A spontaneous proton transfer is key for enzymatic C-glycosylation and restricts the scope of natural C-glycosides

C -glycosides are valuable compounds containing hydrolytically stable C-C bonds. However, their scarcity in nature and their complex synthesis limit their availability. Enzymes represent an environmentally mild paradigm for the synthesis of C -glycosides, but only few enzymes with C -glycosylation activity are known and their catalytic mechanism remains unclear. In this work, we study the intricacies of a C -glycosyltransferase using X-ray crystallography, biochemical assays, and atomistic simulations. We identify two dynamic gates that control substrate access and reactivity, and investigate the molecular mechanism of C -glycosylation, identifying an S _E Ar stepwise process along a critical intermediate that stabilizes through a spontaneous water-mediated proton transfer. This stabilization is related to the chemical properties of the substrate, which dictate whether a compound can be C -glycosylated. Our results provide detailed knowledge and enhance our understanding of this class of enzymes, paving the way for their widespread utilization and engineering.