Rapid purification and properties of potassium-activated aldehyde dehydrogenase from <i>Saccharomyces cerevisiae</i>

Bostian, Karen A.; Betts, Graham F.

doi:10.1042/bj1730773

Cited by 65 publications

(50 citation statements)

References 32 publications

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“…To distinguish this activity from the classical aldehyde dehydrogenase (ALDH) isolated first in 1951 by Black (3), the assay was done in the presence of 50 mM Mg2+ and in the absence of K+. Under these conditions the latter enzyme is completely inactive (5,11,19; unpublished data). The NAD+-specific aldehyde oxidation activity was 142 ± 11 mU/mg in mitochondrial preparations from glucose-grown cells.…”

Section: Resultsmentioning

confidence: 80%

Biochemical basis of mitochondrial acetaldehyde dismutation in Saccharomyces cerevisiae

Thielen

Ciriacy

1991

J Bacteriol

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The reduction of acetaldehyde to ethanol plays a key role in sugar metabolism of baker's yeast, Saccharomyces cerevisiae, by allowing regeneration of NAD+ from glycolytic NADH+H+. The in vivo significance of the reaction catalyzed by alcohol dehydrogenase (ADH) has been verified by mutants (adhl) deficient in ADH I, one of the four known ADH isozymes. Such mutant cells grow slowly on glucose, and growth is entirely dependent on mitochondrial functions (23). Despite this requirement for a functional respiratory chain in adhi mutants, glucose is largely converted to the major fermentation products, glycerol and ethanol, and to minor products such as acetaldehyde and acetate. We showed recently that the residual ethanol production in adhl mutant cells (approximately 30% of wild-type level) cannot be attributed to any of the known ADH isozymes (ADH II through ADH IV) by using mutant cells carrying null alleles at the four ADH loci (9). The existence of another cytoplasmic NAD+-dependent ADH isozyme was also ruled out since adh°cells cannot grow in a medium containing ethanol as the sole source of carbon. Rather, acetaldehyde reduction in adh/ cells occurs inside mitochondria. In fact, mitochondrial preparations from glucose-grown cells can quantitatively convert acetaldehyde to ethanol and acetate. This dismutation of acetaldehyde requires a functional respiratory chain, which is consistent with the finding that ethanol formation in adh/ cells is blocked by antimycin A.

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Section: Resultsmentioning

confidence: 80%

Biochemical basis of mitochondrial acetaldehyde dismutation in Saccharomyces cerevisiae

Thielen

Ciriacy

1991

J Bacteriol

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“…not by aldehyde oxidation) and when this is itself likely to be dramatically lowered by Mg2+, which is present at high intracellular concentrations. It is, however, interesting and perhaps pertinent to note that yeast alcohol dehydrogenase also exhibits a Vm.ax some 150-fold greater than that of its liver counterpart (Dickinson & Monger, 1973 The procedure used for preparing enzyme was essentially that described by Bostian & Betts (1978a), starting with an acetone-dried powder and with phenylmethanesulphonyl fluoride as a proteinase inhibitor. The procedure was taken as far as fraction IV without Vol.…”

Section: Mg2+mentioning

confidence: 99%

“…Enzyme active-site concentrations were calculated by assuming A1°m = 10.0 and taking an Mr of 240000 with four NAD+-binding sites and therefore four active sites/ molecule (Bostian & Betts, 1978a).…”

Section: Mg2+mentioning

confidence: 99%

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The role of the metal ion in the mechanism of the K+-activated aldehyde dehydrogenase of Saccharomyces cerevisiae

Dickinson

Haywood

1987

Biochemical Journal

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The effect of K+ on assays of the enzyme was studied and it appears that the activation occurs slowly by a two-step process. Kinetic measurements suggest that the enzyme-catalysed reaction can proceed slowly (0.4 %) in the complete absence of K+. The enzyme exhibits a K+-activated esterase activity, which is further activated by NAD+ or NADH. Stopped-flow studies indicated that the principal effect of K+ on the dehydrogenase reaction is to accelerate a step (possibly acyl-enzyme hydrolysis) associated with a fluorescence and small absorbance transient that occurs after hydride transfer and before NADH dissociation from the terminal E-NADH complex. The variation of activity of the enzyme with pH was

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“…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.…”

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confidence: 99%

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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Rapid purification and properties of potassium-activated aldehyde dehydrogenase from Saccharomyces cerevisiae

Cited by 65 publications

References 32 publications

Biochemical basis of mitochondrial acetaldehyde dismutation in Saccharomyces cerevisiae

Biochemical basis of mitochondrial acetaldehyde dismutation in Saccharomyces cerevisiae

The role of the metal ion in the mechanism of the K+-activated aldehyde dehydrogenase of Saccharomyces cerevisiae

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

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