Mechanistic elucidation of enzymatic <i>C</i> -glycosylation: facilitation by proton transfer to UDP-glucose

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