Structural Superimposition

Abstact

Benzylisoquinoline alkaloids (BIAs) hold broad pharmaceutical potential, yet poor water solubility and low bioavailability limit their application. Glycosylation improves these properties, but glycosyltransferases (GTs) efficiently catalyzing diverse BIAs remain scarce. Here, we identify UGT74AN1 from Asclepias curassavica, capable of glycosylating various BIAs. Structure-guided semirational engineering yielded the double mutant UGT74AN1M2, exhibiting a 341-fold increase in catalytic efficiency. Molecular dynamics simulations revealed that these mutations widen the substrate channel and strengthen binding. To overcome UDP-glucose dependency, we designed the fusion enzyme AtSuSy-L12-UGT74AN1M2 via the iMARS platform, enabling in situ UDP-glucose generation coupled with highly efficient BIA glycosylation. Consequently, the synthesized dihydrojatrorrhizine-3-O-beta-d-glucoside (1b) demonstrated superior antitumor activity compared to its aglycone through stronger proliferation inhibition and apoptosis induction. This work provides an efficient enzymatic toolkit for green BIA glycoside synthesis and identifies promising candidates for drug development.