skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Molecular Basis for the Catalytic Specificity of the CTX-M Extended-Spectrum β-Lactamases

Journal Article · · Biochemistry
DOI:https://doi.org/10.1021/bi501195g· OSTI ID:1257352
 [1];  [2];  [1];  [3];  [4];  [5];  [1];  [6]
  1. Baylor College of Medicine, Houston, TX (United States). Verna and Marrs McLean Department of Biochemistry and Molecular Biology
  2. Baylor College of Medicine, Houston, TX (United States). Department of Molecular Virology and Microbiology
  3. Baylor College of Medicine, Houston, TX (United States). Department of Molecular Virology and Microbiology
  4. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  5. Baylor College of Medicine, Houston, TX (United States). Verna and Marrs McLean Department of Biochemistry and Molecular Biology; Baylor College of Medicine, Houston, TX (United States). Department of Molecular Virology and Microbiology
  6. Baylor College of Medicine, Houston, TX (United States). Verna and Marrs McLean Dept. of Biochemistry and Molecular Biology; Baylor College of Medicine, Houston, TX (United States). Dept. of Molecular Virology and Microbiology; Baylor College of Medicine, Houston, TX (United States). Dept. of Pharmacology
+ Show Author Affiliations

We report that 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 increased specificity for cefotaxime hydrolysis. To test this hypothesis, site-directed mutagenesis of these positions was performed in the CTX-M-14 β-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. In conclusion, 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.

Research Organization:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
Grant/Contract Number:
AC02- 05CH11231; AI32956; Q1279; 1 R90 DA023418-03
OSTI ID:
1257352
Journal Information:
Biochemistry, Vol. 54, Issue 2; ISSN 0006-2960
Publisher:
American Chemical Society (ACS)Copyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 42 works
Citation information provided by
Web of Science

References (30)

Bacterial Resistance to β-Lactam Antibiotics:  Compelling Opportunism, Compelling Opportunity † journal February 2005
A standard numbering scheme for the class A β-lactamases journal May 1991
Epidemiological Expansion, Structural Studies, and Clinical Challenges of New β-Lactamases from Gram-Negative Bacteria journal October 2011
Growing Group of Extended-Spectrum  -Lactamases: the CTX-M Enzymes journal December 2003
Atomic Resolution Structures of CTX-M β-Lactamases: Extended Spectrum Activities from Increased Mobility and Decreased Stability journal April 2005
CTX-M Enzymes: Origin and Diffusion journal January 2012
Crystal structure of the E166A mutant of extended-spectrum β-lactamase toho-1 at 1.8 Å resolution 1 1Edited by R. Huber journal February 1999
Crystal Structure of Extended-Spectrum β-Lactamase Toho-1:  Insights into the Molecular Mechanism for Catalytic Reaction and Substrate Specificity Expansion † , ‡ journal September 2003
Structural Insights into Substrate Recognition and Product Expulsion in CTX-M Enzymes journal July 2010
Structure and Dynamics of CTX-M Enzymes Reveal Insights into Substrate Accommodation by Extended-spectrum β-Lactamases journal January 2008
Structure-function studies of arginine at position 276 in CTX-M  -lactamases journal February 2008
Analysis of the plasticity of location of the Arg244 positive charge within the active site of the TEM-1 β-lactamase journal October 2009
Molecular structure of the acyl-enzyme intermediate in β-lactam hydrolysis at 1.7 Å resolution journal October 1992
Elucidation of the role of arginine-224 in the turnover processes of class A .beta.-lactamases journal April 1992
Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis book March 2000
iMOSFLM : a new graphical interface for diffraction-image processing with MOSFLM journal March 2011
The CCP4 suite programs for protein crystallography journal September 1994
MOLREP an Automated Program for Molecular Replacement journal December 1997
Phaser crystallographic software journal July 2007
[20] Processing of X-ray diffraction data collected in oscillation mode book January 1997
Coot model-building tools for molecular graphics journal November 2004
LigPlot+: Multiple Ligand–Protein Interaction Diagrams for Drug Discovery journal October 2011
UCSF Chimera?A visualization system for exploratory research and analysis journal January 2004
Additivity of mutational effects in proteins journal September 1990
Evolution of an Antibiotic Resistance Enzyme Constrained by Stability and Activity Trade-offs journal June 2002
Identification of the β-Lactamase Inhibitor Protein-II (BLIP-II) Interface Residues Essential for Binding Affinity and Specificity for Class A β-Lactamases journal June 2013
The Structural Bases of Antibiotic Resistance in the Clinically Derived Mutant β-Lactamases TEM-30, TEM-32, and TEM-34 journal August 2002
Effect of substitution of Asn for Arg-276 in the cefotaxime-hydrolyzing class A β-lactamase CTX-M-4 journal December 1998
Sequence of the Gene Encoding a Plasmid-Mediated Cefotaxime-Hydrolyzing Class A β-Lactamase (CTX-M-4): Involvement of Serine 237 in Cephalosporin Hydrolysis journal May 1998
Natural Antibiotic Resistance and Contamination by Antibiotic Resistance Determinants: The Two Ages in the Evolution of Resistance to Antimicrobials journal January 2012

