Six conserved cysteines of the membrane protein DsbD are required for the transfer of electrons from the cytoplasm to the periplasm of Escherichia coli.
The active-site cysteines of the Escherichia coli periplasmic protein disulfide bond isomerase (DsbC) are kept reduced by the cytoplasmic membrane protein, DsbD. DsbD, in turn, is reduced by cytoplasmic thioredoxin, indicating that DsbD transfers disulfidereducing potential from the cytoplasm to the periplasm. To understand the mechanism of this unusual mode of electron transfer, we have undertaken a genetic analysis of DsbD. In the process, we discovered that the previously suggested start site for the DsbD protein is incorrect. Our results permit the formulation of a model of DsbD membrane topology. Also, we show that six cysteines of DsbD conserved among DsbD homologs are essential for the reduction of DsbC, DsbG and for a reductive pathway leading to c-type cytochrome assembly in the periplasm. Our findings suggest a testable model for the DsbD-dependent transfer of electrons across the membrane, involving a cascade of disulfide bond reduction steps.
Stewart
E J
EJ
Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.
Katzen
F
F
Beckwith
J
J
D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D019897:Periplasm / Q000201:enzymology; D014176:Protein Biosynthesis; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins
D004220:Disulfides; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase; D019704:Protein Disulfide-Isomerases; D003545:Cysteine