Structure-function studies of arginine at position 276 in CTX-M  -lactamases

Pérez-Llarena, Francisco José; Cartelle, Mónica; Mallo, Susana; Beceiro, Alejandro; Pérez, Astrid; Villanueva, Rosa; Romero, Antonio; Bonnet, Richard; Bou, Germán

doi:10.1093/jac/dkn031

Cited by 23 publications

(24 citation statements)

References 22 publications

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“…15 Site-directed mutagenesis studies in the CTX-M-4 and CTX-M-1 enzymes indicate Arg276 in these enzymes contributes to cefotaxime hydrolysis although the impact of substitutions is relatively low and K m values are largely unaffected. 21,22 In addition, the IC 50 for clavulanic acid inhibition for substituted enzymes was similar to the wild type value. Therefore, despite the fact that an arginine at position 276 can largely compensate for the defect in the TEM-1 Arg244Ala mutant, Arg276 does not appear to play a similar role in the CTX-M enzymes.…”

Section: Discussionmentioning

confidence: 53%

“…However, catalytic parameters of the Arg276 substituted enzymes were only moderately affected with the substrates tested. 21,22 Taken together, the site-directed mutagenesis experiments suggest that the arginines found at positions 220, 244, or 276 provide a contribution toward b-lactamase activity, and the variation in position from which this positively charged residue extends indicates a selective pressure for maintaining positive charge within this region of the active site.…”

Section: Resultsmentioning

confidence: 92%

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Analysis of the plasticity of location of the Arg244 positive charge within the active site of the TEM‐1 β‐lactamase

2009

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A large number of b-lactamases have emerged that are capable of conferring bacterial resistance to b-lactam antibiotics. Comparison of the structural and functional features of this family has refined understanding of the catalytic properties of these enzymes. An arginine residue present at position 244 in TEM-1 b-lactamase interacts with the carboxyl group common to penicillin and cephalosporin antibiotics and thereby stabilizes both the substrate and transition state complexes. A comparison of class A b-lactamase sequences reveals that arginine at position 244 is not conserved, although a positive charge at this structural location is conserved and is provided by an arginine at positions 220 or 276 for those enzymes lacking arginine at position 244. The plasticity of the location of positive charge in the b-lactamase active site was experimentally investigated by relocating the arginine at position 244 in TEM-1 b-lactamase to positions 220, 272, and 276 by site-directed mutagenesis. Kinetic analysis of the engineered b-lactamases revealed that removal of arginine 244 by alanine mutation reduced catalytic efficiency against all substrates tested and restoration of an arginine at positions 272 or 276 partially suppresses the catalytic defect of the Arg244Ala substitution. These results suggest an evolutionary mechanism for the observed divergence of the position of positive charge in the active site of class A b-lactamases.

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Section: Discussionmentioning

confidence: 53%

Section: Resultsmentioning

confidence: 92%

Analysis of the plasticity of location of the Arg244 positive charge within the active site of the TEM‐1 β‐lactamase

2009

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“…In TEM-1 and SHV-1, Arg244 is important in the mechanism of inactivation of carbapenems (imipenem and meropenem), clavulanic acid, sulbactam, and tazobactam (27,28,51,53,63). CTX-M-9 does not contain Arg244, and mutagenesis of a potential corresponding position, Arg276, does not firmly establish this amino acid as an "Arg244 equivalent" (42). Given the differences among class A enzymes, we wondered what the intermediates of inactivation by inhibitors would be for CTX-M-9.…”

