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.
Dihydroflavonol 4-reductase (DFR), a member of the short-chain dehydrogenase family, catalyzes the last common step in the biosynthesis of flavan-3-ols and condensed tannins. Initial rates of DFR were measured by monitoring the 340-nm absorbance decrease resulting from the joint consumption of dihydroquercetin (DHQ) and NADPH, as a function of pH, temperature and ionic strength. At pH 6.5 and 30˚C, substrate inhibition was observed above 30 µM DHQ. At lower/non-inhibitory DHQ concentrations, NADP + behaves as a competitive inhibitor with respect to NADPH and as a mixed inhibitor with respect to DHQ, which supports a sequential ordered mechanism, with NADPH binding first and NADP + released last. Binding-equilibrium data obtained by means of the chromatographic method of Hummel and Dreyer at pH 7.5 and by isothermal calorimetric titration at pH 6.5 led to the conclusion that ligands of the apoenzyme included NADPH, NADP + and DHQ. The mechanism which best accounts for substrate inhibition at pH 6.5 in the absence of product involves the formation of a binary non-productive E.DHQ complex. Thus, a productive ternary complex cannot be formed when DHQ binds first. This mechanism of inhibition may prevent the accumulation of unstable leucoanthocyanidins within cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.