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. <i>N</i>-methylation is a common structural feature influencing the BIA bioactivity and bioavailability. Here, we report the characterization and rational engineering of two novel BIA <i>N</i>-methyltransferases (NMTs), SyNMT1 and SyNMT2 from <i>Stephania yunnanensis</i>. 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 <i>N</i>- to <i>O</i>-methylation. This strategy was further extended to PsCNMT from <i>Papaver somniferum</i>, where the E197A/L198F mutation altered its substrate specificity and enhanced catalytic efficiency, increasing the <i>K</i>_cat and <i>K</i>_cat/<i>K</i>_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.