Randomization of the entire active-site helix α1 of the thiol-disulfide oxidoreductase DsbA from Escherichia coli

Abstract

DsbA from Escherichia coli is the most oxidizing member of the thiol-disulfide oxidoreductase family (Eo′ = -122 mV) and is required for efficient disulfide bond formation in the periplasm. The reactivity of the catalytic disulfide bond (Cys30-Pro31-His32-Cys33) is primarily due to an extremely low pKa value (3.4) of Cys30, which is stabilized by the partial positive dipole charge of the active-site helix α1 (residues 30-37). We have randomized all non-cysteine residues of helix α1 (residues 31, 32, and 34-37) and found that two-thirds of the resulting variants complement DsbA deficiency in a dsbA deletion strain. Sequencing of 98 variants revealed a large number of non-conservative replacements in active variants, even at well conserved positions. This indicates that tertiary structure context strongly determines α-helical secondary structure formation of the randomized sequence. A subset of active and inactive variants was further characterized. All these variants were more reducing than wild type DsbA, but the redox potentials of active variants did not drop below -210 mV. All inactive variants had redox potentials lower than -210 mV, although some of the inactive proteins were still re-oxidized by DsbB. This demonstrates that efficient oxidation of substrate polypeptides is the crucial property of DsbA in vivo.