Insights into the Enhanced Ceftazidime Hydrolysis by Ent385 AmpC β‑Lactamase from Multiscale Simulations

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.