Tooke, C. L. | Search bioRxiv

bioRxiv2022

Tautomer-specific deacylation and {Omega}-loop flexibility explain carbapenem-hydrolyzing, broad-spectrum activity of the KPC-2 β-lactamase

KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-{beta}-lactamase (SBL) responsible for extensive {beta}-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate {beta}-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent {beta}-lactams, evade activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently catalyze deacylation of carbapenem-derived acyl-enzymes. We present high-resolution (1.25-1.4 [A]) 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). Mobility of the {Omega}-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 {beta}-lactams. Carbapenem-derived acyl-enzyme structures reveal predominance of the {Delta}1-(2R) imine tautomer, except for the imipenem acyl-enzyme, which is present in dual occupancy in both {Delta}1-(2R) and (2S) configurations. Quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations of deacylation of the KPC-2:meropenem acyl-enzyme, using an adaptive string method (ASM), show that the {Delta}1-(2R) isomer has a 7 kcal/mol higher barrier for the (rate-determining) formation of the tetrahedral deacylation intermediate than the {Delta}2 tautomer. The simulations identify tautomer-specific differences in hydrogen bonding networks involving the carbapenem C-3 carboxylate and the deacylating water, that, together with stabilization by protonated N-4 of accumulating negative charge during oxyanion formation, accelerate deacylation of the {Delta}2-enamine acyl-enzyme compared to the {Delta}1-imine. Taken together, our data show how the flexible {Omega}-loop helps confer broad spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the {Delta}2-enamine acyl-enzyme tautomer. Differentiation of the barriers associated with deacylation of these subtly different {beta}-lactam isomers further identifies ASM as a sensitive method for calculation of reaction energetics that can accurately model turnover and, potentially, predict the impact of substrate modifications or point mutations upon activity.

biochemistry↗

bioRxiv2021

Allosteric communication in Class A β-lactamases occurs via Cooperative Coupling of Loop Dynamics

Allosteric effects control protein (e.g. enzyme) activity in ways that are not fully understood. Better understanding of allosteric effects, and tools to identify them, would offer promising alternative strategies to inhibitor development. Through a combination of equilibrium and nonequilibrium molecular dynamics simulations, we identify allosteric effects and communication pathways from two distant ligand binding sites to important active site structural elements that control enzymatic activity in two prototypical class A {beta}-lactamases, TEM-1 and KPC-2. Both of these enzymes are important determinants of antibiotic resistance in widespread bacterial pathogens. The simulations show that the allosteric sites are connected to the active site in both enzymes, (e.g. affecting the conformation of the {Omega}-loop) highlighting how allosteric inhibitors may exert their effects. Nonequilibrium simulations reveal pathways of communication operating over distances of 30 [A] or more. In these identified signaling pathways, the propagation of the signal occurs through cooperative coupling of loop dynamics. Notably, 50% or more clinically relevant amino acid substitutions in each enzyme map onto the identified signal transduction pathways. This suggests that clinically important variation may affect, or be driven by, differences in allosteric behavior, providing a mechanism by which amino acid substitutions may affect the relationship between spectrum of activity, catalytic turnover and potential allosteric behavior in this clinically important enzyme family. Simulations of the type presented here will help in identifying and analyzing such differences.

biophysics↗