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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.

Dipeptide and tripeptide permease A (DtpA) transporter is a bacterial homologue of the human PepT that is responsible for the uptake of di- and tripeptides from the small intestine and transports them...

Class D β-lactamases (DBLs) form a large family with nearly 1,300 identified members in the β-Lactamase Database (Fig. S1, www.bldb.euhttp://www.bldb.eu/) (1). They are produced mainly by Gram-negative bacteria, with some by Gram-positive bacteria (2). DBLs are an important mechanism of resistance to β-lactam antibiotics, including carbapenems, one of last resort treatments to infections in hospitalized patients (3). The production of class D β-lactamases is a major public health concern, as they can often transfer between different bacterial species, allowing resistance to spread quickly and widely. Some members were found to be membrane-bound and are secreted via outer membrane vesicles (OMVs) to contribute further in antimicrobial resistance dissemination (4). Their mechanism of action involves a catalytic serine, as in classes A and C β-lactamases, since all share the conserved SXXK tetrad and KXG triad (3).

Class D β-lactamases (DBLs) form a large family with nearly 1,300 identified members in the β-Lactamase Database (Fig. S1, www.bldb.euhttp://www.bldb.eu/) (1). They are produced mainly by Gram-negative bacteria, with some by Gram-positive bacteria (2). DBLs are an important mechanism of resistance to β-lactam antibiotics, including carbapenems, one of last resort treatments to infections in hospitalized patients (3). The production of class D β-lactamases is a major public health concern, as they can often transfer between different bacterial species, allowing resistance to spread quickly and widely. Some members were found to be membrane-bound and are secreted via outer membrane vesicles (OMVs) to contribute further in antimicrobial resistance dissemination (4). Their mechanism of action involves a catalytic serine, as in classes A and C β-lactamases, since all share the conserved SXXK tetrad and KXG triad (3).