Randomization of the entire active-site helix alpha 1 of the thiol-disulfide oxidoreductase DsbA from Escherichia coli.
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 (Cys(30)-Pro(31)-His(32)-Cys(33)) is primarily due to an extremely low pK(a) value (3.4) of Cys(30), which is stabilized by the partial positive dipole charge of the active-site helix alpha1 (residues 30-37). We have randomized all non-cysteine residues of helix alpha1 (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 alpha-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.
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, CH-8093 Zürich, Switzerland.
Bjorn
B
Philipps
Rudi
R
Glockshuber
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D014644:Genetic Variation; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D019704:Protein Disulfide-Isomerases