Functional characterization and molecular engineering of a O-methyltransferase involved in bis-benzylisoquinoline alkaloids biosynthesis from Nelumbo nucifera

Nelumbo nucifera (lotus), a traditional aquatic plant in Asia, is valued for its nutritional and therapeutic properties. Benzylisoquinoline alkaloids (BIAs) are its major bioactive components, with significant bioactivities and pharmacological values. O-methyltransferases (OMTs) play a crucial role in shaping the structural and functional diversity of BIAs. Herein, we characterized a specific OMT, designated Nn7OMT, which exhibits a novel catalytic function: methylating the C7 position of the bisbenzylisoquinoline (bisBIA) skeleton. Nn7OMT also displayed substrate promiscuity, catalyzing O-methylation at the C7 position of bisBIAs, C6/C7 positions of 1-benzylisoquinolines, C7 position of aporphines, and C9 position of protoberberines, with isoliensinine as its preferred substrate. The expression profile of Nn7OMT correlates with bisBIA accumulation in planta, supporting its involvement in bisBIA biosynthesis in N. nucifera. Protein engineering guided by molecular docking and molecular dynamics simulations identified key residues critical for Nn7OMT activity. Mutants M158A and V306A retained catalytic activity toward isoliensinine while showing nearly undetectable activity against 1-benzylisoquinoline alkaloids, thereby significantly improving Nn7OMTs substrate specificity. These findings advance our understanding of BIAs biosynthesis in lotus and provide valuable biocatalysts with enhanced specificity. SIGNIFICANCEIn this study, we report the innovative discovery and characterization of Nn7OMT, the first O-methyltransferase isolated from Nelumbo nucifera that harbors a novel catalytic activity toward the bisbenzylisoquinoline alkaloid (bisBIA) backbone. Nn7OMT displayed substrate promiscuity, catalyzing methylation at the C7 position of the bisbenzylisoquinoline skeleton (isoliensinine), C6/C7 positions of the 1-benzylisoquinoline skeleton (norcoclaurine, coclaurine, N-methylcoclaurine), C7 position of the aporphine skeleton (lirinidine), and C9 position of the protoberberine skeleton (scoulerine)--with isoliensinine as its most preferred substrate. Innovatively, the catalysis of isoliensinine by Nn7OMT was a novel function and the first O-methyltransferase identified to catalyze the bisbenzylisoquinoline backbone, which may be involved in the biosynthesis of neferine. Nn7OMTs expression profile correlated with bisBIA accumulation in planta, further supporting its role in bisBIA biosynthesis in N. nucifera. We further engineered Nn7OMT using molecular docking and MD simulation-guided strategies. Mutants M158A and V306A retained catalytic activity for isoliensinine while showing nearly undetectable activity against 1-benzylisoquinoline alkaloid substrates. This targeted engineering significantly improved the specificity of Nn7OMT, rendering it highly suitable for methylated isoliensinine biosynthesis by reducing by-product accumulation and simplifying purification processes. Our work deepens understanding of the unique BIA metabolic pathway in N. nucifera through the identification of the first bisBIA-catalyzing O-methyltransferase, and provides valuable biocatalysts with enhanced substrate specificity.