Structure‐Guided Engineering of a Promiscuous <i>O</i> ‐Methyltransferase for a SAM Regeneration Biocatalysis Platform of Methylated Pharmaceuticals

ABSTRACT O ‐Methylation catalyzed by plant O ‐methyltransferase plays a crucial role in both drug design and biosynthesis of natural products. However, their practical applications are often restricted by strict substrate specificity and a strong dependence on the expensive methyl donor S‐adenosyl‐L‐methionine (SAM). Herein, an O ‐methyltransferase, SmOMT, is identified from the medicinal plant Selaginella moellendorffii , exhibiting substrate promiscuity and regioselectivity. SmOMT catalyzed the methylation of 25 structurally diverse substrates and demonstrated detectable N ‐methylation activity. Combined ternary complex structure and molecular dynamics studies of SmOMT elucidate its catalytic and regioselectivity mechanisms. A double mutant, SmOMT M2 , with enhanced catalytic activity is obtained based on structural analysis. To overcome SAM dependence, a cascade system for SAM regeneration is successfully constructed by coupling SmOMT M2 with a mutant halide methyltransferase, AtHMT V140T . Employing the iMARS platform, a highly active fusion enzyme, AtHMT V140T ‐L 95 ‐SmOMT M2 , is designed. This fusion enzyme outperforms the free‐enzyme cascade system and facilitates the gram‐scale synthesis of a series of methylated compounds with enhanced anti‐inflammatory activity. This work provides a versatile methylating biocatalyst and establishes an efficient SAM regeneration methylation platform, overcoming limitations in enzymatic methylation and enabling the sustainable production of high‐value pharmaceuticals.