Abstract C-glycosylation catalyzed by C-glycosyltransferases affords chemically robust C-glycosides, yet the mechanistic origin of the remarkable selectivity for C- over O-glycosylation remains poorly understood. In this study, we present a comprehensive QM/MM investigation of the GgCGT-catalyzed mono-C-glycosylation of phloretin using UDP-glucose, extending our previous cluster-model analysis to explicitly include the full enzymatic environment. The calculated pathway involves an initial proton transfer from phloretin to UDP-glucose, followed by an SN2-type C-glycoside bond forming step accompanied by dissociation of UDP. The activation barrier of the rate-determining SN2 step is in good agreement with the experimental estimates. Although the alternative O-glycosylation pathway exhibits a lower activation barrier, it is thermodynamically disfavored due to the instability of the O-glycosylated intermediate, which readily undergoes the reverse reaction. In contrast, the C-glycosylated intermediate is strongly stabilized, rendering C-glycosylation effectively irreversible. Structural and natural bond orbital analyses reveal that this stability originates from preservation of a planar His27-Asp122 dyad and a favorable hydrogen-bonding network involving the phosphate group. Furthermore, the C6-OH group of glucose directly stabilizes the key transition state, explaining the reduced reactivity observed experimentally with UDP-xylose. These results establish a dual catalytic strategy in C-glycosyltransferases, combining thermodynamic control for C-selectivity with transition-state stabilization for rate acceleration.
Terada et al. (Mon,) studied this question.