A Two-Water-Assisted Deacylation Pathway in Class C β-Lactamase ACC-1 Revealed by High-Resolution Crystallography and QM/MM Calculations

Abstract β-Lactam antibiotics are among the most widely prescribed antibacterial agents, yet their clinical efficacy is increasingly compromised by β-lactamase-mediated resistance. Class C β-lactamase ACC-1 hydrolyzes third-generation cephalosporins such as cefotaxime through a covalent acyl-enzyme intermediate, but the molecular basis of deacylation remains unresolved. Here, we report high-resolution synchrotron and room-temperature X-ray free-electron laser (XFEL) crystal structures of the cefotaxime-acylated ACC-1. These structures reveal a previously unrecognized two-water configuration near the acyl center, comprising the previously proposed deacylating water, Wat1, and an additional ordered water molecule, Wat2, positioned at a Bürgi–Dunitz angle suitable for nucleophilic attack. Quantum mechanics/molecular mechanics (QM/MM) calculations identify a previously unrecognized two-water-assisted deacylation pathway in which the substrate C4-carboxylate and Wat1 cooperatively activate Wat2 as the nucleophilic deacylating water. This pathway exhibits a substantially lower activation free-energy barrier than the alternative substrate-assisted and Tyr150–Lys67 general-base pathways examined. Together, these findings revise the deacylation mechanism of class C ACC-1 β-lactamase by revealing how an ordered water network and the substrate C4 carboxylate can cooperate to promote acyl-enzyme hydrolysis. This atomic-level mechanism provides a foundation for rational design strategies aimed at developing β-lactam antibiotics and β-lactamase inhibitors that resist or disrupt enzymatic hydrolysis.