Multiscale simulations identify origins of differential carbapenem hydrolysis by the OXA-48 β-lactamase

Hirvonen, Viivi H. A.; Weizmann, Tal Moshe; Mulholland, Anthony J.; Spencer, James; Kamp, M. van der

doi:10.26434/chemrxiv-2021-bv7tb

Cited by 3 publications

(3 citation statements)

References 52 publications

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“…When the 6α-1R-hydroxyethyl group instead accepts a H-bond from the DW (as observed with OXA-48/imipenem), hydrolysis is more efficient. 48 Role of Asn132. The most significant difference between BlaC and other class A β-lactamase active sites is the lack of Asn132 in the former, in which it is replaced by Gly.…”

Section: ■ Resultsmentioning

confidence: 99%

QM/MM Simulations Reveal the Determinants of Carbapenemase Activity in Class A β-Lactamases

et al. 2022

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β-lactam antibiotic resistance in Gram-negative bacteria, primarily caused by β-lactamase enzymes that hydrolyze the β-lactam ring, has become a serious clinical problem. Carbapenems were formerly considered “last resort” antibiotics because they escaped breakdown by most β-lactamases, due to slow deacylation of the acyl-enzyme intermediate. However, an increasing number of Gram-negative bacteria now produce β-lactamases with carbapenemase activity: these efficiently hydrolyze the carbapenem β-lactam ring, severely limiting the treatment of some bacterial infections. Here, we use quantum mechanics/molecular mechanics (QM/MM) simulations of the deacylation reactions of acyl-enzyme complexes of eight β-lactamases of class A (the most widely distributed β-lactamase group) with the carbapenem meropenem to investigate differences between those inhibited by carbapenems (TEM-1, SHV-1, BlaC, and CTX-M-16) and those that hydrolyze them (SFC-1, KPC-2, NMC-A, and SME-1). QM/MM molecular dynamics simulations confirm the two enzyme groups to differ in the preferred acyl-enzyme orientation: carbapenem-inhibited enzymes favor hydrogen bonding of the carbapenem hydroxyethyl group to deacylating water (DW). QM/MM simulations of deacylation give activation free energies in good agreement with experimental hydrolysis rates, correctly distinguishing carbapenemases. For the carbapenem-inhibited enzymes, free energies for deacylation are significantly higher than for the carbapenemases, even when the hydroxyethyl group was restrained to prevent interaction with the DW. Analysis of these simulations, and additional simulations of mutant enzymes, shows how factors including the hydroxyethyl orientation, the active site volume, and architecture (conformations of Asn170 and Asn132; organization of the oxyanion hole; and the Cys69-Cys238 disulfide bond) collectively determine catalytic efficiency toward carbapenems.

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Section: ■ Resultsmentioning

confidence: 99%

QM/MM Simulations Reveal the Determinants of Carbapenemase Activity in Class A β-Lactamases

et al. 2022

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“…Most of the variants in the OXA-48 family are carbapenemases, with elevated IME hydrolysis rates when compared against other carbapenems. 59 For OXA-48, experimental measurements of k cat values for IME hydrolysis vary between 1.5 and 22.5 s −1 , which can be converted to free energy barriers for activation (Δ ⧧ G) from 15.7 to 17.3 kcal/mol using the Eyring equation. For MER, the measured k cat values range between 0.07 and 0.16 s −1 , which converts to barriers from 18.7 to 19.2 kcal/mol.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Multiscale Simulations Identify Origins of Differential Carbapenem Hydrolysis by the OXA-48 β-Lactamase

et al. 2022

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OXA-48 β-lactamases are frequently encountered in bacterial infections caused by carbapenem-resistant Gram-negative bacteria. Due to the importance of carbapenems in the treatment of healthcare-associated infections and the increasingly wide dissemination of OXA-48-like enzymes on plasmids, these β-lactamases are of high clinical significance. Notably, OXA-48 hydrolyzes imipenem more efficiently than other commonly used carbapenems, such as meropenem. Here, we use extensive multiscale simulations of imipenem and meropenem hydrolysis by OXA-48 to dissect the dynamics and to explore differences in the reactivity of the possible conformational substates of the respective acylenzymes. Quantum mechanics/molecular mechanics (QM/MM) simulations of the deacylation reaction for both substrates demonstrate that deacylation is favored when the 6α-hydroxyethyl group is able to hydrogen bond to the water molecule responsible for deacylation but disfavored by the increasing hydration of either oxygen of the carboxylated Lys73 general base. Differences in free energy barriers calculated from the QM/MM simulations correlate well with the experimentally observed differences in hydrolytic efficiency between meropenem and imipenem. We conclude that the impaired breakdown of meropenem, compared to imipenem, which arises from a subtle change in the hydrogen bonding pattern between the deacylating water molecule and the antibiotic, is most likely induced by the meropenem 1β-methyl group. In addition to increased insights into carbapenem breakdown by OXA β-lactamases, which may aid in future efforts to design antibiotics or inhibitors, our approach exemplifies the combined use of atomistic simulations in determining the possible different enzyme–substrate substates and their influence on enzyme reaction kinetics.

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“…Similar to acylation and reverse acylation reactions, a general base-catalyzed mechanism was also proposed for this reaction. The deprotonated carbamylated Lys73 was presumed to be the general base for activating the hydrolytic water molecule. , Lahiri et al suggested that the carbamylated Lys73 is in a protonated form in the EI1 state formed by avibactam. Thus, it fails to activate the hydrolytic water molecule and thereby inhibits the hydrolysis .…”

Section: Resultsmentioning

confidence: 99%

Inhibition Mechanism of Class D β-Lactamases by Avibactam

2022

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Escalation of antibiotic resistance due to class D β-lactamases (DBLs) carrying bacteria is a matter of grave concern. This class of enzymes can efficiently hydrolyze the carbapenem group of antibiotics that are the last-reserved therapeutics for infections caused by multidrug-resistant bacteria. The development of efficient inhibitors against DBLs calls for a molecular-level understanding of the hydrolysis mechanism. Here, we investigate the mechanism of inhibition of OXA-48 DBL enzyme by one of the diazobicyclooctane class of inhibitors, namely avibactam, through molecular dynamics simulations and free energy calculations. Hydrolysis as well as inhibition mechanisms are expected to be intricate due to the presence of N-carbamylated lysine (Lys73), multiple acidic and basic active site residues, and active site water molecules. Our extensive mechanistic study characterizes the most probable reaction route and critical reaction intermediates starting from the acylation to the full hydrolysis of the covalent intermediate. This study discerns the residues that act as the general bases at different steps. Free energies and reaction intermediate structures are corroborated with the available experimental kinetics data and crystal structures. We also simulated the deacylation of a β-lactam drug, namely meropenem, which is known to be hydrolyzed efficiently by the enzyme. By comparing the mechanism of meropenem hydrolysis with that of avibactam, our study reveals the important chemical features that are useful for designing inhibitors.

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Multiscale simulations identify origins of differential carbapenem hydrolysis by the OXA-48 β-lactamase

Cited by 3 publications

References 52 publications

QM/MM Simulations Reveal the Determinants of Carbapenemase Activity in Class A β-Lactamases

QM/MM Simulations Reveal the Determinants of Carbapenemase Activity in Class A β-Lactamases

Multiscale Simulations Identify Origins of Differential Carbapenem Hydrolysis by the OXA-48 β-Lactamase

Inhibition Mechanism of Class D β-Lactamases by Avibactam

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