Tautomer-Specific Deacylation and Ω-Loop Flexibility Explain the Carbapenem-Hydrolyzing Broad-Spectrum Activity of the KPC-2 β-Lactamase

Abstract

KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-β-lactamase (SBL) responsible for extensive β-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate β-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent β-lactams, evade the activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently deacylate carbapenem acyl-enzymes. We present high-resolution (1.25-1.4 Å) crystal structures of KPC-2 acyl-enzymes with representative penicillins (ampicillin), cephalosporins (cefalothin), and carbapenems (imipenem, meropenem, and ertapenem) obtained utilizing an isosteric deacylation-deficient mutant (E166Q). The mobility of the Ω-loop (residues 165-170) negatively correlates with antibiotic turnover rates (kcat), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different β-lactams. Carbapenem-derived acyl-enzyme structures reveal the predominance of the Δ1-(2R) imine rather than the Δ2 enamine tautomer. Quantum mechanics/molecular mechanics molecular dynamics simulations of KPC-2:meropenem acyl-enzyme deacylation used an adaptive string method to differentiate the reactivity of the two isomers. These identify the Δ1-(2R) isomer as having a significantly (7 kcal/mol) higher barrier than the Δ2 tautomer for the (rate-determining) formation of the tetrahedral deacylation intermediate. Deacylation is therefore likely to proceed predominantly from the Δ2, rather than the Δ1-(2R) acyl-enzyme, facilitated by tautomer-specific differences in hydrogen-bonding networks involving the carbapenem C-3 carboxylate and the deacylating water and stabilization by protonated N-4, accumulating a negative charge on the Δ2 enamine-derived oxyanion. Taken together, our data show how the flexible Ω-loop helps confer broad-spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the Δ2-enamine acyl-enzyme tautomer.