Dataset Information

Molecular basis for the catalytic specificity of the CTX-M extended-spectrum ?-lactamases.

ABSTRACT: Extended-spectrum ?-lactamases (ESBLs) pose a threat to public health because of their ability to confer resistance to extended-spectrum cephalosporins such as cefotaxime. The CTX-M ?-lactamases are the most widespread ESBL enzymes among antibiotic resistant bacteria. Many of the active site residues are conserved between the CTX-M family and non-ESBL ?-lactamases such as TEM-1, but the residues Ser237 and Arg276 are specific to the CTX-M family, suggesting that they may help to define the increased specificity for cefotaxime hydrolysis. To test this hypothesis, site-directed mutagenesis of these positions was performed in the CTX-M-14 ?-lactamase. Substitutions of Ser237 and Arg276 with their TEM-1 counterparts, Ala237 and Asn276, had a modest effect on cefotaxime hydrolysis, as did removal of the Arg276 side chain in an R276A mutant. The S237A:R276N and S237A:R276A double mutants, however, exhibited 29- and 14-fold losses in catalytic efficiency for cefotaxime hydrolysis, respectively, while the catalytic efficiency for benzylpenicillin hydrolysis was unchanged. Therefore, together, the Ser237 and Arg276 residues are important contributors to the cefotaximase substrate profile of the enzyme. High-resolution crystal structures of the CTX-M-14 S70G, S70G:S237A, and S70G:S237A:R276A variants alone and in complex with cefotaxime show that residues Ser237 and Arg276 in the wild-type enzyme promote the expansion of the active site to accommodate cefotaxime and favor a conformation of cefotaxime that allows optimal contacts between the enzyme and substrate. The conservation of these residues, linked to their effects on structure and catalysis, imply that their coevolution is an important specificity determinant in the CTX-M family.

SUBMITTER: Adamski CJ

PROVIDER: S-EPMC4303298 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Molecular basis for the catalytic specificity of the CTX-M extended-spectrum β-lactamases.

Adamski Carolyn J CJ Cardenas Ana Maria AM Brown Nicholas G NG Horton Lori B LB Sankaran Banumathi B Prasad B V Venkataram BV Gilbert Hiram F HF Palzkill Timothy T

Biochemistry 20141224 2

Extended-spectrum β-lactamases (ESBLs) pose a threat to public health because of their ability to confer resistance to extended-spectrum cephalosporins such as cefotaxime. The CTX-M β-lactamases are the most widespread ESBL enzymes among antibiotic resistant bacteria. Many of the active site residues are conserved between the CTX-M family and non-ESBL β-lactamases such as TEM-1, but the residues Ser237 and Arg276 are specific to the CTX-M family, suggesting that they may help to define the incre ...[more]

PMID: 25489790

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Project description:The most common mechanism of resistance to β-lactam antibiotics in Gram-negative bacteria is the production of β-lactamases that hydrolyze the drugs. Class A β-lactamases are serine active-site hydrolases that include the common TEM, CTX-M, and KPC enzymes. The TEM enzymes readily hydrolyze penicillins and older cephalosporins. Oxyimino-cephalosporins, such as cefotaxime and ceftazidime, however, are poor substrates for TEM-1 and were introduced, in part, to circumvent β-lactamase-mediated resistance. Nevertheless, the use of these antibiotics has lead to evolution of numerous variants of TEM with mutations that significantly increase the hydrolysis of the newer cephalosporins. The CTX-M enzymes emerged in the late 1980s and hydrolyze penicillins and older cephalosporins and derive their name from the ability to also hydrolyze cefotaxime. The CTX-M enzymes, however, do not efficiently hydrolyze ceftazidime. Variants of CTX-M enzymes, however, have evolved that exhibit increased hydrolysis of ceftazidime. Finally, the KPC enzyme emerged in the 1990s and is characterized by its broad specificity that includes penicillins, most cephalosporins, and carbapenems. The KPC enzyme, however, does not efficiently hydrolyze ceftazidime. As with the TEM and CTX-M enzymes, variants have recently evolved that extend the spectrum of KPC β-lactamase to include ceftazidime. This review discusses the structural and mechanistic basis for the expanded substrate specificity of each of these enzymes that result from natural mutations that confer oxyimino-cephalosporin resistance. For the TEM enzyme, extended-spectrum mutations act by establishing new interactions with the cephalosporin. These mutations increase the conformational heterogeneity of the active site to create sub-states that better accommodate the larger drugs. The mutations expanding the spectrum of CTX-M enzymes also affect the flexibility and conformation of the active site to accommodate ceftazidime. Although structural data are limited, extended-spectrum mutations in KPC may act by mediating new, direct interactions with substrate and/or altering conformations of the active site. In many cases, mutations that expand the substrate profile of these enzymes simultaneously decrease the thermodynamic stability. This leads to the emergence of additional global suppressor mutations that help correct the stability defects leading to increased protein expression and increased antibiotic resistance.

Dataset Information

Molecular basis for the catalytic specificity of the CTX-M extended-spectrum ?-lactamases.

Publications

Molecular basis for the catalytic specificity of the CTX-M extended-spectrum β-lactamases.

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