Coclaurine N-methyltransferase-like enzymes drive the final biosynthetic reaction of the anti-Alzheimer’s drug galanthamine in Amaryllidaceae

Galanthamine, an isoquinoline alkaloid used to treat symptoms of Alzheimer's disease, is predominantly extracted from Amaryllidaceae plants, yet its supply remains limited. In this study, we identified, isolated, and characterized N-methyltransferases (NMTs) from three galanthamine-producing species: Leucojum aestivum, Lycoris radiata, and Hippeastrum papilio. The transcriptomic analysis identified five unique NMT isoforms, among which LaLrHpNMT1, an isoform highly conserved across all three species, exhibited the highest catalytic activity. Phylogenetic and structural analyses revealed that these enzymes share high sequence conservation and maintain the class I methyltransferase Rossmann fold with key catalytic residues, paralleling known NMTs from benzylisoquinoline alkaloid pathways. Flexible docking simulations confirmed that norgalanthamine, a crucial precursor, fits within the enzyme's active site and interacts with conserved residues Glu204 and His208. In vitro and in planta assays demonstrated that LaLrHpNMT1 efficiently catalyzes the N-methylation of norgalanthamine to galanthamine. Site-directed mutagenesis confirmed the key role of Glu204 and the participation of Phe residues in substrate stabilization. Additional enzyme assays revealed that LaLrHpNMT1 is promiscuous towards various alkaloid intermediates, while subcellular localization using eGFP-tagged constructs exposed a dual distribution in the cytosol and endoplasmic reticulum, suggesting that NMT activity occurs at the cytosol-ER interface where other biosynthetic enzymes reside. Environmental stress experiments in H. papilio shoots culture showed significant upregulation of NMT expression under heat and other stress conditions associated with AA levels modulation, indicating a potential link between stress responses and alkaloid biosynthesis. These findings deepen our understanding of galanthamine biosynthesis and provide a foundation for metabolic engineering strategies aimed at improving production yields.