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

C-Glycosyltransferases have garnered attention owing to their ability to synthesize C-glycosides with high conversion and selectivity in one-pot reactions. Their potential in rational enzyme engineering makes them valuable for the synthesis of diverse C-glycosides. However, the detailed reaction mechanism remains unclear. To address this, we investigated the C-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 C-glycosylation. The reaction proceeds via 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 S_N2-like manner. The S_N2 step yields a non-aromatic intermediate, which can be rapidly converted to C-glycoside even without an enzymatic environment. The key residue that facilitates the rate-determining S_N2 step is His-27, which stabilizes phloretin via hydrogen bonding. Additionally, to clarify why alternative products such as O-glycosides are not formed, we also investigated the O-glycosylation pathway. Our calculations revealed that O-glycosylation was promoted by proton transfer from UDP-glucose, like C-glycosylation, but was suppressed by structural fixation due to hydrogen bonding among phloretin, glucose, and GgCGT.