Enzymatic and structural basis of lignan methylation diversity in Schisandra chinensis accelerates the discovery of anticancer drug resistance reversers

Multidrug resistance severely limits chemotherapy efficacy in lung adenocarcinoma. Dibenzocyclooctadiene (DBCOD) lignans isolated from Schisandra chinensis have been reported to reverse drug resistance, and our findings demonstrate that polymethoxylated DBCOD lignans with varying methylation patterns differ in their ability to enhance the sensitivity of paclitaxel-resistant lung adenocarcinoma cells. However, the enzymatic mechanisms governing the formation of polymethylated lignans remain largely elusive. Herein, we characterized three functional O -methyltransferases (OMTs) from S. chinensis : SchOMT2 functions as a promiscuous catalyst that mediates both mono- and di-methylation of diverse lignan scaffolds, including dibenzocyclooctadiene-type ( e.g. , gomisin J) and dibenzylbutane-type ( e.g. , pregomisin) lignans, whereas SchOMT6 and SchOMT7 exhibit strict substrate specificity. Virus-Induced Gene Silencing (VIGS) of SchOMT2 in S. chinensis leaves reduced the accumulation of multiple polymethylated lignans, confirming its catalytic role in S . chinensis . Moreover, the structures of SchOMT2 and its complex with SAM and SAM-gomisin J elucidates the molecular basis of substrate recognition. Mutagenesis studies revealed that key residues responsible for divergent substrate specificity are closely correlated with the size and hydrophobicity of the catalytic pockets, governing regioselectivity and playing a critical role in the catalytic promiscuity. These results establish a mechanistic link between lignan methylation diversity and anticancer drug-resistance reversal, and provide a structural roadmap for engineering O -methyltransferases to optimize lignan derivatives. Schisandra chinensis O -methyltransferases (OMTs) mediate lignan polymethylation, producing polymethylated products that inhibit P-glycoprotein (P-gp) and reverse drug resistance in lung adenocarcinoma. The crystal structure of SchOMT2 reveals the molecular basis of its substrate scope and regioselectivity, and provides structural insights informing enzyme engineering.