Discovery of Novel Plant <i>O</i> ‐Methyltransferases for Directed Methylation of Flavonoids and Stilbenoids

Flavonoid O ‐methylation (FOMT) is a major tailoring reaction that alters physicochemical properties and generates valuable methoxylated derivatives, but preparative biocatalysis remains limited by scarce enzymes and insufficient regioselective control. Here, we targeted a defined branch within a previously established phylogenetic tree of 3,873 putative flavonoid O ‐methyltransferases (OMTs) and used active‐site pocket residue patterns to guide enzyme selection. Heterologous expression of plant‐derived candidates in Escherichia coli yielded three soluble FOMTs from Carpinus fangiana (CfOMT), Glycine soja (GsOMT), and Rhodamnia argentea (RaOMT), for functional characterization. Using a substrate panel spanning flavones, flavanones, flavonols, a dihydrochalcone, a flavan‐3‐ol, and stilbenoids, we compared substrate scope, conversion, and regioselectivity. CfOMT showed high activity toward polyhydroxylated flavonoids and favored 7‐OH/4′‐OH methylation, affording single monomethylated products. GsOMT preferred polyphenolic substrates and produced defined 3′‐ and 2′‐ O ‐methylated products, including monomethylated resveratrol. RaOMT displayed broader substrate tolerance and supported sequential methylation of selected flavonols. Structural modeling and docking revealed conserved overall scaffolds but distinct pocket features that explain these selectivities. These findings expand the plant OMT toolbox and provide structure‐guided leads for engineering biocatalysts for preparative methoxylated polyphenols.