Mechanistic elucidation of enzymatic -glycosylation: facilitation by proton transfer to UDP-glucose.

-Glycosyltransferases have garnered attention owing to their ability to synthesize -glycosides with high conversion and selectivity in one-pot reactions. Their potential in rational enzyme engineering makes them valuable for the synthesis of diverse -glycosides. However, the detailed reaction mechanism remains unclear. To address this, we investigated the -glycosylation of phloretin catalyzed by the glycosyltransferase GgCGT in the presence of the coenzyme UDP-glucose. Using density functional theory (DFT) calculations on a cluster model, we identified the most favorable pathway for -glycosylation. The reaction proceeds an initial proton transfer from phloretin to UDP-glucose, followed by the nucleophilic attack of phloretin on the glucose moiety and subsequent dissociation of UDP in an S2-like manner. The S2 step yields a non-aromatic intermediate, which can be rapidly converted to -glycoside even without an enzymatic environment. The key residue that facilitates the rate-determining S2 step is His-27, which stabilizes phloretin hydrogen bonding. Additionally, to clarify why alternative products such as -glycosides are not formed, we also investigated the -glycosylation pathway. Our calculations revealed that -glycosylation was promoted by proton transfer from UDP-glucose, like -glycosylation, but was suppressed by structural fixation due to hydrogen bonding among phloretin, glucose, and GgCGT.