Engineering Substrate Promiscuity and Catalytic Efficiency of Benzylisoquinoline Alkaloids <i>N</i> -Methyltransferases

Benzylisoquinoline alkaloids (BIAs) represent a diverse class of plant-derived secondary metabolites with significant medicinal and agricultural values. N -methylation is a common structural feature influencing the BIA bioactivity and bioavailability. Here, we report the characterization and rational engineering of two novel BIA N -methyltransferases (NMTs), SyNMT1 and SyNMT2 from Stephania yunnanensis . Key residues governing substrate promiscuity and catalytic efficiency were identified (A216 / F217 in SyNMT1; E218 / L219 / L223 in SyNMT2). Notably, the SyNMT2-L219F variant was engineered into a specific BIA 6OMT, representing the first instance of functional conversion from N - to O -methylation. This strategy was further extended to PsCNMT from Papaver somniferum, where the E197A/L198F mutation altered its substrate specificity and enhanced catalytic efficiency, increasing the K cat and K cat / K m values by 1.22- and 1.07-fold, respectively. This work provides a blueprint for engineering both the substrate promiscuity and catalytic efficiency of BIA NMTs, enriching the enzymatic toolkit for producing medicinally and agriculturally relevant BIAs.