Homology of Saccharomyces cerevisiae ADH4 to an iron-activated alcohol dehydrogenase from Zymomonas mobilis

Williamson, Valerie M.; Paquin, Charlotte E.

doi:10.1007/bf00329668

Cited by 148 publications

(107 citation statements)

References 38 publications

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“…Finally, YLR070C has recently been shown to code for a xylitol dehydrogenase (15). The ADH4 gene (16) codes for ADHIV, which is considered a member of the "iron-activated" ADH family (17).…”

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

González¹,

Fernández²,

Larroy³

et al. 2000

Journal of Biological Chemistry

116

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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.

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confidence: 99%

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

González¹,

Fernández²,

Larroy³

et al. 2000

Journal of Biological Chemistry

116

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“…Additional classes, IV and V (3)(4)(5)(6)(7)(8), of these medium-chain alcohol dehydrogenases and further isozymes in some species (9,10) have since been added. Still more enzymes (11,12) and another protein family, short-chain dehydrogenases (11), also belong to this system in vertebrates, and additional families and forms occur in prokaryotes, plants, yeasts, insects, and other organisms (13)(14)(15)(16). The medium-chain alcohol dehydrogenase family is now a well-established entity with functionally, structurally, and evolutionarily distinctive features (17).…”

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Mammalian class IV alcohol dehydrogenase (stomachalcohol dehydrogenase): structure, origin, and correlation withenzymology.

Parés

Cederlund

Moreno

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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The structure of a mammalian class IV alcohol dehydrogenase has been determined by peptide analysis of the protein isolated from rat stomach. The structure indicates that the enzyme constitutes a separate alcohol dehydrogenase class, in agreement with the distinct enzymatic properties; the class IV enzyme is somewhat closer to class I (the "classical" liver alcohol dehydrogenase; -68% residue identities) than to the other classes (II, m, and V; -60% residue identities), suggesting that class IV might have originated through duplication of an early vertebrate class I gene. The activity of the class IV protein toward ethanol is even higher than that of the classical liver enzyme. Both Km and kalt values are high, the latter being the highest of any class characterized so far. Structurally, these properties are correlated with replacements at the active site, affecting both substrate and coenzyme binding. In particular, Ala-294 (instead of valine) results in increased space in the middle section of the substrate cleft, Gly-47 (instead of a basic residue) results in decreased charge interactions with the coenzyme pyrophosphate, and Tyr-363 (instead of a basic residue) may also affect coenzyme binding. In combination, these exchanges are compatible with a promotion of the off dissociation and an increased turnover rate. In contrast, residues at the inner part of the substrate cleft are bulky, accounting for low activity toward secondary alcohols and cyclohexanol. Exchanges at positions 259-261 involve minor shifts in glycine residues at a reverse turn in the coenzyme-binding fold. Clearly, class IV is distinct in structure, ethanol turnover, stomach expression, and possible emergence from class I.

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“…In the liver, cytosolic alcohol dehydrogenase (ADH) oxidizes ethanol to acetaldehyde and then mitochondrial aldehyde dehydrogenase (ALDH) oxidizes the intermediate to acetate (14). In S. cerevisiae, it appears that a mitochondrial alcohol dehydrogenase (ADH3) is responsible for the initial oxidation (65,67). Little is known about the role of ALDHs in the next step.…”

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Molecular cloning of the mitochondrial aldehyde dehydrogenase gene of Saccharomyces cerevisiae by genetic complementation

et al. 1991

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Mutants of Saccharomyces cerevisiae deficient in mitochondrial aldehyde dehydrogenase (ALDH) activity were isolated by chemical mutagenesis with ethyl methanesulfonate. The mutants were selected by their inability to grow on ethanol as the sole carbon source. The ALDH mutants were distinguished from alcohol dehydrogenase mutants by an aldehyde indicator plate test and by immunoscreening. The ALDH gene was isolated from a yeast genomic DNA library on a 5.7-kb insert of a recombinant DNA plasmid by functional complementation of the aldh mutation in S.cerevisiae. An open reading frame which specifies 533 codons was found within the 2.0-kb BamHI-BstEII fragment in the 5.7-kb genomic insert which can encode a protein with a molecular weight of 58,630. The N-terminal portion of the protein contains many positively charged residues which may serve as a signal sequence that targets the protein to the mitochondria. The amino acid sequence of the proposed mature yeast enzyme shows 30% identity to each of the known ALDH sequences from eukaryotes or prokaryotes. The amino acid residues corresponding to mammalian cysteine 302 and glutamates 268 and 487, implicated to be involved at the active site, were conserved. S. cerevisiae ALDH was found to be localized in the mitochondria as a tetrameric enzyme. Thius, that organelle is responsible for acetaldehyde oxidation, as was found in mammalian liver.Saccharomyces cerevisiae, unlike higher eukaryotes, can metabolize as well as produce ethanol. During fermentation, the consumption of sugars results in the accumulation of ethanol. When glucose or any other fermentable sugar is absent from the culture medium, S. cerevisiae can utilize ethanol as the carbon source aerobically. The metabolism of ethanol is well understood in mammals. In the liver, cytosolic alcohol dehydrogenase (ADH) oxidizes ethanol to acetaldehyde and then mitochondrial aldehyde dehydrogenase (ALDH) oxidizes the intermediate to acetate (14). In S. cerevisiae, it appears that a mitochondrial alcohol dehydrogenase (ADH3) is responsible for the initial oxidation (65,67). Little is known about the role of ALDHs in the next step.Various groups have purified and characterized yeast ALDH (12,49,54). It was reported to be a tetrameric enzyme with a subunit molecular mass of 60 kDa and a low Km for acetaldehyde. Unlike the liver enzymes, the yeast enzyme is activated by K+ ions (4, 32). Whereas the mammalian enzymes have been sequenced at the cDNA (16,19,29,34) as well as the protein (24, 33, 60) level, no sequence work has been reported for the yeast enzyme.One of our interests was to study the subcellular localization of acetaldehyde metabolism in S. cerevisiae. In the mammalian liver tissue, this was studied by selectively inhibiting the cytosolic or the mitochondrial isozyme of ALDH (11,52). An alternative approach would be to introduce the enzyme into a cell deficient in ALDH activity. S. cerevisiae would be a suitable model system, as it can metabolize ethanol and could serve as a host for the expression of foreign g...

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Homology of Saccharomyces cerevisiae ADH4 to an iron-activated alcohol dehydrogenase from Zymomonas mobilis

Cited by 148 publications

References 38 publications

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

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

Mammalian class IV alcohol dehydrogenase (stomachalcohol dehydrogenase): structure, origin, and correlation withenzymology.

Molecular cloning of the mitochondrial aldehyde dehydrogenase gene of Saccharomyces cerevisiae by genetic complementation

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