Insights into the catalytic and regulatory mechanisms of dihydroflavonol 4-reductase, a key enzyme of anthocyanin synthesis in Zanthoxylum bungeanum

Abstract Accumulation of anthocyanins largely determines the fruit color, and dihydroflavonol 4-reductase (DFR) is a key enzyme involved in the formation of anthocyanins. However, the catalytic and regulatory mechanisms of DFR are unclear. In this study, the gene encoding DFR from Zanthoxylum bungeanum Maxim. was cloned and ZbDFR was analyzed in detail. The ZbDFR accepted dihydrokaempferol, dihydroquercetin and dihydromyricetin as substrates. Flavonols such as myricetin, quercetin and kaempferol significantly inhibited the activity of ZbDFR, while quercitrin and isoquercitrin slightly increased the activity. Quercetin was a competitive inhibitor at low concentrations, and it had a combined effect of competitive and noncompetitive inhibition at high concentrations, which was consistent with ZbDFR having two inhibitor binding sites. In addition, the content of different types of flavonoids in Z. bungeanum peel at green, semi-red and red stage was analyzed, and the in vivo results could be explained by the regulation of ZbDFR activity in vitro. Site-directed mutagenesis combined with enzyme activity experiments showed that Ser128, Tyr163, Phe164 and Lys167 are the key catalytic amino acid residues. The Ser128, Tyr163 and Lys167 were crucial for the hydrogen transfer reaction, and mutation of these amino acids resulted in the loss of all or most of the activity. Phe164 was found to be important for the regulation of ZbDFR by flavonols. Accordingly, ZbDFR is a node at which flavonoids regulate the synthesis of anthocyanins and proanthocyanins.