Cited By (12)

Crystallographic Snapshots of Class A β-Lactamase Catalysis Reveal Structural Changes That Facilitate β-Lactam Hydrolysis journal January 2017
Emergence of NDM-1- and CTX-M-3-Producing Raoultella ornithinolytica in Human Gut Microbiota journal November 2019
Structural and Mechanistic Basis for Extended-Spectrum Drug-Resistance Mutations in Altering the Specificity of TEM, CTX-M, and KPC β-lactamases journal February 2018
Structural Insights into the TLA-3 Extended-Spectrum β-Lactamase and Its Inhibition by Avibactam and OP0595 journal July 2017
Defining Substrate Specificity in the CTX-M Family: the Role of Asp240 in Ceftazidime Hydrolysis journal April 2018
Designing of inhibitors against CTX-M-15 type β-lactamase: potential drug candidate against β-lactamases-producing multi-drug-resistant bacteria journal June 2017
Natural Variants of the KPC-2 Carbapenemase have Evolved Increased Catalytic Efficiency for Ceftazidime Hydrolysis at the Cost of Enzyme Stability journal June 2015
Prevalence and antibiotic susceptibility pattern of CTX-M type extended-spectrum β-lactamases among clinical isolates of gram-negative bacilli in Jimma, Ethiopia journal October 2018
blaCTX-M-152, a Novel Variant of CTX-M-group-25, Identified in a Study Performed on the Prevalence of Multidrug Resistance among Natural Inhabitants of River Yamuna, India journal February 2016
Designing of inhibitors against CTX-M-15 type β-lactamase: potential drug candidate against β-lactamases-producing multi-drug-resistant bacteria text January 2017
Designing of inhibitors against CTX-M-15 type β-lactamase: potential drug candidate against β-lactamases-producing multi-drug-resistant bacteria text January 2019
Designing of inhibitors against CTX-M-15 type β-lactamase: potential drug candidate against β-lactamases-producing multi-drug-resistant bacteria text January 2019

Similar Records

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
Journal Article · Wed Jun 14 00:00:00 EDT 2017 · Biochemistry · OSTI ID:1257352
+3 more
Mapping the determinants of catalysis and substrate specificity of the antibiotic resistance enzyme CTX-M β-lactamase
Journal Article · Thu Jan 12 00:00:00 EST 2023 · Communications Biology · OSTI ID:1257352
+3 more
Removal of the Side Chain at the Active-Site Serine by a Glycine Substitution Increases the Stability of a Wide Range of Serine β-Lactamases by Relieving Steric Strain
Journal Article · Tue Apr 12 00:00:00 EDT 2016 · Biochemistry · OSTI ID:1257352
+5 more

Related Subjects

59 BASIC BIOLOGICAL SCIENCES