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Cloning and active site mutagenesis of Vibrio cholerae DsbA, a periplasmic enzyme that catalyzes disulfide bond formation.

1993; Elsevier BV; Volume: 268; Issue: 6 Linguagem: Inglês

10.1016/s0021-9258(18)53613-9

1083-351X

Jiaao Yu, Stephen H. McLaughlin, Robert B. Freedman, Timothy R. Hirst,

Protein Structure and Dynamics

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 thioldisulfide oxidoreductases (Yu, J., Webb, H., and Hirst, T. R (1992) Mol.Microbiol.6, 1949Microbiol.6, -1958)).Here, we report the cloning of the dsbA gene of V. cholerae and the demonstration that the encoded periplasmic 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.' The abbreviations used are: EtxB, B subunit of heat-labile enterotoxin from E. coli; PDI,