González E, Fernández M R, Larroy C, Solà L, Pericàs M A, Parés X, Biosca J A
Department of Biochemistry and Molecular Biology, Faculty of Sciences, Universitat Autònoma de Barcelona, E-08193 Bellaterra (Barcelona),and Spain.
J Biol Chem. 2000 Nov 17;275(46):35876-85. doi: 10.1074/jbc.M003035200.
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 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.
1996年酿酒酵母基因组计划的完成表明，该基因组中近60%的潜在开放阅读框没有通过实验确定的功能。利用含锌中链醇脱氢酶中存在的保守序列基序，我们发现了几个功能未明确的潜在醇脱氢酶基因。其中一个基因YAL060W，使用多拷贝诱导型载体进行了过量表达，其蛋白质产物被纯化至同质。发现该酶是一种同二聚体，在有NAD(+)而不是NADP存在时，它可以催化(2R,3R)-2,3-丁二醇（K(m)=14 mM，k(cat)=78,000 min⁻¹）和内消旋丁二醇（K(m)=65 mM，k(cat)=46,000 min⁻¹）分别立体特异性氧化为(3R)-乙偶姻和(3S)-乙偶姻。然而，它无法将这些乙偶姻进一步氧化为双乙酰。在有NADH存在时，它可以催化外消旋乙偶姻（(3R/3S)-乙偶姻；K(m)=4.5 mM，k(cat)=98,000 min⁻¹）分别立体特异性还原为(2R,3R)-2,3-丁二醇和内消旋丁二醇。通过气液色谱分析产物来确定底物立体特异性。因此，YAL060W基因产物可归类为NAD依赖性(2R,3R)-2,3-丁二醇脱氢酶（BDH）。酿酒酵母可以以2,3-丁二醇作为唯一的碳源和能源生长。在这些条件下，在总细胞提取物中观察到(2R,3R)-2,3-丁二醇脱氢酶活性增加了3.5倍。诱导酶的等电聚焦图谱与纯BDH（pI 6.9）的图谱一致。在实验室条件下，YAL060W基因的破坏对酵母不是致命的。 disrupted菌株也可以在2,3-丁二醇上生长，尽管其细胞密度低于野生型菌株。考虑到YAL060W基因产物的底物特异性，我们提议将该基因命名为BDH。相应的酶是中链脱氢酶/还原酶家族中第一个被表征的真核(2R,3R)-2,3-丁二醇脱氢酶。
