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Stereochemistry of furfural reduction by a Saccharomyces cerevisiae aldehyde reductase that contributes to in situ furfural detoxification.

ABSTRACT: Ari1p from Saccharomyces cerevisiae, recently identified as an intermediate-subclass short-chain dehydrogenase/reductase, contributes in situ to the detoxification of furfural. Furfural inhibits efficient ethanol production by yeast, particularly when the carbon source is acid-treated lignocellulose, which contains furfural at a relatively high concentration. NADPH is Ari1p's best known hydride donor. Here we report the stereochemistry of the hydride transfer step, determined by using (4R)-[4-(2)H]NADPD and (4S)-[4-(2)H]NADPD and unlabeled furfural in Ari1p-catalyzed reactions and following the deuterium atom into products 2-furanmethanol or NADP(+). Analysis of the products demonstrates unambiguously that Ari1p directs hydride transfer from the si face of NADPH to the re face of furfural. The singular orientation of substrates enables construction of a model of the Michaelis complex in the Ari1p active site. The model reveals hydrophobic residues near the furfural binding site that, upon mutation, may increase specificity for furfural and enhance enzyme performance. Using (4S)-[4-(2)H]NADPD and NADPH as substrates, primary deuterium kinetic isotope effects of 2.2 and 2.5 were determined for the steady-state parameters k(cat)(NADPH) and k(cat)/K(m)(NADPH), respectively, indicating that hydride transfer is partially rate limiting to catalysis.

SUBMITTER: Bowman MJ

PROVIDER: S-EPMC2916452 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Stereochemistry of furfural reduction by a Saccharomyces cerevisiae aldehyde reductase that contributes to in situ furfural detoxification.

Bowman Michael J MJ Jordan Douglas B DB Vermillion Karl E KE Braker Jay D JD Moon Jaewoong J Liu Z Lewis ZL

Applied and environmental microbiology 20100604 15

Ari1p from Saccharomyces cerevisiae, recently identified as an intermediate-subclass short-chain dehydrogenase/reductase, contributes in situ to the detoxification of furfural. Furfural inhibits efficient ethanol production by yeast, particularly when the carbon source is acid-treated lignocellulose, which contains furfural at a relatively high concentration. NADPH is Ari1p's best known hydride donor. Here we report the stereochemistry of the hydride transfer step, determined by using (4R)-[4-(2 ...[more]

PMID: 20525870

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Project description:Quinone reductases are flavin-containing enzymes that have been implicated in protecting organisms from redox stress and, more recently, as redox switches controlling the action of the proteasome. The reactions of the catalytic cycle of the dimeric quinone reductase Lot6p from Saccharomyces cerevisiae were studied in anaerobic stopped-flow experiments at 4 degrees C. Both NADH and NADPH reacted similarly, reducing the FMN prosthetic group rapidly at saturation but binding with very low affinity. The enzyme stereospecifically transferred the proS-hydride of NADPH with an isotope effect of 3.6, indicating that hydride transfer, and not an enzyme conformational change, is rate-determining in the reductive half-reaction. No intermediates such as charge-transfer complexes were detected. In the oxidative half-reaction, reduced enzyme reacted in a single phase with the six quinone substrates tested. The observed rate constants increased linearly with quinone concentration up to the limits allowed by solubility, indicating either a bimolecular reaction or very weak binding. The logarithm of the bimolecular rate constant increases linearly with the reduction potential of the quinone, consistent with the notion that quinone reductases strongly disfavor radical intermediates. Interestingly, both half-reactions of the catalytic cycle strongly resemble bioorganic model reactions; the reduction of Lot6p by NAD(P)H is moderately faster than nonenzymatic models, while the oxidation of Lot6p by quinones is actually slower than nonenzymatic reactions. This curious situation is consistent with the structure of Lot6p, which has a crease we propose to be the binding site for pyridine nucleotides and a space, but no obvious catalytic residues, near the flavin allowing the quinone to react. The decidedly suboptimized catalytic cycle suggests that selective pressures other than maximizing quinone consumption shaped the evolution of Lot6p. This may reflect the importance of suppressing other potentially deleterious side reactions, such as oxygen reduction, or it may indicate that the role Lot6p plays as a redox sensor in controlling the proteasome is more important than its role as a detoxifying enzyme.

Dataset Information

Stereochemistry of furfural reduction by a Saccharomyces cerevisiae aldehyde reductase that contributes to in situ furfural detoxification.

Publications

Stereochemistry of furfural reduction by a Saccharomyces cerevisiae aldehyde reductase that contributes to in situ furfural detoxification.

Similar Datasets

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