Genetic and biochemical characterization of a short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.
The gene encoding a short-chain alcohol dehydrogenase, AdhA, has been identified in the hyperthermophilic archaeon Pyrococcus furiosus, as part of an operon that encodes two glycosyl hydrolases, the beta-glucosidase CelB and the endoglucanase LamA. The adhA gene was functionally expressed in Escherichia coli, and AdhA was subsequently purified to homogeneity. The quaternary structure of AdhA is a dimer of identical 26-kDa subunits. AdhA is an NADPH-dependent oxidoreductase that converts alcohols to the corresponding aldehydes/ketones and vice versa, with a rather broad substrate specificity. Maximal specific activities were observed with 2-pentanol (46 U x mg(-1)) and pyruvaldehyde (32 U x mg(-1)) in the oxidative and reductive reaction, respectively. AdhA has an optimal activity at 90 degrees C, at which temperature it has a half life of 22.5 h. The expression of the adhA gene in P. furiosus was demonstrated by activity measurements and immunoblot analysis of cell extracts. A role of this novel type of archaeal alcohol dehydrogenase in carbohydrate fermentation is discussed.
van der Oost
J
J
Laboratory of Microbiology, Wageningen University, the Netherlands. john.vanderoost@algemeen.micr.wag-ur.nl
Voorhorst
W G
WG
Kengen
S W
SW
Geerling
A C
AC
Wittenhorst
V
V
Gueguen
Y
Y
de Vos
W M
WM
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics*; D000429:Alcohol Oxidoreductases / Q000235:genetics*; D000595:Amino Acid Sequence; D015153:Blotting, Western; D050260:Carbohydrate Metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005285:Fermentation; D007700:Kinetics; D008969:Molecular Sequence Data; D009876:Operon; D010957:Plasmids / Q000378:metabolism; D020836:Protein Structure, Quaternary; D020027:Pyrococcus furiosus / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase