[Cloning and functional characterization of O-methyltransferase genes in lignan synthesis pathway of Schisandrae Sphenantherae Fructus].

Dibenzocyclooctene lignans are the primary bioactive constituents of the TCM Schisandrae Sphenantherae Fructus,exhibiting diverse pharmacological activities including hepatoprotective,anti-inflammatory,and antioxidant effects. Their biosynthesis in plants involves the conversion of pregomisin catalyzed by various enzymes,a process that includes multiple O-methylation steps,which are predominantly catalyzed by O-methyltransferase(OMT). This study cloned an OMT gene,designated Ssp OMT13,from Schisandra sphenanthera. The open reading frame of Ssp OMT13 was 1 053 bp in length,encoding a protein of 350 amino acids. Bioinformatics analysis revealed that Ssp OMT13 contained a characteristic methyltransferase domain,classifying it as a type Ⅰ methyltransferase. The recombinant prokaryotic expression plasmid was constructed,and the protein was expressed in Escherichia coli and purified. In vitro enzymatic assays demonstrated that Ssp OMT13 possessed dual catalytic activities: it catalyzed the conversion of gomisin J to gomisin K1,and further catalyzed the conversion of gomisin K1 to schisandrin A. Molecular docking combined with sitedirected mutagenesis identified key amino acid residues Pro27,Leu30,Val110,Met113,and Trp299,which were critical for its catalytic activity. This study expands the understanding of key enzyme genes involved in lignan secondary metabolism and provides a significant research basis for elucidating the mechanism of quality formation of Schisandrae Sphenantherae Fructus.