A Golgi‐localized N‐methyltransferase and reversible aldo–keto reductases coordinate dual terminal routes in galanthamine biosynthesis

SUMMARY

Galanthamine, a therapeutic Amaryllidaceae alkaloid produced exclusively by species within the Amaryllidoideae subfamily, is a key treatment for early‐stage symptoms of Alzheimer's disease. Elucidating its biosynthetic pathway is essential for strategies aimed at enhancing production through metabolic engineering. Galanthamine 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 aldo–keto reductase (AKR) candidates ( La AKR1, La AKR2, and La AKR3) and three N ‐methyltransferase (NMT) candidates from Leucojum aestivum : La NMT, homologous to coclaurine N‐methyltransferase‐like (NMT‐like), and two γ‐tocopherol methyltransferases (TMT) homologs, La TMT1 and La TMT2. Subcellular localization studies revealed distinct compartmentalization, with La NMT targeted to the ER‐cytosol, La TMT1 to plastids, and La TMT2 to the Golgi apparatus. In vitro, La TMT2 methylated both nornarwedine and norgalanthamine, with a kinetic preference for nornarwedine. La TMT1 methylated γ‐tocopherol to α‐tocopherol (vitamin E). All three AKRs catalyzed reversible interconversions between nornarwedine and norgalanthamine, and between narwedine and galanthamine, with La AKR3 favoring the reduction reaction whereas La AKR1 the oxidation reaction. These findings identify La TMT2 and La AKRs as key branch‐enabling enzymes, reconcile long‐standing models of galanthamine biosynthesis, and provide a strategic target for metabolic engineering strategies to enhance galanthamine production.