Conversion of a catalytic into a structural disulfide bond by circular permutation.

The thiol-disulfide oxidoreductase DsbA from Escherichia coli is the strongest oxidant of the enzyme family and required for disulfide bond formation in the bacterial periplasm. The catalytic domain of this 189-residue protein has a thioredoxin-like fold and contains a catalytic disulfide bridge that is located within the sequence Cys30-Pro31-His32-Cys33 at the N-terminus of an alpha-helix. The Cys30-Cys33 disulfide bond destabilizes DsbA by about 16 kJ/mol at pH 7.0, which appears to be caused by the extremely low pKa value of approximately 3.4 of the nucleophilic Cys30 thiol. Here we report the characterization of a circularly permuted variant of DsbA, termed H32-P31, in which the natural termini are connected by a Gly3-Thr-Gly linker and the new termini are located between the active-site cysteines (first residue His32, last residue Pro31). The disulfide bond in the variant thus connects the second with the penultimate residue. H32-P31 adopts a wild-type-like structure and folds reversibly and cooperatively in both redox forms. However, the permuted variant is catalytically inactive as dithiol oxidase in vivo and in vitro. Both cysteine thiols have pKa values > 8; the variant is 500-fold more reducing than the wild type and more stable in its oxidized form. Thus, the Cys30-Cys33 disulfide in the variant H32-P31 has adopted properties of a structural disulfide bond.