Rational Engineering of a Sucrose Phosphorylase for Switchable Regioselective Synthesis of Antioxidant Gallic Acid α-Glucosides.

Abstract

Gallic acid is a bioactive polyphenol with poor solubility and stability. Enzymatic α-glucosylation can address these limitations, but regioselective synthesis among multiple phenolic hydroxyls remains challenging. Here, we engineer a sucrose phosphorylase from Bifidobacterium adolescentis (BaSP) for the switchable synthesis of gallic acid 4-O- and 3-O-α-d-glucopyranosides. Structure-guided rational design identifies four key regions controlling substrate orientation. The double mutant I231A/D400E achieves >90% selectivity for the 4-O isomer with 82% yield in 2 h. Strikingly, the mutant L341 V/T157G completely reverses regioselectivity, producing the 3-O isomer with >98% yield and >99% selectivity. Molecular docking reveals that these mutations remodel the active pocket and substrate channel, positioning the target hydroxyl group adjacent to the catalytic center. The resulting α-glucosides show up to 12-fold improved water solubility and superior cytoprotective, anti-inflammatory (IL-1β), and antioxidant (HO-1/NQO1) activities compared to gallic acid. This work provides two high-performance, regiocomplementary biocatalysts for green synthesis of pharmaceutically relevant gallic acid glycosides and offers mechanistic insights into regioselectivity control in sucrose phosphorylases.