Aerobic activity of Escherichia coli alcohol dehydrogenase is determined by a single amino acid

Expression of the alcohol dehydrogenase gene, adhE, in Escherichia coli is anaerobically regulated at both the transcriptional and the translational l evels. To study the AdhE protein, the adhE(+) structural gene was cloned in to expression vectors under the control of the lacZ and trp(c) promoters. W ild-type AdhE protein produced under aerobic conditions from these construc ts was inactive. Constitutive mutants (adhC) that produced high levels of A dhE under both aerobic and anaerobic conditions were previously isolated. W hen only the adhE structural gene from one of the adhC mutants was cloned i nto expression vectors, highly functional AdhE protein was isolated under b oth aerobic and anaerobic conditions. Sequence analysis revealed that the a dhE gene from the adhC mutant contained two mutations resulting in two amin o acid substitutions, Ala267Thr and Glu568Lys. Thus, adhC strains contain a promoter mutation and two mutations in the structural gene. The mutant str uctural gene from adhC strains was designated adhE*. Fragment exchange expe riments revealed that the substitution responsible for aerobic expression i n the adhE* clones is Glu568Lys. Genetic selection and site-directed mutage nesis experiments showed that virtually any amino acid substitution for Glu 568 produced AdhE that was active under both aerobic and anaerobic conditio ns. These findings suggest that adhE expression is also regulated posttrans lationally and that strict regulation of alcohol dehydrogenase activity in E. coli is physiologically significant.