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.

A new NADP(H)-dependent alcohol dehydrogenase (the YCR105W gene product, ADHVII) has been identified in Saccharomyces cerevisiae. The enzyme has been purified to homogeneity and found to be a homodimer of 40 kDa subunits and a pI of 6.2-6.4. ADHVII shows a broad substrate specificity similar to the recently characterized ADHVI (64% identity), although they show some differences in kinetic properties. ADHVI and ADHVII are the only members of the cinnamyl alcohol dehydrogenase family in yeast. Simultaneous deletion of ADH6 and ADH7 was not lethal for the yeast. Both enzymes could participate in the synthesis of fusel alcohols, ligninolysis and NADP(H) homeostasis.Keywords: cinnamyl alcohol dehydrogenase; fusel alcohols; NADP(H) homeostasis; ligninolysis.The current version of the Yeast Proteome Database (http://www.proteome.com) lists approximately 260 oxidoreductases (160 of them have been characterized experimentally, and the rest predicted by sequence similarity or by other analysis) [1]. Our group is interested in the identification and characterization of novel alcohol dehydrogenase (ADH) gene products from Saccharomyces cerevisiae [2,3]. ADHs are oxidoreductases that catalyze the reversible oxidation of alcohols to aldehydes or ketones, with the corresponding reduction of NAD or NADP. ADHs constitute a large group of enzymes that can be subdivided into at least three distinct enzyme superfamilies: medium-chain and short-chain dehydrogenases/reductases, and iron-activated alcohol dehydrogenases [4,5]. The medium-chain dehydrogenase/ reductase (MDR) superfamily consists of enzymes with a subunit size of approximately 350 residues, dimeric or tetrameric, with two domains in each subunit: one catalytic and one responsible for the binding of the nucleotide (NAD or NADP). Many enzymes of the MDR family have zinc in their active site, and have a sequence motif known as the zinc-containing ADH signature: GHEX 2 GX 5 (G,A)X 2 (I,V,A,C,S) [6]. According to the Pfam and COG databases [7,8], the S. cerevisiae genome codes for 21 potential MDR enzymes, with 12 of them showing the zinc ADH signature described above. These 12 zinc-containing yeast MDR include ADH1, ADH2, ADH3, ADH5, ADH6, SFA1, SOR1 and its 99% identical YDL246C, XYL2, BDH1, YAL061W and YCR105W. All these yeast MDRs, except YCR105W and YAL061W, have enzymatic activities experimentally determined. In the present study, we report the characterization of the YCR105W gene from S. cerevisiae as a new alcohol dehydrogenase. The gene was overexpressed in yeast cells and the corresponding protein product purified to homogeneity. The enzyme showed a wide substrate specificity, using NADP(H) as coenzyme. Given the similar substrate specificities and the sequence identity (64%) between the Ycr105p and the recently characterized ADHVI [3], we propose the name of ADH7, for the YCR105W gene and ADHVII for its coded protein. A null adh7 yeast strain and a double mutant adh6D adh7D were constructed and their growths compared with a wild-type strain. M A T E R I A L S A N ...

YMR318C represents an open reading frame from Saccharomyces cerevisiae with unknown function. It possesses a conserved sequence motif, the zinc-containing alcohol dehydrogenase (ADH) signature, specific to the medium-chain zinc-containing ADHs. In the present study, the YMR318C gene product has been purified to homogeneity from overexpressing yeast cells, and found to be a homodimeric ADH, composed of 40 kDa subunits and with a pI of 5.0-5.4. The enzyme was strictly specific for NADPH and was active with a wide variety of substrates, including aliphatic (linear and branched-chain) and aromatic primary alcohols and aldehydes. Aldehydes were processed with a 50-fold higher catalytic efficiency than that for the corresponding alcohols. The highest k(cat)/K(m) values were found with pentanal>veratraldehyde > hexanal > 3-methylbutanal >cinnamaldehyde. Taking into consideration the substrate specificity and sequence characteristics of the YMR318C gene product, we have proposed this gene to be called ADH6. The disruption of ADH6 was not lethal for the yeast under laboratory conditions. Although S. cerevisiae is considered a non lignin-degrading organism, the catalytic activity of ADHVI can direct veratraldehyde and anisaldehyde, arising from the oxidation of lignocellulose by fungal lignin peroxidases, to the lignin biodegradation pathway. ADHVI is the only S. cerevisiae enzyme able to significantly reduce veratraldehyde in vivo, and its overexpression allowed yeast to grow under toxic concentrations of this aldehyde. The enzyme may also be involved in the synthesis of fusel alcohols. To our knowledge this is the first NADPH-dependent medium-chain ADH to be characterized in S. cerevisiae.

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The TRP1 marker has been commonly used for gene disruption experiments and subsequent phenotypic analysis. However, introduction of the TRP1 gene into a trp1 strain markedly affects growth under many conditions used for phenotypic profiling. Therefore, its use in the past should be revisited and utilization of this marker should be avoided in future analyses.