Conserved amino acid residues and gene expression patterns associated with the substrate preferences of the competing enzymes FLS and DFR

Abstract Flavonoids, an important class of specialized metabolites, are synthesized from phenylalanine and present in almost all plant species. Different branches of flavonoid biosynthesis lead to products like flavones, flavonols, anthocyanins, and proanthocyanidins. Dihydroflavonols form the branching point towards the production of non-coloured flavonols via flavonol synthase (FLS) and coloured anthocyanins via dihydroflavonol 4-reductase (DFR). An angiosperm-wide comparison of FLS and DFR sequences revealed the substrate specifying amino acids in both enzymes. We identified the patterns of these residue occurrences in angiosperms and revealed that monocots generally possess FLS with Y132 residue (FLS Y ) and DFR with N133 residue (DFR N ). In contrast, dicots generally possess FLS H and DFR N , DFR D and DFR A . Generally, angiosperm FLS prefers dihydrokaempferol, whereas DFR favors dihydroquercetin or dihydromyricetin. This study illustrates the importance of substrate-specifying residues in resolving competition between the FLS and DFR enzymes, delineates the evolution of these enzymes, and provides insights into mechanisms directing the metabolicflux of theflavonoid biosynthesis, with potential implications for ornamental plants and molecular breeding strategies.