The influence of His94 and Pro149 in modulating the activity of V-cholerae DsbA

DsbA is the primary catalyst of disulfide bond formation in the periplasm of gram-negative bacteria. Numerous theoretical and experimental studies have been undertaken to determine the molecular mechanisms by which DsbA acts as a potent oxidant, whereas the homologous cytoplasmic protein, thioredoxin, acts as a reductant. Many of these studies have focused on the nature of the two residues that lie between the active-site cysteines. Although these are clearly important, they are not solely responsible for the differences in activity between these thiol-disulfide oxidoreductases. Q97 in the helical domain of E. coli DsbA has been implicated in influencing the redox potential of E. coli DsbA. In V cholerae DsbA, the analogous residue is H94. In this study, the effect of H94 on the oxidase activity of DsbA is examined, along with the role of the conserved cis-proline residue P149. The DsbA mutant H94L shows a nearly fourfold increase in activity over the wild-type enzyme. To our knowledge, this is the first time an increase in the normal activity of a thiol-disulfide oxidoreductase has been reported. Potential reasons for this increase in activity are discussed.