Distant and New Mutations in CTX-M-1 β-Lactamase Affect Cefotaxime Hydrolysis

Pérez-Llarena, Francisco José; Kerff, Frédéric; Abián, Olga; Mallo, Susana; Fernández, M.C.; Galleni, Moreno; Sancho, Javier; Bou, Germán

doi:10.1128/aac.00298-11

Cited by 24 publications

(24 citation statements)

References 26 publications

(31 reference statements)

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“…GES-18 differs from GES-5 by a single Val80Ile substitution located on helix H2 in a hydrophobic core distant from the catalytic center. The Val80 residue in CTX-M-1 is known to be involved in cefotaxime hydrolysis, and a Val80Ala substitution may affect the dynamics and flexibility of this enzyme (41). We have shown that the Val80Ile substitution in GES-18 does not significantly modify the substrate profile in comparison to GES-5, but the k cat value was modified for some substrates.…”

Section: Discussionmentioning

confidence: 73%

GES-18, a New Carbapenem-Hydrolyzing GES-Type β-Lactamase from Pseudomonas aeruginosa That Contains Ile80 and Ser170 Residues

Bebrone

Bogaerts

Delbrück

et al. 2013

Antimicrob Agents Chemother

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A clinical isolate of Pseudomonas aeruginosa recovered from the lower respiratory tract of an 81-year-old patient hospitalized in Belgium was sent to the national reference center to determine its resistance mechanism. PCR sequencing identified a new GES variant, GES-18, which differs from the carbapenem-hydrolyzing enzyme GES-5 by a single amino acid substitution (Val80Ile, in the numbering according to Ambler) and from GES-1 by two substitutions (Val80Ile and Gly170Ser). Detailed kinetic characterization showed that GES-18 and GES-5 hydrolyze imipenem and cefoxitin with similar kinetic parameters and that GES-18 was less susceptible than GES-1 to classical ␤-lactamase inhibitors such as clavulanate and tazobactam. The overall structure of GES-18 is similar to the solved structures of GES-1 and GES-2, the Val80Ile and Gly170Ser substitutions causing only subtle local rearrangements. Notably, the hydrolytic water molecule and the Glu166 residue were slightly displaced compared to their counterparts in GES-1. Our kinetic and crystallographic data for GES-18 highlight the pivotal role of the Gly170Ser substitution which distinguishes GES-5 and GES-18 from GES-1.

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Section: Discussionmentioning

confidence: 73%

GES-18, a New Carbapenem-Hydrolyzing GES-Type β-Lactamase from Pseudomonas aeruginosa That Contains Ile80 and Ser170 Residues

Bebrone

Bogaerts

Delbrück

et al. 2013

Antimicrob Agents Chemother

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“…They form a rapidly growing family that comprises more than 100 variants (http://www.lahey.org/studies) and are divided into five groups according to amino acid sequence identity, with different groups being prevalent in different countries [6], [7]. CTX-M ESBLs (derived its name from being highly active on C efo T a X ime and isolated in M unich) are characterized by displaying greater hydrolytic activity against cefotaxime than against ceftazidime [8].…”

Section: Introductionmentioning

confidence: 99%

Biochemical Characterization of CTX-M-15 from Enterobacter cloacae and Designing a Novel Non-β-Lactam-β-Lactamase Inhibitor

et al. 2013

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The worldwide dissemination of CTX-M type β-lactamases is a threat to human health. Previously, we have reported the spread of bla CTX-M-15 gene in different clinical strains of Enterobacteriaceae from the hospital settings of Aligarh in north India. In view of the varying resistance pattern against cephalosporins and other β-lactam antibiotics, we intended to understand the correlation between MICs and catalytic activity of CTX-M-15. In this study, steady-state kinetic parameters and MICs were determined on E. coli DH5α transformed with bla CTX-M-15 gene that was cloned from Enterobacter cloacae (EC-15) strain of clinical background. The effect of conventional β-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) on CTX-M-15 was also studied. We have found that tazobactam is the best among these inhibitors against CTX-M-15. The inhibition characteristic of tazobactam is defined by its very low IC50 value (6 nM), high affinity (K i = 0.017 µM) and better acylation efficiency (k +2/K′ = 0.44 µM−1s−1). It forms an acyl-enzyme covalent complex, which is quite stable (k +3 = 0.0057 s−1). Since increasing resistance has been reported against conventional β-lactam antibiotic-inhibitor combinations, we aspire to design a non-β-lactam core containing β-lactamase inhibitor. For this, we screened ZINC database and performed molecular docking to identify a potential non-β-lactam based inhibitor (ZINC03787097). The MICs of cephalosporin antibiotics in combination with this inhibitor gave promising results. Steady-state kinetics and molecular docking studies showed that ZINC03787097 is a reversible inhibitor which binds non-covalently to the active site of the enzyme through hydrogen bonds and hydrophobic interactions. Though, it’s IC50 (180 nM) is much higher than tazobactam, it has good affinity for CTX-M-15 (K i = 0.388 µM). This study concludes that ZINC03787097 compound can be used as seed molecule to design more efficient non-β-lactam containing β-lactamase inhibitor that could evade pre-existing bacterial resistance mechanisms.

