Mechanism of Bacterial Arginine N‑Glycosylation: A Chemically Challenging Post-Translational Modification

Arginine N-glycosylation is a post-translational modification that bacterial pathogens use to subvert host immunity, yet the catalytic activation of the intrinsically weak guanidinium nucleophile has remained unresolved. Based on structural data, a direct inverting S_N2 mechanism had been suggested, but alternative, more stepwise routes and the identity of the catalytic base could not be firmly established. Here, we delineate the molecular mechanism by which the nonlocus of enterocyte effacement (non-LEE)-encoded effector protein B1 (NleB1), a promising virulence factor of enteropathogens, transfers N-acetylglucosamine (GlcNAc) to arginine residues of host substrates. Using structural modeling, extensive molecular dynamics, and state-of-the-art QM/MM free-energy simulations combined with kinetic experiments, we elucidate the catalytic mechanism of NleB1. The reaction proceeds through a single-step, dissociative S_N2-type mechanism, with no stable intermediate. Proton transfer to the catalytic base occurs immediately after the transition state, and is preceded by distortion (loss of planarity) of the acceptor guanidinium that primes nucleophilic attack. The simulations unambiguously identify Glu253, rather than Asp186, as the general base, and reveal that Glu253 plays multiple roles: it disrupts the planar guanidinium conformation of the acceptor arginine to enhance nucleophilicity, orients Arg117, accepts its proton, and subsequently promotes product relaxation via guanidinium replanarization, while Asp186 acts structurally to stabilize the donor substrate. Together, these residues enable a chemically demanding transformation that challenges chemical expectations for guanidinium reactivity. This study provides a comprehensive mechanistic study of arginine N-glycosylation, resolving its long-standing mechanistic conundrum and establishing catalytic rules likely conserved among Arg-specific glycosyltransferases.