Functional characterization of the methyltransferase PcOMT4 from Pogostemon cablin (Blanco) Benth.

BACKGROUND: Pogostemon cablin (Blanco) Benth, a traditional Chinese medicine, contains flavonoids with approximately 64% specifically methylated at the C-7 position. However, the specific O-methyltransferase (OMT) responsible for this modification remains uncharacterized. METHODS AND RESULTS: Five PcOMT genes were cloned from P. cablin via genomic screening and BLAST analysis. The functional characterization of PcOMT4 was conducted using in vitro enzymatic assays with 18 individual compounds and total flavonoid extracts. Virus-induced gene silencing (VIGS) combined with non-targeted metabolomic analysis was performed to verify its in vivo function. In vitro enzymatic assays and virus-induced gene silencing (VIGS) demonstrated that PcOMT4 exhibits high regioselectivity for C-7 methylation as well as catalytic promiscuity. Molecular docking in conjunction with site-directed mutagenesis was employed to identify key amino acid residues critical for catalysis and substrate recognition, including Phe142, Met146, Leu200, Val216, Asn219, Met220, Pro225, His237, Asn238, Ala282, and Glu296. CONCLUSIONS: This work represents the first functional characterization of an OMT in P. cablin, providing critical insights for genetic improvement aimed at enhancing the production of clinically valuable flavonoid glycosides in this medicinal plant.