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

01 April 2025, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

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 the 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 SN2-like manner. The SN2 step yields non-aromatic intermediate, which can be rapidly converted to C-glycoside even without an enzymatic environment. The key residue that facilitates the rate-determining SN2 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.

Supplementary materials

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Supporting Information
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Detalis of calculated results and Cartesian coordinates of intermediates and transition states.
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