PMID:12193604

Citation	Philipps, B and Glockshuber, R (2002) Randomization of the entire active-site helix alpha 1 of the thiol-disulfide oxidoreductase DsbA from Escherichia coli. J. Biol. Chem. 277:43050-7
Abstract	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.
Links	PubMed Online version:10.1074/jbc.M207638200
Keywords	Base Sequence; Binding Sites; Catalysis; Circular Dichroism; DNA Primers; Escherichia coli/enzymology; Genetic Variation; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Disulfide-Isomerases/chemistry; Protein Disulfide-Isomerases/genetics; Protein Disulfide-Isomerases/metabolism; Protein Structure, Secondary; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism
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