Alteration of substrate specificity of Zymomonas mobilis alcohol dehydrogenase-2 using in vitro random mutagenesis.
Random mutagenesis of the gene encoding Zymomonas mobilis alcohol dehydrogenase-2 has enabled isolation of variants of the enzyme that have substrate specificities different from that of the wild-type enzyme. After amino acids responsible for the changes were identified, directed mutation at these sites was also carried out. Variants that are active on butanol have been investigated in detail. Changes at residue 161 and other changes at residues 155 and 165 cause enhanced activity with longer-chain alcohols. The 165 change also induces a marked alcohol-activation phenomenon that is observed not only with ethanol, but also with a nonsubstrate alcohol, 2-propanol, and with low concentrations of Triton X-100. These alterations to the alcohol binding pocket mainly introduce larger, more hydrophobic residues, suggesting that it is not the size but the hydrophobicity of the pocket that affects the substrate specificity. Variants active with NADP were isolated, and, as with similar variants of the yeast enzyme, they were found to have an Asp residue replaced by a neutral amino acid. However, unlike the yeast examples in which the affinity was substantially reduced, the affinity for NAD+ in these variants was little changed, and the affinity for NADP+ was higher than that for NAD+. As this enzyme is naturally ferrous ion-activated, and inactive with zinc, attempts were made to find variants that had activity with zinc. One was found, but the screening method also isolated other variants with altered metal ion preferences due to a mutation affecting amino acid 330.
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School of Biochemistry, La Trobe University, Melbourne, Victoria, Australia.
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D020001:1-Butanol; D000426:Alcohol Dehydrogenase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D000440:Butanols / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D014644:Genetic Variation; D007527:Isoenzymes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D019216:Metals, Heavy / Q000494:pharmacology; D016296:Mutagenesis; D009249:NADP / Q000378:metabolism; D013379:Substrate Specificity; D017355:Zymomonas / Q000201:enzymology*
D000440:Butanols; D002413:Cations, Divalent; D007527:Isoenzymes; D019216:Metals, Heavy; D009249:NADP; D020001:1-Butanol; D000426:Alcohol Dehydrogenase