Functional Characterization of Two Methyltransferases Involved in Benzylisoquinoline Alkaloid Biosynthesis in Sinomenium acutum.

Benzylisoquinoline alkaloids (BIAs) are a diverse group of plant secondary metabolites with significant pharmacological activities, where methylation reactions catalyzed by methyltransferases play crucial roles in structural diversification and bioactivity modulation. In this study, we identified and functionally characterized two key methyltransferases, Sinomenium acutum 6-O-methyltransferase (Sa6OMT) and S. acutum coclaurine N-methyltransferase (SaCNMT), involved in BIA biosynthesis in S. acutum, a traditional Chinese medicinal plant with anti-rheumatoid arthritis properties. Candidate genes, including SaOMT3 and SaNMT4, were selected through transcriptome analysis of different tissues combined with phylogenetic analysis and expression pattern evaluation. Functional validation demonstrated that SaOMT3 encodes a 6OMT that catalyzes the conversion of (S)-norcoclaurine to (S)-coclaurine, while SaNMT4 encodes a CNMT that mediates the N-methylation of (S)-coclaurine to (S)-N-methylcoclaurine. Biochemical characterization revealed optimal activity at 40°C for both enzymes, with distinct optimal pH levels (8.5 for Sa6OMT and 8.0 for SaCNMT) and higher catalytic efficiency for SaCNMT. This study provides valuable insights into the molecular mechanisms underlying BIA biosynthesis in S. acutum and enhances our understanding of plant specialized metabolism.