Identification and characterization of two -methyltransferases methylating -glycosylflavonoids.

accumulates several active substances, including -glycosylflavonoids, which have important pharmacological activities and health benefits. However, enzymes catalyzing the methylation step of -glycosylflavonoids in remain unknown. In this study, three -methyltransferases (OMTs) were identified from that have the capacity for -methylation of the -glycosylflavonoid isoorientin. The Type II OMTs IiOMT1 and IiOMT2 efficiently catalyze isoorientin to form isoscoparin, and decorate one of the aromatic vicinal hydroxyl groups on flavones and methylate the C6, C8, and 3'-hydroxyl positions to form oroxylin A, wogonin, and chrysoeriol, respectively. However, the Type I OMT IiOMT3 exhibited broader substrate promiscuity and methylated the C7 and 3'-hydroxyl positions of flavonoids. Further site-directed mutagenesis studies demonstrated that five amino acids of IiOMT1/IiOMT2 (D121/D100, D173/D149, A174/A150R, N200/N176, and D248/D233) were critical residues for their catalytic activity. Additionally, only transient overexpression of Type II OMTs and in significantly increased isoscoparin accumulation, indicating that the Type II OMTs and could catalyze the methylation step of -glycosylflavonoid, isoorientin at the 3'-hydroxyl position. This study provides insights into the biosynthesis of methylated -glycosylflavonoids, and IiOMTs could be promising catalysts in the synthesis of bioactive compounds.