In vitro and in vivo redox states of the Escherichia coli periplasmic oxidoreductases DsbA and DsbC.
DsbC is a periplasmic protein of Escherichia coli that was previously identified by a genetic selection that rescued sensitivity to dithiothreitol in Tn10 mutagenized cells. The Erwinia chrysanthemi dsbC gene was identified in a previous genetic screen to restore motility in a dsbA null strain. In order to analyze the biochemical role of E. coli DsbC, the protein was overexpressed, purified, and compared with DsbA in terms of disulfide isomerization, thiol oxidation, and in vivo redox state. In vitro, DsbC and DsbA have an equivalent kcat for disulfide isomerization with the model substrate, misfolded insulin-like growth factor-1. However, DsbA is a more effective oxidant than DsbC of protein dithiols. In vivo, DsbA is found exclusively in the oxidized state in wild-type strains grown in rich media. On the other hand, in vivo DsbC has one pair of cysteines oxidized and one pair reduced. DsbD is required to maintain this reduced pair of cysteines, confirming previous genetic results. A dsbC deletion strain showed decreases in the production of some, but not all, heterologous proteins containing multiple disulfide bonds. Notably, those proteins affected by the dsbC deletion do not have the cysteines paired consecutively.
Joly
J C
JC
Department of Fermentation and Cell Culture R&D, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, California 94080, USA. joly@gene.com
Swartz
J R
JR
D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D007334:Insulin-Like Growth Factor I / Q000737:chemistry / Q000378:metabolism; D007535:Isomerases / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding
D004220:Disulfides; D007334:Insulin-Like Growth Factor I; D007535:Isomerases; D019704:Protein Disulfide-Isomerases