Structural evidence for the inhibition of grape dihydroflavonol 4-reductase by flavonols

Dihydroflavonol 4-reductase (DFR) is a key enzyme of the flavonoid biosynthesis pathway which catalyses the NADPH-dependent reduction of 2R,3R-trans-dihydroflavonols to leuco­anthocyanidins. The latter are the precursors of antho­cyans and condensed tannins, two major classes of phenolic com­pounds that strongly influence the organoleptic properties of wine. DFR has been investigated in many plant species, but little was known about its structural properties until the three-dimensional structure of the Vitis vinifera enzyme complexed with NADP⁺ and its natural substrate dihydroquercetin (DHQ) was described. In the course of the study of substrate specificity, crystals of DFR-NADP⁺-flavonol (myricetin and quercetin) complexes were obtained. Their structures exhibit major changes with respect to that of the abortive DFR-NADP⁺-DHQ complex. Two flavonol molecules bind to the catalytic site in a stacking arrangement and alter its geometry, which becomes incompatible with enzymatic activity. The X-­ray structures of both DFR-NADP⁺-myricetin and DFR-NADP⁺-quercetin are reported together with preliminary spectroscopic data. The results suggest that flavonols could be inhibitors of the activity of DFR towards dihydroflavonols.