Discovery of a novel flavonol O-methyltransferase possessing sequential 4'- and 7-O-methyltransferase activity from Camptotheca acuminata Decne.

The biosynthetic route for flavonol in Camptotheca acuminata has been recently elucidated from a chemical point of view. However, the genes involved in flavonol methylation remain unclear. It is a critical step for fully uncovering the flavonol metabolism in this ancient plant. In this study, the multi-omics resource of this plant was utilized to perform flavonol O-methyltransferase-oriented mining and screening. Two genes, CaFOMT1 and CaFOMT2 are identified, and their recombinant CaFOMT proteins are purified to homogeneity. CaFOMT1 exhibits strict substrate and catalytic position specificity for quercetin, and selectively methylates only the 4'-OH group. CaFOMT2 possesses sequential O-methyltransferase activity for the 4'-OH and 7-OH of quercetin. These CaFOMT genes are enriched in the leaf and root tissues. The catalytic dyad and critical substrate-binding sites of the CaFOMTs are determined by molecular docking and further verified through site-mutation experiments. PHE181 and MET185 are designated as the critical sites for flavonol substrate selectivity. Genomic environment analysis indicates that CaFOMTs evolved independently and that their ancestral genes are different from that of the known Ca10OMT. This study provides molecular insights into the substrate-binding pockets of two new CaFOMTs responsible for flavonol metabolism in C. acuminata.