Differential in vivo roles played by DsbA and DsbC in the formation of protein disulfide bonds.

Several Escherichia coli proteins participate in protein disulfide bond formation. Among them, DsbA is the primary factor that oxidizes target cysteines. Biochemical evidence indicates that DsbC has disulfide isomerization activity. To study intracellular functions of DsbA and DsbC, we used an alkaline phosphatase mutant, PhoA[SCCC], with the most amino-terminal cysteine replaced by serine. It was found that the remaining 3 cysteines in PhoA[SCCC] form a disulfide bond of incorrect as well as correct combinations. An aberrant disulfide bond was preferentially formed in wild-type cells, which was converted slowly to the normal disulfide bond. This conversion did not occur in the dsbC-disrupted cells. Overproduction of DsbC stimulated the formation of the correct disulfide bond. In contrast, the inefficiently formed disulfide bonds in the dsbA-disrupted cells, and the more efficiently formed disulfide bonds in the same strain in the presence of oxidized glutathione were mostly in the correct form. These results suggest that the DsbA-catalyzed reaction can be too rapid for some proteins. DsbA may simply oxidize available pairs of cysteines, which happen to be in an incorrect combination in the case of PhoA[SCCC]. In contrast, DsbC stimulates the formation of correct disulfide bonds and corrects previously introduced aberrant ones. Thus, DsbC acts to isomerize disulfide bonds in vivo.