Reaction mechanism and regioselectivity of uridine diphosphate glucosyltransferase RrUGT3: a combined experimental and computational study

Abstract

Uridine diphosphate glucosyltransferase (UGT) from Rhodiola rosea shows high selectivity towards the phenolic hydroxyl group in the glucosylation of p-hydroxybenzyl alcohol (HBA). However, the detailed catalytic mechanism, including the substrate binding mechanism and the origins of the regioselectivity remain unclear. In this work, we solved the crystal structure of the enzyme and elucidated the molecular basis of the catalytic mechanism using site-directed mutagenesis and multiscale computational studies. Our results indicate that the conformational change of a loop region from the “open” to the “closed” state is crucial for the formation of the active enzyme–substrate ternary complex, and the loop conformation change is favored by substrate binding. The quantum mechanical/molecular mechanical (QM/MM) and quantum chemical calculation results suggest that the catalytic power of RrUGT3 mainly originates from the preorganized protein environment that stabilizes the catalytically active state of the sugar donor uridine-5′-diphosphate-glucose (UDPG). Moreover, the favored substrate binding and lower activation barrier of the phenolic glycosylation dictate the regiopreference of the enzyme. These results expand our knowledge on the catalytic mechanism of UGTs, and will facilitate the rational modification and design of highly active and regioselective UGT variants for the practical production of glycosides.