With the widespread use and abuse of antibiotics for the past decades, antimicrobial resistance poses a serious threat to public health nowadays. β-Lactams are the most used antibiotics, and β-lactamases the most widespread resistance mechanism. Class C β-lactamases, also known as cephalosporinases, usually do not hydrolyse the latest and most potent β-lactams, expanded spectrum cephalosporins and carbapenems. However, the recent emergence of extended-spectrum AmpC cephalosporinases, their resistance to inhibition by classic β-lactamase inhibitors, and the fact that they can contribute to carbapenem resistance when paired with impermeability mechanisms, means that these enzymes may still prove worrisome in the future.<br/><br/>Here we report a new class C β-lactamase, CMY-136, and characterize its structure, dynamics and hydrolytic properties. CMY-136 features a Y221H point mutation compared with CMY-2, which confers an increased level of resistance to ticarcillin, cefuroxime, cefotaxime and ceftolozane/tazobactam. CMY-136 is capable of hydrolysing ticarcillin and cloxacillin, which act as inhibitors of CMY-2. The X-ray structure of the apo enzyme shows an unusual backbone conformation of the Ω-loop residues 204-215 and the His221 residue partially occupying the active site. Covalent docking calculations evidenced steric clashes that might explain the reduced activity of some β-lactams on CMY-136 compared with CMY-2. Molecular dynamics simulations highlighted an increased flexibility around the R1 and R2 pockets in the active site, which may be correlated with increased kcat values, allowing the acyl-enzyme complexes to adopt more often a conformation competent for attack by the hydrolytic water. Overall, crystallographic and molecular modeling experiments suggest that the hydrolytic profile alterations are the result of an increased flexibility and a different conformation of the Ω-loop, caused by the Y221H mutation. These results constitute the structural basis for the design of more versatile β-lactamase inhibitors, adapted to the increased diversity of hydrolytic profiles of β-lactamases encountered in clinical settings.

Structure, dynamics, and hydrolytic properties of the class C CMY-136 β-lactamase

2019 Spring National Meeting

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