The epimerase activity of anthocyanidin reductase from Vitis vinifera and its regiospecific hydride transfers
| Autor: | Katell Bathany, Claude Manigand, Chloé Maugé, Jean Chaudiere, Bernard Gallois, Mahmoud Gargouri, Jean-Marie Schmitter |
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| Rok vydání: | 2010 |
| Předmět: |
Flavonoids
Spectrometry Mass Electrospray Ionization Stereochemistry Hydride Clinical Biochemistry Stereoisomerism Photochemistry Biochemistry Quinone methide Pelargonidin Anthocyanidin reductase Anthocyanins Anthocyanidins chemistry.chemical_compound Stereospecificity chemistry Tandem Mass Spectrometry Biocatalysis NADH NADPH Oxidoreductases Vitis Anthocyanidin 3-O-glucosyltransferase Molecular Biology Chromatography High Pressure Liquid Hydrogen Anthocyanidin |
| Zdroj: | Biological Chemistry. 391:219-227 |
| ISSN: | 1437-4315 1431-6730 |
| DOI: | 10.1515/bc.2010.015 |
| Popis: | Anthocyanidin reductase (ANR) from Vitis vinifera catalyzes an NADPH-dependent double reduction of anthocyanidins producing a mixture of (2S,3R)- and (2S,3S)-flavan-3-ols. At pH 7.5 and 30°C, the first hydride transfer to anthocyanidin is irreversible, and no intermediate is released during catalysis. ANR reverse activity was assessed in the presence of excess NADP+. Analysis of products by reverse phase and chiral phase HPLC demonstrates that ANR acts as a flavan-3-ol C3-epimerase under such conditions, but this is only observed with 2R-flavan-3-ols, not with 2S-flavan-3-ols produced by the enzyme in the forward reaction. In the presence of deuterated coenzyme 4S-NADPD, ANR transforms anthocyanidins into dideuterated flavan-3-ols. The regiospecificity of deuterium incorporation into catechin and afzelechin – derived from cyanidin and pelargonidin, respectively – was analyzed by liquid chromatography coupled with electro- spray ionization-tandem mass spectrometry (LC/ESI-MS/MS), and it was found that deuterium was always incorporated at C2 and C4. We conclude that C3-epimerization should be achieved by tautomerization between the two hydride transfers and that this produces a quinone methide intermediate which serves as C4 target of the second hydride transfer, thereby avoiding any stereospecific modification of carbon 3. The inversion of C2 stereochemistry required for ‘reverse epimerization’ suggests that the 2S configuration induces an irreversible product dissociation. |
| Databáze: | OpenAIRE |
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