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“…33 In addition, the carboxylate group at the C3/C4 position of β-lactam substrates forms hydrogen bonds to Ser/Thr235 in class A enzymes including wild-type CTX-M-14. 34–37 The structure of the non-covalent complex (S70G/P167S/CAZ) shows the carbonyl oxygen of the β-lactam ring present in the oxyanion hole forming hydrogen bonds with the main chain NH of residues 70 and 237. In addition, the carboxylate group on the dihydrothiazine ring of ceftazidime forms hydrogen bonds with the side chain hydroxyl groups of Ser130 and Thr235 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…38–40 The hydrogen bond interactions between ceftazidime and key residues in the active site are highlighted in Figure 4B. 34 The entire ceftazidime molecule is accommodated in the active site of E166A/P167S/CAZ (Fig. 3B & 4B).…”

Section: Resultsmentioning

confidence: 99%

The Drug-Resistant Variant P167S Expands the Substrate Profile of CTX-M β-Lactamases for Oxyimino-Cephalosporin Antibiotics by Enlarging the Active Site upon Acylation

Patel¹,

Hu²,

Stojanoski³

et al. 2017

Biochemistry

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β-Lactamases are enzymes produced by bacterial cells that provide resistance to β-lactam antibiotics. The CTX-M class of β-lactamases provides resistance against the antibiotic, cefotaxime, but not a related oxyimino-cephalosporin antibiotic, ceftazidime. β-lactamases that carry the P167S substitution, however, have been reported to provide ceftazidime resistance. The mechanism by which the P167S substitution expands the substrate profile of CTX-M enzymes is not known. In this study, CTX-M-14 was used as the model enzyme to study the structural changes caused by the P167S mutation that may accelerate ceftazidime turnover. X-ray crystallography was used to determine the structures of the CTX-M-14 P167S apo-enzyme along with the structures of the S70G/P167S, E166A/P167S and E166A mutant enzymes complexed with ceftazidime as well as the E166A/P167S apo-enzyme. The S70G and E166A mutations allow the capture of the enzyme-substrate complex and acylated forms of the ceftazidime molecule, respectively. The results showed a large conformational change in the Ω-loop of the CTX-M-14 ceftazidime acyl-enzyme complex of the P167S mutant but not in the enzyme-substrate complex suggesting the conformational change occurs upon acylation. The conformational change results in a larger active site cavity that prevents steric clash between the aminothiazole ring of ceftazidime and the Asn170 residue in the Ω-loop, allowing for accommodation of ceftazidime for hydrolysis. In addition, the conformational change in the Ω-loop was not observed in the E166A/P167S apo-enzyme, suggesting the presence of acylated ceftazidime influences the conformational change. Finally, the E166A acyl-enzyme structure with ceftazidime did not exhibit the altered Ω-loop conformation, indicating the P167S substitution is required for the change. Taken together, the results reveal that the P167S substitution and the presence of acylated ceftazidime both drive the structure towards a conformational change of the Ω-loop and that in CTX-M P167S enzymes found in drug-resistant bacteria this will lead to increased ceftazidime hydrolysis. This study demonstrates how a naturally occurring substitution can dramatically alter the active site to expand the substrate profile of an enzyme due to antibiotic selective pressure.

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Distant and New Mutations in CTX-M-1 β-Lactamase Affect Cefotaxime Hydrolysis

Cited by 24 publications

References 26 publications

GES-18, a New Carbapenem-Hydrolyzing GES-Type β-Lactamase from Pseudomonas aeruginosa That Contains Ile80 and Ser170 Residues

GES-18, a New Carbapenem-Hydrolyzing GES-Type β-Lactamase from Pseudomonas aeruginosa That Contains Ile80 and Ser170 Residues

Biochemical Characterization of CTX-M-15 from Enterobacter cloacae and Designing a Novel Non-β-Lactam-β-Lactamase Inhibitor

The Drug-Resistant Variant P167S Expands the Substrate Profile of CTX-M β-Lactamases for Oxyimino-Cephalosporin Antibiotics by Enlarging the Active Site upon Acylation

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