Two O-methyltransferases are responsible for multiple O-methylation steps in the biosynthesis of furanocoumarins from Angelica decursiva

Angelica decursiva, an important traditional medicinal plant, possesses a unique pharmacological activity. Its principal active ingredients are coumarins, including scopoletin, bergapten, and imperatorin. However, the enzymes catalyzing the critical step of coumarins biosynthesis pathway remain unidentified. This study initially screened 14 candidate O-methyltransferases (OMTs) through transcriptomics and metabolic determination. Combined with gene expression profile and biochemical assays, two OMTs (AdOMT1 and AdOMT2) were identified to be responsible for the O-methylation of coumarins in A. decursiva. AdOMT1 showed higher catalytic efficiency for bergaptol (Kcat/Km = 3123.70), while AdOMT2 exhibited higher substrate and catalytic promiscuity, allowing it to catalyze the methylation of various coumarins, phenylpropanes, and flavonoids. Based on molecular docking and site-specific mutagenesis determined that His126/Asn132, Phe171/Phe177, Trp261/Trp267, and Asn312/Ile317 were the key catalytic residues of AdOMT1 and AdOMT2 for the O-methylation of bergaptol and xanthotoxol. Further phylogenetic analysis confirmed the reasons for the catalytic functional differentiation of AdOMT1 and AdOMT2. This study provides a basis for exploring the coumarins O-methylation mechanism and plays a critical role in diversifying the structures used in coumarins drug discovery.