Enhancing resistance to cephalosporins in class C β-lactamases: Impact of Gly214Glu in CMY-2

The biochemical properties of CMY-32, a class C enzyme possessing a single-amino acid substitution in the Ω loop (Gly214Glu), were compared to those of the parent enzyme, CMY-2, a widespread class C β-lactamase. In parallel with our microbiological characterization, the Gly214Glu substitution in CMY-32 reduced catalytic efficiency (k_cat/K_m) by 50-70% against "good" substrates (i.e., cephalothin) while increasing k _cat/K_m against "poor" substrates (i.e., cefotaxime). Additionally, CMY-32 was more susceptible to inactivation by sulfone β-lactamase inhibitors (i.e., sulbactam and tazobactam) than CMY-2. Timed electrospray ionization mass spectrometry (ESI-MS) analysis of the reaction of CMY-2 and CMY-32 with different substrates and inhibitors suggested that both β-lactamases formed similar intermediates during catalysis and inactivation. We next showed that the carbapenems (imipenem, meropenem, and doripenem) form long-lived acyl-enzyme intermediates and present evidence that there is β-lactamase-catalyzed elimination of the C₆ hydroxyethyl substituent. Furthermore, we discovered that the monobactam aztreonam and BAL29880, a new β-lactamase inhibitor of the monobactam class, inactivate CMY-2 and CMY-32 by forming an acyl-enzyme intermediate that undergoes elimination of SO₃^2-.Molecular modeling and dynamics simulations suggest that the Ω loop is more constrained in CMY-32 than CMY-2. Our model also proposes that Gln120 adopts a novel conformation in the active site while new interactions form between Glu214 and Tyr221, thus explaining the increased level of cefotaxime hydrolysis.When it is docked in the active site,we observe that BAL29880 exploits contacts with highly conserved residues Lys67 and Asn152 in CMY-2 and CMY-32. These findings highlight (i) the impact of single-amino acid substitutions on protein evolution in clinically important AmpC enzymes and (ii) the novel insights into the mechanisms by which carbapenems andmonobactams interact with CMY-2 and CMY-32 β-lactamases.