Glycyrrhiza inflata O-methyltransferases characterization for one-step production of trimethyl myricetin/dihydromyricetin

This study lays a foundation for trimethylated flavonoid biosynthesis. • Two novel flavonoid OMTs with 3′,4′,5′- O -trimethylation were identified and characterized. • Key residue driving trimethylation was identified and verified. • Trimethylation enhanced the antibacterial activity of myricetin. Flavonoids are plant specialized metabolites widely distributed in plants with many health benefits. O -methyltransferases (OMTs) are pivotal in plant specialized metabolism by producing methylated derivatives with enhanced biological activity. In this study, we identified two novel flavonoid OMTs, GiOMT4 and GiOMT9, from Glycyrrhiza inflata , a medicinal licorice species commonly used in Chinese medicine. These enzymes could catalyze myricetin and dihydromyricetin to produce 3′,4′,5′- O -trimethyl derivatives in a reaction. Structural modeling and site-directed mutagenesis identified Trp193 as critical residue determining the specificity of trimethylation of GiOMT4. Notably, trimethylation markedly enhanced the antibacterial activity of flavonoids against Gram-positive bacteria, including Methicillin-Resistant Staphylococcus aureus and Vancomycin-Resistant Enterococcus . Furthermore, trimethyl dihydromyricetin was identified as a novel compound with unexplored pharmacological potential. Collectively, this study expands the enzymatic repertoire of licorice OMTs, provides new insights into the structure-function relationships underling multiple methylations, and establishes a foundation or sustainable biocatalytic production of highly bioactive methylated flavonoids in G. inflata and related species.