Functional analysis of the dihydroflavonol 4-reductase family of : exploiting key amino acids to reconstruct reduction activity.

Anthocyanins and proanthocyanidins (PAs) are important types of flavonoids, plant secondary metabolites with a wide range of industrial and pharmaceutical applications. DFR (dihydroflavonol 4-reductase) is a pivotal enzyme that plays an important role in the flavonoid pathway. Here, four genes were isolated from , and their overexpression was analyzed and Based on transcription and metabolic analyses, expression was closely consistent with catechins and PAs accumulation. Moreover, enzyme activity analyses revealed that the two recombinant proteins CsDFRa and CsDFRc exhibited DFR activity, converting dihydroflavonols into leucoanthocyanins , but CsDFRb1 and CsDFRb3 did not. and overexpression in mutants () revealed that are involved in the biosynthesis of anthocyanins and PAs, as and restored not only the purple petiole phenotype but also the seed coat color. Site-directed mutagenesis revealed that the two amino acid residues S117 and T123 of CsDFRa play a prominent role in controlling DFR reductase activity. Enzymatic assays indicated that CsDFRa and CsDFRc exhibited a higher affinity for DHQ and DHK, respectively, whereas CsDFRb1 and CsDFRb1 exhibited a higher affinity for DHM. Our findings comprehensively characterize the DFRs from and shed light on their critical role in metabolic engineering.