Molecular characterization of two O-methyltransferases involved in benzylisoquinoline alkaloid biosynthesis in Aristolochia debilis

Aristolochia plants contain pharmacologically important benzylisoquinoline alkaloids (BIAs) used in the formulation of traditional herbal drugs. However, owing to the nephrotoxicity of aristolochic acids (AAs) present in Aristolochiaceae plants, the use of herbal medicines derived from Aristolochia plants is prohibited. To modulate metabolic pathways, the characterization of critical biosynthetic enzymes responsible for the biosynthesis of AAs and other BIAs is indispensable. In this study, we investigated the enzymatic properties of two O-methyltransferases (OMTs) isolated from Aristolochia debilis, designated AdOMT1 and AdOMT2. The evaluation of OMT activity using purified recombinant proteins across a wide range of substrates, along with kinetic studies and docking simulations, revealed that AdOMT1 primarily catalyzed the 6-O-methylation of (R,S)-norcoclaurine, (R,S)-norlaudanosoline, and (R,S)-laudanosoline, whereas AdOMT2 was highly active in catalyzing the 7-O-methylation of (S)-coclaurine. Additionally, the 7-O-methylation activity of AdOMT1 against 6-O-methylated substrates and the sequential dual-O-methylation activity of AdOMT2 against (R,S)-norcoclaurine were observed. Our results offer important perspectives on the molecular evolution and substrate recognition of OMTs in plants and identify the key enzymes responsible for BIA biosynthesis in Aristolochia species.