D007535:Isomerases / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011490:Protein Disulfide Reductase (Glutathione) / Q000235:genetics* / Q000378:metabolism; D019704:Protein Disulfide-Isomerases; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D004220:Disulfides; D008565:Membrane Proteins; D011490:Protein Disulfide Reductase (Glutathione); D007535:Isomerases; D019704:Protein Disulfide-Isomerases