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Título:
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Evolution of the adhE Gene Product of Escherichia coli from a Functional Reductase to a Dehydrogenase
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Autor/a:
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Membrillo-Hernández, Jorge; Echave Lozano, Pedro; Cabiscol Català, Elisa; Tamarit Sumalla, Jordi; Ros Salvador, Joaquim; Lin, Edmund C. C.
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Notas:
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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:NAD1 oxidoreductases, whereas
the COOH-terminal region is homologous to a family of
Fe21-dependent ethanol:NAD1 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 randomly chosen mutant revealed
an Ala-267 3 Thr substitution in the acetaldehyde:NAD1
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 AdhEA267T/
E568K. Purified AdhEA267T and AdhEA267T/E568K showed
highly elevated acetaldehyde dehydrogenase activities.
It therefore appears that when AdhE catalyzes the two
sequential reactions in the counter-physiological direction,
acetaldehyde dehydrogenation is the rate-limiting
step. Both mutant proteins were more thermosensitive
than the wild-type protein, but AdhEA267T/E568K was
more thermal stable than AdhEA267T. Since both mutant
enzymes exhibited similar kinetic properties, the second
mutation probably conferred an increased growth
rate on ethanol by stabilizing AdhEA267T
This work was supported by United States Public Health Service Grant GM40993 from the NIGMS of the National Institutes of Health and Dirección General de Enseñanza Superior e Investigación Científica Project PB97-1456. |
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Derechos:
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(c) The American Society for Biochemistry and Molecular Biology, 2000
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Tipo de documento:
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article
publishedVersion |
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Editor:
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American Society for Biochemistry and Molecular Biology
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