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Abstract
The emergence of multidrug-resistant bacteria poses a significant threat to public health. Particularly, they are becoming increasingly resistant to β-lactam antibiotics, 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 C E. cloacae AmpC 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. First, 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 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 was in very good agreement with the difference deduced from experimental kinetic data. Detailed analysis of the transition state ensembles, alongside additional simulations, show 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. This can contribute to understand other reported Class C beta-lactamase variants that confer reduced susceptibility to antibiotic treatment.
Supplementary materials
Title
Supporting Information
Description
Details of molecular mechanics molecular dynamics simulations, description of QM regions in QM/MM simulations (Figure S1), free and potential energy surfaces, including DFT corrected energies (Figure S2), AmpC Ent385 vs. AmpC P99 sequence alignment (Figure S3), histograms of key interaction distances (Figure S4-S5, S7 and S9), and structural representations of reaction intermediates in Path 2 (Figure S6). We also provide interaction fingerprints for ceftazidime in the AmpC P99 and Ent385 acyl-enzymes (Figure S8), and distances relevant to catalytic activity (Figure S10).
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