Abstract. c-Myc is an oncogene that is dysregulated in ∼70% of cancers. Its multifaceted function complicates effective drug targeting of the protein. i-Mo

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The emergence of multidrug-resistant bacteria poses a significant threat to public health. Particularly, they are becoming increasingly resistant to β-lactam antibiotics, which are one of the most important drug classes for the treatment of bacterial infections. Ceftazidime-avibactam has shown promising activity against highly drug-resistant bacteria, including carbapenem-resistant Enterobacterales. However, an Ala294-Pro295 deletion in the Class CE. cloacaeAmpC β-lactamase can confer reduced susceptibility to these agents. In this study, we investigated the molecular mechanisms underlying the enhanced hydrolysis of ceftazidime by E. cloacae Ent385 AmpC β-lactamase with the deletion using quantum mechanics/molecular mechanics (QM/MM) simulations. We used constant pH molecular dynamics simulations of the β-lactamase-ceftazidime acyl-enzyme complex to verify the likely protonation states, confirming Tyr150 primarily exists as a tyrosinate. We then used QM/MM (DFTB2/ff14SB) umbrella sampling to calculate the reaction-free energy barriers (Δ‡G) for the deacylation step of cephalosporin hydrolysis. This reveals that Tyr150 (rather than the substrate) acts as the base. Importantly, the difference in Δ‡G between the canonical E. cloacae AmpC (P99) and the Ent385 variant with Ala294-Pro295 reinserted, on the one hand, and the Ent385 variant, on the other, was in very good agreement with the difference deduced from experimental kinetic data. Detailed analysis of the transition state ensembles, alongside additional simulations, shows that the Ala294-Pro295 deletion allows the entrance of an additional water molecule that helps stabilize the tetrahedral intermediate. Overall, our QM/MM simulations provide valuable insights into the reaction mechanism and reasons for enhanced ceftazidime breakdown. The protocol used in this study successfully captures the kinetic differences observed among the studied variants. This approach can be employed to investigate other Class C β-lactamase variants with similar features, providing insights into their mechanisms and potential contributions to reduced susceptibility to antibiotic treatments.