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

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-L₉₅-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.