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J. Biol. Chem., Vol. 275, Issue 46, 35876-35885, November 17, 2000

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Characterization of a (2R,3R)-2,3-Butanediol Dehydrogenase as the Saccharomyces cerevisiae YAL060W Gene Product
DISRUPTION AND INDUCTION OF THE GENE^*

Eva González, M. Rosario Fernández, Carol Larroy, Lluís Solà^§, Miquel A. Pericàs^§, Xavier Parés^¶, and Josep A. Biosca

From the  Department of Biochemistry and Molecular Biology, Faculty of Sciences, Universitat Autònoma de Barcelona, E-08193 Bellaterra (Barcelona) and the ^§ Unitat de Recerca en Síntesi Asimètrica, Departament de Química Orgànica, Universitat de Barcelona, E-08028 Barcelona, Spain

The completion of the Saccharomyces cerevisiae genome project in 1996 showed that almost 60% of the potential open reading frames of the genome had no experimentally determined function. Using a conserved sequence motif present in the zinc-containing medium-chain alcohol dehydrogenases, we found several potential alcohol dehydrogenase genes with no defined function. One of these, YAL060W, was overexpressed using a multicopy inducible vector, and its protein product was purified to homogeneity. The enzyme was found to be a homodimer that, in the presence of NAD⁺, but not of NADP, could catalyze the stereospecific oxidation of (2R,3R)-2,3-butanediol (K_m = 14 mM, k_cat = 78,000 min¹) and meso-butanediol (K_m = 65 mM, k_cat = 46,000 min¹) to (3R)-acetoin and (3S)-acetoin, respectively. It was unable, however, to further oxidize these acetoins to diacetyl. In the presence of NADH, it could catalyze the stereospecific reduction of racemic acetoin ((3R/3S)- acetoin; K_m = 4.5 mM, k_cat = 98,000 min¹) to (2R,3R)-2,3-butanediol and meso-butanediol, respectively. The substrate stereospecificity was determined by analysis of products by gas-liquid chromatography. The YAL060W gene product can therefore be classified as an NAD-dependent (2R,3R)-2,3-butanediol dehydrogenase (BDH). S. cerevisiae could grow on 2,3-butanediol as the sole carbon and energy source. Under these conditions, a 3.5-fold increase in (2R,3R)-2,3-butanediol dehydrogenase activity was observed in the total cell extracts. The isoelectric focusing pattern of the induced enzyme coincided with that of the pure BDH (pI 6.9). The disruption of the YAL060W gene was not lethal for the yeast under laboratory conditions. The disrupted strain could also grow on 2,3-butanediol, although attaining a lesser cell density than the wild-type strain. Taking into consideration the substrate specificity of the YAL060W gene product, we propose the name of BDH for this gene. The corresponding enzyme is the first eukaryotic (2R,3R)-2,3-butanediol dehydrogenase characterized of the medium-chain dehydrogenase/reductase family.

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