A Set of Regioselective O-Methyltransferases Gives Rise to the Complex Pattern of Methoxylated Flavones in Sweet Basil

Polymethoxylated flavonoids occur in a number of plant families, including the Lamiaceae. To date, the metabolic pathways giving rise to the diversity of these compounds have not been studied. Analysis of our expressed sequence tag database for four sweet basil (Ocimum basilicum) lines afforded identification of candidate flavonoid O-methyltransferase genes. Recombinant proteins displayed distinct substrate preferences and product specificities that can account for all detected 7-/6-/4'-methylated, 8-unsubstituted flavones. Their biochemical specialization revealed only certain metabolic routes to be highly favorable and therefore likely in vivo. Flavonoid O-methyltransferases catalyzing 49- and 6-O-methylations shared high identity (approximately 90%), indicating that subtle sequence changes led to functional differentiation. Structure homology modeling suggested the involvement of several amino acid residues in defining the proteins' stringent regioselectivities. The roles of these individual residues were confirmed by site-directed mutagenesis, revealing two discrete mechanisms as a basis for the switch between 6- and 4'-O-methylation of two different substrates. These findings delineate major pathways in a large segment of the flavone metabolic network and provide a foundation for its further elucidation.