Identification and characterization of a new disulfide isomerase-like protein (DsbD) in Escherichia coli.
Previous studies have established that DsbA and DsbC, periplasmic proteins of Escherichia coli, are two key players involved in disulfide bond formation. A search for extragenic mutations able to compensate for the lack of dsbA function in vivo led us to the identification of a new gene, designated dsbD. Lack of DsbD protein leads to some, but not all, of the phenotypic defects observed with other dsb mutations, such as hypersensitivity to dithiothreitol and to benzylpenicillin. In addition, unlike the rest of the dsb genes, dsbD is essential for bacterial growth at temperatures above 42 degrees C. Cloning of the wild-type gene and sequencing and overexpression of the protein show that dsbD is part of an operon and encodes an inner membrane protein. A 138 amino acid subdomain of the protein was purified and shown to possess an oxido-reductase activity in vitro. Expressing this subdomain in the periplasmic space helped restore the phenotypic defects associated with a dsbD null mutation. Interestingly, this domain shares 45% identity with the portion of the eukaryotic protein disulfide isomerase carrying the active site. We further show that in dsbD mutant bacteria the dithiol active sites of DsbA and DsbC proteins are mostly oxidized, as compared with wild-type bacteria. Our results argue that DsbD generates a reducing source in the periplasm, which is required for maintaining proper redox conditions. The finding that overexpression of DsbD leads to a Dsb- phenotype, very similar to that exhibited by dsbA null mutants, is in good agreement with such a model.
Missiakas
D
D
Département de Biochimie Médicale, Centre Médical Universitaire, Switzerland.
Schwager
F
F
Raina
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007535:Isomerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D019704:Protein Disulfide-Isomerases; D014158:Transcription, Genetic
D017931:DNA Primers; D010088:Oxidoreductases; D007535:Isomerases; D019704:Protein Disulfide-Isomerases