Production and characterization of a thermostable alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily.
The gene encoding a novel alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The gene, referred to as adhD, was functionally expressed in Escherichia coli and subsequently purified to homogeneity. The enzyme has a monomeric conformation with a molecular mass of 32 kDa. The catalytic activity of the enzyme increases up to 100 degrees C, and a half-life value of 130 min at this temperature indicates its high thermostability. AdhD exhibits a broad substrate specificity with, in general, a preference for the reduction of ketones (pH optimum, 6.1) and the oxidation of secondary alcohols (pH optimum, 8.8). Maximal specific activities were detected with 2,3-butanediol (108.3 U/mg) and diacetyl-acetoin (22.5 U/mg) in the oxidative and reductive reactions, respectively. Gas chromatrography analysis indicated that AdhD produced mainly (S)-2-pentanol (enantiomeric excess, 89%) when 2-pentanone was used as substrate. The physiological role of AdhD is discussed.
Machielsen
Ronnie
R
Laboratory of Microbiology, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands. Ronnie.machielsen@wur.nl
Uria
Agustinus R
AR
Kengen
Servé W M
SW
van der Oost
John
J
D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000145:classification*; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli; D006358:Hot Temperature; D007536:Isomerism; D007700:Kinetics; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000426:Alcohol Dehydrogenase; D000449:Aldehyde Reductase