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confidence: 99%

Exploring the Inhibition of CTX-M-9 by β-Lactamase Inhibitors and Carbapenems

Bethel

Taracila

Shyr

et al. 2011

Antimicrob Agents Chemother

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Currently, CTX-M ␤-lactamases are among the most prevalent and most heterogeneous extended-spectrum ␤-lactamases (ESBLs). In general, CTX-M enzymes are susceptible to inhibition by ␤-lactamase inhibitors. However, it is unknown if the pathway to inhibition by ␤-lactamase inhibitors for CTX-M ESBLs is similar to TEM and SHV ␤-lactamases and why bacteria possessing only CTX-M ESBLs are so susceptible to carbapenems. Here, we have performed a kinetic analysis and timed electrospray ionization mass spectrometry (ESI-MS) studies to reveal the intermediates of inhibition of CTX-M-9, an ESBL representative of this family of enzymes. CTX-M-9 ␤-lactamase was inactivated by sulbactam, tazobactam, clavulanate, meropenem, doripenem, ertapenem, and a 6-methylidene penem, penem 1. CTX-M enzymes are becoming one of the most prevalent extended-spectrum ␤-lactamases (ESBLs) (3,8,9,(30)(31)(32) in the world. The widespread dissemination of CTX-M ␤-lactamases, especially Escherichia coli ST131 possessing CTX-M-15, has had a significant impact on the treatment of hospital-and community-acquired infections caused by E. coli and other enteric bacilli (6, 7, 13, 23, 36, 41, 44-49, 55, 59).As class A family ␤-lactamases, CTX-Ms are the most genetically heterogeneous (5 major divisions, CTX-M-1, -2, -8, -25, and -9-like groups) (1, 24, 35, 44-46, 58, 60). Most CTX-M enzymes expressed in E. coli provide a high level of resistance to the oxyimino-cephalosporins, cefotaxime and ceftriaxone, and variable levels of resistance to cefepime and cefpirome (3,43). Depending on the type of CTX-M expressed by the isolates, the MICs of ceftazidime are also increased (43). In addition, the MICs of combinations of clavulanate with amoxicillin or ticarcillin vary, and in some cases, low-level resistance has been observed (3).As a consequence of their clinical importance, the reaction mechanism of CTX-M ESBLs has been the subject of intense study (10-12, 14, 16, 42). However, the molecular properties and characteristics of CTX-M that determine the level of susceptibility and resistance to ␤-lactam-␤-lactamase inhibitor combinations and carbapenems are still unknown. Of the currently available inhibitors, tazobactam is the most active (50% inhibitory concentrations [IC 50 s] are 2 to 10 nM for tazobactam and 9 to 90 nM for clavulanate), and sulbactam is the least active (IC 50 s are 0.1 to 4.5 M) (3).In order to develop more effective and broader-spectrum ␤-lactamase inhibitors (18), detailed kinetic and biochemical measurements are needed to reveal the important intermediates in the inactivation of the CTX-M family. In TEM-1 and SHV-1, Arg244 is important in the mechanism of inactivation of carbapenems (imipenem and meropenem), clavulanic acid, sulbactam, and tazobactam (27,28,51,53,63). CTX-M-9 does not contain Arg244, and mutagenesis of a potential corresponding position, Arg276, does not firmly establish this amino acid as an "Arg244 equivalent" (42). Given the differences among class A enzymes, we wondered what the intermediates of inactivation by ...

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“…It is known that in class A ␤-lactamases like TEM and SHV variants, Arg244 seems to play an important role in substrate and inhibitor binding (34)(35)(36). This role seems to be fulfilled in CTX-M ␤-lactamases by Arg276 (37). In other class A ␤-lactamases like the Streptomyces albus G ␤-lactamase or KPC-2 and NMC-A carbapenemases (PDB entries 1BSG, 3DW0, and 1BUE, respectively) (38), an arginine residue at position 220 was observed at the ␤4 strand whose side-chain guanidinium group occupies a position equivalent to that of the lateral residue of Arg244/Arg276 in the mentioned class A ␤-lactamases.…”

Section: Resultsmentioning

confidence: 99%

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

Kerff

Herman

et al. 2014

Antimicrob Agents Chemother

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b PER-2 belongs to a small (7 members to date) group of extended-spectrum ␤-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most ␤-lactams. In this study, we determined the X-ray structure of PER-2 at 2.20 Å and evaluated the possible role of several residues in the structure and activity toward ␤-lactams and mechanism-based inhibitors. PER-2 is defined by the presence of a singular trans bond between residues 166 to 167, which generates an inverted ⍀ loop, an expanded fold of this domain that results in a wide active site cavity that allows for efficient hydrolysis of antibiotics like the oxyimino-cephalosporins, and a series of exclusive interactions between residues not frequently involved in the stabilization of the active site in other class A ␤-lactamases. PER ␤-lactamases might be included within a cluster of evolutionarily related enzymes harboring the conserved residues Asp136 and Asn179. Other signature residues that define these enzymes seem to be Gln69, Arg220, Thr237, and probably Arg/Lys240A ("A" indicates an insertion according to Ambler's scheme for residue numbering in PER ␤-lactamases), with structurally important roles in the stabilization of the active site and proper orientation of catalytic water molecules, among others. We propose, supported by simulated models of PER-2 in combination with different ␤-lactams, the presence of a hydrogen-bond network connecting Ser70-Gln69-water-Thr237-Arg220 that might be important for the proper activity and inhibition of the enzyme. Therefore, we expect that mutations occurring in these positions will have impacts on the overall hydrolytic behavior.

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Structure-function studies of arginine at position 276 in CTX-M -lactamases

Abstract: Arg-276 in CTX-M-type beta-lactamases is not equivalent to Arg-244 in IRT-type enzymes. Position Arg-276 appears to be important for cefotaxime hydrolysis in CTX-M-type enzymes, although different effects were obtained regarding the replaced amino acid.

Cited by 23 publications

References 22 publications

Analysis of the plasticity of location of the Arg244 positive charge within the active site of the TEM‐1 β‐lactamase

Analysis of the plasticity of location of the Arg244 positive charge within the active site of the TEM‐1 β‐lactamase

Exploring the Inhibition of CTX-M-9 by β-Lactamase Inhibitors and Carbapenems

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

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