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Titolo:
Evolution of the adhE gene product of Escherichia coli from a functional reductase to a dehydrogenase - Genetic and biochemical studies of the mutantproteins
Autore:
Membrillo-Hernandez, J; Echave, P; Cabiscol, E; Tamarit, J; Ros, J; Lin, ECC;
Indirizzi:
Univ Lleida, Fac Med, Dept Ciencies Med Basiques, Lleida 25198, Spain UnivLleida Lleida Spain 25198 iencies Med Basiques, Lleida 25198, Spain Harvard Univ, Sch Med, Dept Microbiol & Mol Genet, Boston, MA 02115 USA Harvard Univ Boston MA USA 02115 robiol & Mol Genet, Boston, MA 02115 USA
Titolo Testata:
JOURNAL OF BIOLOGICAL CHEMISTRY
fascicolo: 43, volume: 275, anno: 2000,
pagine: 33869 - 33875
SICI:
0021-9258(20001027)275:43<33869:EOTAGP>2.0.ZU;2-O
Fonte:
ISI
Lingua:
ENG
Soggetto:
FERMENTATIVE ALCOHOL-DEHYDROGENASE; ENCODING ETHANOL OXIDOREDUCTASE; PROPANEDIOL OXIDOREDUCTASE; ZYMOMONAS-MOBILIS; OXIDATIVE STRESS; ENZYMES; L-1,2-PROPANEDIOL; SEQUENCE; CLONING; FUCOSE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
35
Recensione:
Indirizzi per estratti:
Indirizzo: Ros, J Univ Lleida, Fac Med, Dept Ciencies Med Basiques, Lleida 25198, Spain Univ Lleida Lleida Spain 25198 Med Basiques, Lleida 25198, Spain
Citazione:
J. Membrillo-Hernandez et al., "Evolution of the adhE gene product of Escherichia coli from a functional reductase to a dehydrogenase - Genetic and biochemical studies of the mutantproteins", J BIOL CHEM, 275(43), 2000, pp. 33869-33875

Abstract

The multifunctional AdhE protein of Escherichia coli (encoded by the adhE gene) physiologically catalyzes the sequential reduction of acetyl-CoA to acetaldehyde and then to ethanol under fermentative conditions. The NH2-terminal region of the AdhE protein is highly homologous to aldehyde:NAD(+) oxidoreductases, whereas the COOH-terminal region is homologous to a family ofFe2+-dependent ethanol:NAD(+) oxidoreductases. This fusion protein also functions as a pyruvate formate lyase deactivase. E. coli cannot grow aerobically on ethanol as the sole carbon and energy source because of inadequate rate of adhE transcription and the vulnerability of the AdhE protein to metal-catalyzed oxidation. In this study, we characterized 16 independent two-step mutants with acquired and improved aerobic growth ability on ethanol, The AdhE proteins in these mutants catalyzed the sequential oxidation of ethanol to acetaldehyde and to acetyl-CoA All first stage mutants grew on ethanol with a doubling time of about 240 min. Sequence analysis of a randomlychosen mutant revealed an Ala-267 --> Thr substitution in the acetaldehyde:NAD(+) oxidoreductase domain of AdhE. All second stage mutants grew on ethanol 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 elevated acetaldehyde dehydrogenase activities. It therefore appears that when AdhE catalyzes the two sequential reactions in the counter-physiologicaldirection, acetaldehyde dehydrogenation is the rate-limiting step. Both mutant proteins were more thermosensitive than the wild-type protein, but AdhE(A267T/E568K) was more thermal stable than AdhE(A267T). Since both mutant enzymes exhibited similar kinetic properties, the second mutation probably conferred an increased growth rate on ethanol by stabilizing AdhE(A267T).

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 06/04/20 alle ore 05:46:18