The epimerase activity of anthocyanidin reductase from Vitis vinifera and its regiospecific hydride transfers

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 degrees 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 C-3-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 electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS), and it was found that deuterium was always incorporated at C-2 and C-4. We conclude that C-3-epimerization should be achieved by tautomerization between the two hydride transfers and that this produces a quinone methide intermediate which serves as C-4 target of the second hydride transfer, thereby avoiding any stereospecific modification of carbon 3. The inversion of C-2 stereochemistry required for 'reverse epimerization' suggests that the 2S configuration induces an irreversible product dissociation.