Evolution of the adhE gene product of Escherichia coli from a functional reductase to a dehydrogenase - Genetic and biochemical studies of the mutantproteins

The multifunctional AdhE protein of Escherichia coli (encoded by the adhE g ene) physiologically catalyzes the sequential reduction of acetyl-CoA to ac etaldehyde and then to ethanol under fermentative conditions. The NH2-termi nal region of the AdhE protein is highly homologous to aldehyde:NAD(+) oxid oreductases, whereas the COOH-terminal region is homologous to a family of Fe2+-dependent ethanol:NAD(+) oxidoreductases. This fusion protein also fun ctions as a pyruvate formate lyase deactivase. E. coli cannot grow aerobica lly on ethanol as the sole carbon and energy source because of inadequate r ate of adhE transcription and the vulnerability of the AdhE protein to meta l-catalyzed oxidation. In this study, we characterized 16 independent two-s tep mutants with acquired and improved aerobic growth ability on ethanol, T he AdhE proteins in these mutants catalyzed the sequential oxidation of eth anol to acetaldehyde and to acetyl-CoA All first stage mutants grew on etha nol with a doubling time of about 240 min. Sequence analysis of a randomly chosen mutant revealed an Ala-267 --> Thr substitution in the acetaldehyde: NAD(+) oxidoreductase domain of AdhE. All second stage mutants grew on etha nol with a doubling time of about 90 min, and all of them produced an AdhE( E568K)(A267T/), Purified AdhE(A267T) and AdhE(A267T/E568K) showed highly el evated acetaldehyde dehydrogenase activities. It therefore appears that whe n AdhE catalyzes the two sequential reactions in the counter-physiological direction, acetaldehyde dehydrogenation is the rate-limiting step. Both mut ant proteins were more thermosensitive than the wild-type protein, but AdhE (A267T/E568K) was more thermal stable than AdhE(A267T). Since both mutant e nzymes exhibited similar kinetic properties, the second mutation probably c onferred an increased growth rate on ethanol by stabilizing AdhE(A267T).