Abstract
An alpha,beta-dicarbonyl reductase activity was purified from Saccharomyces cerevisiae and identified as the cytosolic enzyme D-Arabinose dehydrogenase (ARA1) by MALDI-TOF/TOF. Size exclusion chromatography analysis of recombinant Ara1p revealed that this protein formed a homodimer. Ara1p catalyzed the reduction of the reactive alpha,beta-dicarbonyl compounds methylglyoxal, diacetyl, and pentanedione in a NADPH dependant manner. Ara1p had apparent Km values of approximately 14 mM, 7 mM and 4 mM for methylglyoxal, diacetyl and pentanedione respectively, with corresponding turnover rates of 4.4, 6.9 and 5.9 s(-1) at pH 7.0. pH profiling showed that Ara1p had a pH optimum of 4.5 for the diacetyl reduction reaction. Ara1p also catalyzed the NADP+ dependant oxidation of acetoin; however this back reaction only occurred at alkaline pH values. That Ara1p was important for degradation of alpha,beta-dicarbonyl substrates was further supported by the observation that ara1-Delta knockout yeast mutants exhibited a decreased growth rate phenotype in media containing diacetyl.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Acetoin Dehydrogenase / chemistry
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Acetoin Dehydrogenase / isolation & purification
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Amino Acid Sequence
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Diacetyl / chemistry
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Diacetyl / metabolism
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Kinetics
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Mass Spectrometry
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Molecular Sequence Data
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Oxidation-Reduction
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Oxidoreductases Acting on Aldehyde or Oxo Group Donors / chemistry*
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Oxidoreductases Acting on Aldehyde or Oxo Group Donors / genetics
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Oxidoreductases Acting on Aldehyde or Oxo Group Donors / metabolism
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Pyruvaldehyde / chemistry
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Pyruvaldehyde / metabolism
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Saccharomyces cerevisiae / enzymology*
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins / chemistry*
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism
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Sequence Alignment
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Sugar Alcohol Dehydrogenases / chemistry*
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Sugar Alcohol Dehydrogenases / genetics
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Sugar Alcohol Dehydrogenases / metabolism
Substances
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Ara1 protein, S cerevisiae
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Recombinant Proteins
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Saccharomyces cerevisiae Proteins
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Pyruvaldehyde
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Sugar Alcohol Dehydrogenases
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Acetoin Dehydrogenase
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D-arabinose dehydrogenase
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Oxidoreductases Acting on Aldehyde or Oxo Group Donors
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Diacetyl