Mechanistic Insights into Amide Bond Formation Catalyzed by the Condensation CcbD Enabled by a Single-Backbone Oxyanion Hole

CcbD catalyzes a key condensation step in lincosamide biosynthesis via a Cys-His-Glu catalytic triad, forming an amide bond between an ergothioneine-conjugated thiooctose and an amino acid substrate, either proline (Pro) or its modified derivative 4-propyl-l-proline (PPL), which is covalently linked to a carrier protein (CP). Despite its biological importance, the catalytic mechanism of the enzyme, specifically the elements that stabilize oxyanion intermediates, remains incompletely understood. Here, we combined an AlphaFold3-derived covalent complex model with molecular dynamics refinement and QM/MM umbrella sampling to characterize the acylation and deacylation reaction mechanisms in detail. Both steps proceed via single, concerted yet asynchronous mechanisms, with deacylation identified as the rate-limiting stage (Δ<i>G</i>^⧧ = 17.4 kcal/mol). Acylation involves proton transfer from the nucleophilic Cys17 to His131 coupled to nucleophilic attack, forming a tetrahedral intermediate, followed by thioester formation and CP release. Deacylation is initiated by His131-mediated deprotonation of the NH₂ group of ergothioneine-conjugated thiooctose, followed by nucleophilic attack on the Cys17-Pro covalent intermediate to form the amide bond. Calculations further reveal that the Cys17 backbone N-H serves as the sole hydrogen-bond donor of the oxyanion hole, with His150 stabilizing the local loop conformation. These mechanistic insights not only highlight principles for designing compact catalytic triads exploiting single-backbone oxyanion stabilization, but also provide a foundation for future CcbD protein engineering aimed at producing non-natural lincomamides with diverse acyl and sugar moieties, thereby offering promising avenues for drug discovery.