Cloning and active site mutagenesis of Vibrio cholerae DsbA, a periplasmic enzyme that catalyzes disulfide bond formation.
Recently, a gene (dsbA) involved in the biogenesis of secreted oligomeric enterotoxins in Vibrio cholerae was described, which encodes an exported protein possessing a -Cys-Pro-His-Cys- motif similar to that found in the active sites of eukaryotic and prokaryotic thiol-disulfide oxidoreductases (Yu, J., Webb, H., and Hirst, T. R (1992) Mol. Microbiol. 6, 1949-1958). Here, we report the cloning of the dsbA gene of V. cholerae and the demonstration that the encoded periphlasmic enzyme has disulfide isomerase-like activity. Oligonucleotide-directed mutagenesis of either of the 2 Cys residues to Ala in the putative active site of DsbA abolished both its isomerase activity and its capacity to promote enterotoxin biogenesis. We conclude that the Cys residues constitute the active site domain of DsbA and are essential for its activity in vivo and in vitro.
Yu
J
J
Biological Laboratory, University of Kent, Canterbury, United Kingdom.
McLaughlin
S
S
Freedman
R B
RB
Hirst
T R
TR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D004220:Disulfides / Q000378:metabolism*; D006801:Humans; D007535:Isomerases / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019704:Protein Disulfide-Isomerases; D017386:Sequence Homology, Amino Acid; D014734:Vibrio cholerae / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D004220:Disulfides; D007535:Isomerases; D019704:Protein Disulfide-Isomerases