Characterization and Molecular Engineering of a N-Methyltransferase from Edible Nelumbo nucifera Leaves Involved in Nuciferine Biosynthesis

Lotus leaf, traditionally used as both edible tea and herbal medicine in Asia, contains nuciferine, a lipid-lowering and weight-loss compoud. The biosynthetic pathways of nuciferine in Nelumbo nucifera remain unclear. We characterized a specific N-methyltransferase, NnNMT, which had a novel function and catalyzed only nuciferine synthesis from the aporphine-type alkaloid N-nornuciferine. The expression profile of NnNMT was in agreement with BIA accumulation patterns in four tissues from three varieties, suggesting that NnNMT is involved in nucleiferine biosynthesis in Nelumbo nucifera. Protein engineering based on molecular docking and dynamic simulations revealed key residues (Y98, H208, F256, Y81, F329, G260, P76, and H80) crucial for NnNMT activity, with the F257A mutant showing increased efficiency. These findings enhance our understanding of aporphine alkaloid biosynthesis and support the development of lotus-based functional foods and medicinal applications.