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J. Biol. Chem., Vol. 277, Issue 45, 43050-43057, November 8, 2002

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Randomization of the Entire Active-site Helix 1 of the Thiol-disulfide Oxidoreductase DsbA from Escherichia coli^*,

Björn Philipps and Rudi Glockshuber^§

From the Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, CH-8093 Zürich, Switzerland

DsbA from Escherichia coli is the most oxidizing member of the thiol-disulfide oxidoreductase family (E_o' = 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 pK_a value (3.4) of Cys³⁰, which is stabilized by the partial positive dipole charge of the active-site helix 1 (residues 30-37). We have randomized all non-cysteine residues of helix 1 (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 -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.

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