A single dipeptide sequence modulates the redox properties of a whole enzyme family

Background: Disulfide exchange reactions are catalyzed by thiol/disulfide oxidoreductases. These enzymes possess a thioredoxin fold and contain a catalytic disulfide with the sequence Cys–X–X–Cys at the N terminus of an α helix. Despite these similarities, the various members differ strongly in their redox potentials (-122 mV to -270 mV). Using the strong oxidant DsbA from Escherichia coli as a model system, we investigated whether the redox properties of these enzymes can be modulated rationally by exchange of the X–X dipeptide.

Results: The X–X dipeptide of DsbA (Cys30–Pro31–His32–Cys33) was exchanged by the dipeptides of eukaryotic protein disulfide isomerase (PDI; Gly–His), glutaredoxin (Pro–Tyr), and thioredoxin (Gly–Pro) from E. coli. All variants were less oxidizing than wild-type DsbA and their redox potentials were in the order of the related natural enzymes (DsbA > PDI > glutaredoxin > thioredoxin). The equilibrium constant between glutathione and the thioredoxin-like variant increased 1200-fold compared with wild-type DsbA. The variants also showed a strong increase in the pK_a of the nucleophilic cysteine (Cys30). As for glutaredoxin and thioredoxin, the catalytic disulfide stabilized the corresponding variants while destabilizing wild-type DsbA and the PDI-like variant.

Conclusions: The X–X dipeptide in the active site of thiol/disulfide oxidoreductases appears to be the main determinant of the redox properties of these enzymes. This empirical finding should be very useful for the design of new thiol/disulfide oxidoreductases with altered redox potentials and for studying the function of these enzymes in vivo.