Branch-Enabling N-Methyltransferase in Golgi Reconciles Divergent Models of Galanthamine Biosynthesis

Galanthamine, a therapeutic Amaryllidaceae alkaloid produced exclusively by species within the Amaryllidoideae subfamily, is a key treatment for early-stage symptoms of Alzheimers disease. Elucidating its biosynthetic pathway is essential for strategies aimed at enhancing production through metabolic engineering. Galantamine derives from the metabolic precursor 4'-O-methylnorbelladine, which undergoes cytochrome P450-mediated para-ortho C-C phenol coupling to yield nornarwedine. Two competing terminal routes have been proposed: (i) reduction of nornarwedine to norgalanthamine, followed by N-methylation, or (ii) N-methylation of nornarwedine to narwedine prior to reduction. Here, we identify three N-methyltransferase (NMT) candidates from Leucojum aestivum: LaNMT, related to coclaurine NMTs, and two {gamma}-tocopherol methyltransferases (TMT) homologs, LaTMT1 and LaTMT2. Subcellular localization studies revealed distinct compartmentalization, with LaNMT targeted to the ER-cytosol, LaTMT1 to plastids, and LaTMT2 to the Golgi apparatus. In vitro enzyme assays demonstrated that LaTMT2 methylates both nornarwedine and norgalanthamine, with a kinetic preference for nornarwedine. Agroinfiltration for transient expression in Nicotiana benthamiana further confirmed LaTMT2 as a catalytically efficient and substrate-promiscuous enzyme that supports both terminal routes. These findings identify LaTMT2 as a key branch-enabling N-methyltransferase that reconcile long-standing models of galanthamine biosynthesis and provides a strategic target for metabolic engineering strategies to enhance galanthamine production. Significant statementThis study identifies LaTMT2, a Golgi-localized {gamma}-tocopherol methyltransferase homolog, as a branch-enabling N-methyltransferase that resolves competing models of galanthamine biosynthesis. By revealing an unanticipated Golgi-associated step in Amaryllidaceae alkaloid metabolism, it redefines the subcellular organization of specialized metabolic pathways and provides a strategic enzymatic target for metabolic engineering of high-value therapeutic alkaloids.