Dataset Information

Structural Insights into the Inhibition of the Extended-Spectrum ?-Lactamase PER-2 by Avibactam.

ABSTRACT: The diazabicyclooctane (DBO) avibactam (AVI) reversibly inactivates most serine-?-lactamases. Previous investigations showed that inhibition constants of AVI toward class A PER-2 are reminiscent of values observed for class C and D ?-lactamases (i.e., k 2/K of ?103 M-1 s-1) but lower than other class A ?-lactamases (i.e., k 2/K = 104 to 105 M-1 s-1). Herein, biochemical and structural studies were conducted with PER-2 and AVI to explore these differences. Furthermore, biochemical studies on Arg220 and Thr237 variants with AVI were conducted to gain deeper insight into the mechanism of PER-2 inactivation. The main biochemical and structural observations revealed the following: (i) both amino-acid substitutions in Arg220 and the rich hydrophobic content in the active site hinder the binding of catalytic waters and acylation, impairing AVI inhibition; (ii) movement of Ser130 upon binding of AVI favors the formation of a hydrogen bond with the sulfate group of AVI; and (iii) the Thr237Ala substitution alters the AVI inhibition constants. The acylation constant (k 2/K) of PER-2 by AVI is primarily influenced by stabilizing hydrogen bonds involving AVI and important residues such as Thr237 and Arg220. (Variants in Arg220 demonstrate a dramatic reduction in k 2/K) We also observed that displacement of Ser130 side chain impairs AVI acylation, an observation not made in other extended-spectrum ?-lactamases (ESBLs). Comparatively, relebactam combined with a ?-lactam is more potent against Escherichia coli producing PER-2 variants than ?-lactam-AVI combinations. Our findings provide a rationale for evaluating the utility of the currently available DBO inhibitors against unique ESBLs like PER-2 and anticipate the effectiveness of these inhibitors toward variants that may eventually be selected upon AVI usage.

SUBMITTER: Ruggiero M

PROVIDER: S-EPMC6709463 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Publications

Structural Insights into the Inhibition of the Extended-Spectrum β-Lactamase PER-2 by Avibactam.

Ruggiero Melina M Papp-Wallace Krisztina M KM Brunetti Florencia F Barnes Melissa D MD Bonomo Robert A RA Gutkind Gabriel G Klinke Sebastián S Power Pablo P

Antimicrobial agents and chemotherapy 20190823 9

The diazabicyclooctane (DBO) avibactam (AVI) reversibly inactivates most serine-β-lactamases. Previous investigations showed that inhibition constants of AVI toward class A PER-2 are reminiscent of values observed for class C and D β-lactamases (i.e., k2/K of ≈103 M-1 s-1) but lower than other class A β-lactamases (i.e., k2/K = 104 to 105 M-1 s-1). Herein, biochemical an ...[more]

PMID: 31235626

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Project description:PER ?-lactamases are an emerging family of extended-spectrum ?-lactamases (ESBL) found in Gram-negative bacteria. PER ?-lactamases are unique among class A enzymes as they possess an inverted omega (?) loop and extended B3 ?-strand. These singular structural features are hypothesized to contribute to their hydrolytic profile against oxyimino-cephalosporins (e.g., cefotaxime and ceftazidime). Here, we tested the ability of avibactam (AVI), a novel non-?-lactam ?-lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., k2/K = 2 × 103 ± 0.1 × 103 M-1 s-1, where k2 is the rate constant for acylation (carbamylation) and K is the equilibrium constant) that were obtained when AVI was tested were reminiscent of values observed testing the inhibition by AVI of class C and D ?-lactamases (i.e., k2/K range of ?103 M-1 s-1) and not class A ?-lactamases (i.e., k2/K range, 104 to 105 M-1 s-1). Once AVI was bound, a stable complex with PER-2 was observed via mass spectrometry (e.g., 31,389 ± 3 atomic mass units [amu] ? 31,604 ± 3 amu for 24 h). Molecular modeling of PER-2 with AVI showed that the carbonyl of AVI was located in the oxyanion hole of the ?-lactamase and that the sulfate of AVI formed interactions with the ?-lactam carboxylate binding site of the PER-2 ?-lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 ?-strands) were observed and may affect the binding of necessary catalytic water molecules, thus slowing acylation (k2/K) of AVI onto PER-2. Similar electrostatics and hydrophobicity of the active site were also observed between OXA-48 and PER-2, while CTX-M-15 was more hydrophilic. To demonstrate the ability of AVI to overcome the enhanced cephalosporinase activity of PER-2 ?-lactamase, we tested different ?-lactam-AVI combinations. By lowering MICs to ?2 mg/liter, the ceftaroline-AVI combination could represent a favorable therapeutic option against Enterobacteriaceae expressing blaPER-2 Our studies define the inactivation of the PER-2 ESBL by AVI and suggest that the biophysical properties of the active site contribute to determining the efficiency of inactivation.

Dataset Information

Structural Insights into the Inhibition of the Extended-Spectrum ?-Lactamase PER-2 by Avibactam.

Publications

Structural Insights into the Inhibition of the Extended-Spectrum β-Lactamase PER-2 by Avibactam.

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