Characterization of a (2R,3R)-2,3-Butanediol Dehydrogenase as theSaccharomyces cerevisiae YAL060W Gene Product

Abstract

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 mediumchain 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 min ؊1 ) and meso-butanediol (K m ‫؍‬ 65 mM, k cat ‫؍‬ 46,000 min ؊1 ) 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 min ؊1 ) 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.

Allyl alcohol and garlic (Allium sativum) extract produce oxidative stress in Candida albicans

“…However, decreased respiration, as observed with GPE and other garlic constituents (K. M. Lemar and others, unpublished), was not observed for AA. This may be explained by the fact that the alcohol dehydrogenase function is not implicated in glucose respiration, as when organisms are grown in the presence of excess glucose, alcohol dehydrogenase is inactivated (Gonzalez et al, 2000). The conversion of AA to the toxic aldehyde acrolein would therefore not occur.…”

Distinct Intracellular Localization of Gpd1p and Gpd2p, the Two Yeast Isoforms of NAD+-dependent Glycerol-3-phosphate Dehydrogenase, Explains Their Different Contributions to Redox-driven Glycerol Production

“…Cells were grown in E-flasks under aerobic conditions. generation by reduction of acetoin to 2,3-butanediol (18,36). Following acetoin addition growth of the gpd2⌬cox18⌬ strain resumed (Fig.…”

“…Both of these rescuing activities are likely to take place in the cytosol; acetoin is reduced to 2,3-butanediol by the cytosolic NAD ϩ -linked 2,3-butanediol dehydrogenase, Bdh1p (36,38), and Gpd1p is a cytosolic/peroxisomal enzyme. Based on experimental evidence (51), the reversible ethanol-acetaldehyde shuttle is the most likely pathway for resolving the redox-problem of non-respiring mitochondria.…”

The Catabolism of Amino Acids to Long Chain and Complex Alcohols in Saccharomyces cerevisiae

“…Seven of these can be eliminated after a consideration of their properties. For example, BDH1 (YAL060w) has been shown recently (18) to be involved in the formation of 2,3-butanediol. On the basis of similarity it is safe to assume that YAL061W can be called BDH2 and that it has a similar activity.…”