IN-VITRO AND IN-VIVO REDOX STATES OF THE ESCHERICHIA-COLI PERIPLASMICOXIDOREDUCTASES DSBA AND DSBC

DsbC is a periplasmic protein of Escherichia coli that was previously identified by a genetic selection that rescued sensitivity to dithioth reitol in Tn10 mutagenized cells, The Erwinia chrysanthemi dsbC gene w as identified in a previous genetic screen to restore motility in a ds bA null strain. In order to analyze the biochemical role of E. coli Ds bC, 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 k(cat) for disulfide isomerization with the model substrate, misfolded insulinlike growth factor-1. However, DsbA is a more effective oxidant than DsbC of prote in 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 D sbC has one pair of cysteines oxidized and one pair reduced, DsbD is r equired to maintain this reduced pair of cysteines, confirming previou s genetic results, A dsbC deletion strain showed decreases in the prod uction of some, but not all, heterologous proteins containing multiple disulfide bonds, Notably, those proteins affected by the dsbC deletio n do not have the cysteines paired consecutively.