ALTERATION OF SUBSTRATE-SPECIFICITY OF ZYMOMONAS-MOBILIS ALCOHOL DEHYDROGENASE-2 USING IN-VITRO RANDOM MUTAGENESIS

Random mutagenesis of the gene encoding Zymomonas mobilis alcohol dehy drogenase-a has enabled isolation of variants of the enzyme that have substrate specificities different from that of the wild-type enzyme. A fter amino acids responsible for the changes were identified, directed mutation at these sites was also carried out. Variants that are activ e 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-a ctivation phenomenon that is observed not only with ethanol, but also with a nonsubstrate alcohol, 2-propanol, and with low concentrations o f Triton X-100. These alterations to the alcohol binding pocket mainly introduce larger, more hydrophobic residues, suggesting that it is no t the size but the hydrophobicity of the pocket that affects the subst rate specificity. Variants active with NADP were isolated, and, as wit h similar variants of the yeast enzyme, they were found to have an Asp residue replaced by a neutral amino acid, However, unlike the yeast e xamples 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 fe rrous ion-activated, and inactive with zinc, attempts were made to fin d variants that had activity with zinc. One was found, but the screeni ng method also isolated other variants with altered metal ion preferen ces due to a mutation affecting amino acid 330. (C) 1997 Academic Pres s.