Crystal Structure of the Extended-Spectrum β-Lactamase PER-2 and Insights into the Role of Specific Residues in the Interaction with β-Lactams and β-Lactamase Inhibitors

Ruggiero, Melina; Kerff, Frédéric; Herman, R.; Sapunaric, Frédéric; Galleni, Moreno; Gutkind, Gabriel; Charlier, Paulette; Sauvage, Eric; Power, Pablo

doi:10.1128/aac.00089-14

Cited by 17 publications

(10 citation statements)

References 48 publications

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“…Recently, the crystallographic structure of PER-2 at a 2.2-Å resolution revealed the presence of a unique fold in the ⍀-loop of PER-2. This results in an enlarged and potentially mobile active site cavity, unlike in other class A ␤-lactamases, which allows for the more efficient hydrolysis of ␤-lactams, like the oxyimino-cephalosporins (6). Additionally, a hydrogen bond network connecting Ser70-Gln69-water-Thr237-Arg220 is observed, which is postulated to be important for the enhanced activity and inhibition of this ␤-lactamase (6).…”

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confidence: 99%

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

Ruggiero

Papp-Wallace

Brunetti

et al. 2019

Antimicrob Agents Chemother

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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 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 (k2/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 k2/K.) We also observed that displacement of Ser130 side chain impairs AVI acylation, an observation not made in other extended-spectrum β-lactamases (ESBLs). Comparatively, relebactam combined with a β-lactam is more potent against Escherichia coli producing PER-2 variants than β-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.

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confidence: 99%

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

Ruggiero

Papp-Wallace

Brunetti

et al. 2019

Antimicrob Agents Chemother

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“…3B). Ruggiero et al suggested that these configurations of the double loop structure in PER-2 (and TLA-3) yielded an expanded active site that could accommodate oxyiminocephalosporins harboring bulky R1 side chains (21). We predicted the binding mode between ceftazidime and TLA-3 through in silico analysis and revealed that TLA-3 had sufficient space to accommodate the bulky R1 side chains ( Fig.…”

Section: Resultsmentioning

confidence: 88%

“…3B and C). The overall configuration of on August 5, 2020 by guest http://aac.asm.org/ the ⍀ loop (loop 1) in TLA-3 is most closely related to that of the ⍀ loop in PER-2 (21) but is inverted relative to the ⍀ loops in CTX-M-15 (16) and KPC-2 (15) ( Fig. 3B).…”

Section: Resultsmentioning

confidence: 95%

“…TLA-3 forms one ␣/␤ domain and one ␣ domain, with the active site being located at the interface between these domains ( Fig. 3A), as in other class A ␤-lactamases (15,16,21). The active site is defined by motif I (Ser70-Thr71-Tyr72-Lys73), motif II (Ser130-Asp131-Asn132), motif III (Lys234-Thr235-Gly236), and the ⍀ loop (16 amino acids, Ala164 to Asn179) ( Fig.…”

Section: Resultsmentioning

confidence: 95%

“…Ser237 in TLA-3 corresponds to Thr237 in PER-2, and the hydroxyl group of both residues is predicted to coordinate with the carboxylate group of substrate ␤-lactams (21). This stabilization of the Ser237 side chain by Arg220 potentially contributes to firm recognition of substrate ␤-lactams, which results in the increased ␤-lactam catalytic activity of TLA-3 (21). In fact, the substitutions at Arg220 and Thr237 deteriorate the enzymatic behavior of PER-1 (23).…”

Section: Resultsmentioning

confidence: 99%

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Structural Insights into the TLA-3 Extended-Spectrum β-Lactamase and Its Inhibition by Avibactam and OP0595

Jin

Wachino

Yamaguchi

et al. 2017

Antimicrob Agents Chemother

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The development of effective inhibitors that block extended-spectrum β-lactamases (ESBLs) and restore the action of β-lactams represents an effective strategy against ESBL-producing We evaluated the inhibitory effects of the diazabicyclooctanes avibactam and OP0595 against TLA-3, an ESBL that we identified previously. Avibactam and OP0595 inhibited TLA-3 with apparent inhibitor constants () of 1.71 ± 0.10 and 1.49 ± 0.05 μM, respectively, and could restore susceptibility to cephalosporins in the TLA-3-producing strain. The value of the second-order acylation rate constant (/, where is the acylation rate constant and is the equilibrium constant) of avibactam [(3.25 ± 0.03) × 10 M · s] was closer to that of class C and D β-lactamases (/, <10 M · s) than that of class A β-lactamases (/, >10 M · s). In addition, we determined the structure of TLA-3 and that of TLA-3 complexed with avibactam or OP0595 at resolutions of 1.6, 1.6, and 2.0 Å, respectively. TLA-3 contains an inverted Ω loop and an extended loop between the β5 and β6 strands (insertion after Ser237), which appear only in PER-type class A β-lactamases. These structures might favor the accommodation of cephalosporins harboring bulky R1 side chains. TLA-3 presented a high catalytic efficiency (/ ) against cephalosporins, including cephalothin, cefuroxime, and cefotaxime. Avibactam and OP0595 bound covalently to TLA-3 via the Ser70 residue and made contacts with residues Ser130, Thr235, and Ser237, which are conserved in ESBLs. Additionally, the sulfate group of the inhibitors formed polar contacts with amino acid residues in a positively charged pocket of TLA-3. Our findings provide a structural template for designing improved diazabicyclooctane-based inhibitors that are effective against ESBL-producing .

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Whole Genome Sequence Analysis of Burkholderia contaminans FFH2055 Strain Reveals the Presence of Putative β-Lactamases

et al. 2019

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Burkholderia contaminans is a member of the Burkholderia cepacia complex (Bcc), a pathogen with increasing prevalence among cystic fibrosis (CF) patients and the cause of numerous outbreaks due to the use of contaminated commercial products. The antibiotic resistance determinants, particularly β-lactamases, have been poorly studied in this species. In this work, we explored the whole genome sequence (WGS) of a B. contaminans isolate (FFH 2055) and detected four putative β-lactamase-encoding genes. In general, these genes have more than 93% identity with β-lactamase genes found in other Bcc species. Two β-lactamases, a class A (Pen-like, suggested name PenO) and a class D (OXA-like), were further analyzed and characterized. Amino acid sequence comparison showed that Pen-like has 82% and 67% identity with B. multivorans PenA and B. pseudomallei PenI, respectively, while OXA-like displayed strong homology with class D enzymes within the Bcc, but only 22–44% identity with available structures from the OXA family. PCR reactions designed to study the presence of these two genes revealed a heterogeneous distribution among clinical and industrial B. contaminans isolates. Lastly, blaPenO gene was cloned and expressed into E. coli to investigate the antibiotic resistance profile and confers an extended-spectrum β-lactamase (ESBL) phenotype. These results provide insight into the presence of β-lactamases in B. contaminans, suggesting they play a role in antibiotic resistance of these bacteria.

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Crystal Structure of the Extended-Spectrum β-Lactamase PER-2 and Insights into the Role of Specific Residues in the Interaction with β-Lactams and β-Lactamase Inhibitors

Cited by 17 publications

References 48 publications

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

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

Structural Insights into the TLA-3 Extended-Spectrum β-Lactamase and Its Inhibition by Avibactam and OP0595

Whole Genome Sequence Analysis of Burkholderia contaminans FFH2055 Strain Reveals the Presence of Putative β-Lactamases

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