Evolution of carbapenemase activity in the class C β-lactamase ADC-1

Antibiotic resistance in bacteria poses a significant threat to public health. Among dozens of available antimicrobial agents, carbapenems are used as drugs of choice for the treatment of serious infections caused by pathogens resistant to other antibiotics. However, their usefulness has been severely compromised due to the emergence and wide spread of carbapenem-resistant clinical isolates worldwide. High-level resistance to carbapenems in bacteria is mediated by the production of β-lactamases from three molecular classes, A, B, and D, but not by class C enzymes. In this study, we selected a triple mutant of the intrinsic class C Acinetobacter-derived cephalosporinase ADC-1 (ADC-1_TM) that confers high-level resistance to the carbapenems meropenem, ertapenem, and doripenem. Kinetic experiments demonstrated that the apparent binding affinity, along with the acylation and deacylation rates, were all improved for the mutant enzyme. X-ray crystallography, molecular docking, and molecular dynamics simulations revealed that the amino acid substitutions in ADC-1_TM produce significant changes in the enzyme active site architecture and binding mode of the carbapenem ertapenem. These changes allow for better positioning of a deacylating water for nucleophilic attack, thus explaining the significantly improved rate of ertapenem deacylation by ADC-1_TM. In this study, we showed for the first time that a class C β-lactamase can produce high-level resistance to carbapenem antibiotics, which underlines the potential for enzymes of this class to evolve such resistance and could further exacerbate the problem of antibiotic resistance in bacteria.IMPORTANCECarbapenems belong to the most widely used family of β-lactam antibiotics and are considered drugs of choice for difficult-to-treat and often deadly infections. Widespread carbapenem-resistant isolates have drastically diminished the utility of these important antibiotics and resulted in high mortality rates. Resistance to carbapenems in clinical pathogens is mainly due to the production of β-lactamases, enzymes that destroy these drugs. Out of the four molecular classes of β-lactamases, various enzymes belonging to classes A, B, and D produce high levels of resistance to carbapenems; however, enzymes of class C have failed to evolve such resistance. Here, we demonstrate that the intrinsic ADC-1 β-lactamase of the clinically important pathogen Acinetobacter baumannii can evolve high-level resistance to carbapenems by just three amino acid substitutions and disclose the molecular mechanisms of its carbapenemase activity. This study demonstrates the potential for the evolution of carbapenemase activity in class C β-lactamases.