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 electro- spray 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.
Gargouri
Mahmoud
M
Chimie et Biologie des Membranes et des Nanoobjets UMR CNRS 5248, Bâtiment B8, Avenue des Facultés, Université Bordeaux 1, F-33405 Talence Cedex, France.|Laboratoire de Physiologie Moléculaire des Plantes, Centre de Biotechnologie de Borj-Cedria, B.P. 901, 2050 Hamman-Lif, Tunisia.
Chaudière
Jean
J
Chimie et Biologie des Membranes et des Nanoobjets UMR CNRS 5248, Bâtiment B8, Avenue des Facultés, Université Bordeaux 1, F-33405 Talence Cedex, France.
Manigand
Claude
C
Chimie et Biologie des Membranes et des Nanoobjets UMR CNRS 5248, Bâtiment B8, Avenue des Facultés, Université Bordeaux 1, F-33405 Talence Cedex, France.
Maugé
Chloé
C
Chimie et Biologie des Membranes et des Nanoobjets UMR CNRS 5248, Bâtiment B8, Avenue des Facultés, Université Bordeaux 1, F-33405 Talence Cedex, France.
Bathany
Katell
K
Chimie et Biologie des Membranes et des Nanoobjets UMR CNRS 5248, Bâtiment B8, Avenue des Facultés, Université Bordeaux 1, F-33405 Talence Cedex, France.
Schmitter
Jean-Marie
JM
Chimie et Biologie des Membranes et des Nanoobjets UMR CNRS 5248, Bâtiment B8, Avenue des Facultés, Université Bordeaux 1, F-33405 Talence Cedex, France.
Gallois
Bernard
B
Chimie et Biologie des Membranes et des Nanoobjets UMR CNRS 5248, Bâtiment B8, Avenue des Facultés, Université Bordeaux 1, F-33405 Talence Cedex, France.
D000872:Anthocyanins / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D002851:Chromatography, High Pressure Liquid; D005419:Flavonoids / Q000737:chemistry / Q000378:metabolism*; D006859:Hydrogen / Q000737:chemistry / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry; D027843:Vitis / Q000201:enzymology*
D000872:Anthocyanins; D005419:Flavonoids; C404987:flavan-3-ol; D006859:Hydrogen; D009247:NADH, NADPH Oxidoreductases