The asparagine to aspartic acid substitution at position 276 of TEM-35 and TEM-36 is involved in the beta-lactamase resistance to clavulanic acid. 1995

I Saves, and O Burlet-Schiltz, and P Swarén, and F Lefèvre, and J M Masson, and J C Promé, and J P Samama
Groupe d'Ingénierie des Protéines, CNRS, Toulouse, France.

TEM-35 (inhibitor resistant TEM (IRT)-4) and TEM-36 (IRT-7) clavulanic acid-resistant beta-lactamases have evolved from TEM-1 beta-lactamase by two substitutions: a methionine to a leucine or a valine at position 69 and an asparagine to an aspartic acid at position 276. The substitutions at position 69 have previously been shown to be responsible for the resistance to clavulanic acid, and they are the only mutations encountered in TEM-33 (IRT-5) and TEM-34 (IRT-6). However, the N276D substitution has never been found alone in inhibitor-resistant beta-lactamases, and its role in resistance to clavulanic acid was thus unclear. The N276D mutant was constructed, purified, and kinetically characterized. It was shown that the substitution has a direct effect on substrate affinities and leads to slightly decreased catalytic efficiencies and that clavulanic acid becomes a poor substrate of the enzyme. Electrospray mass spectrometry demonstrated the simultaneous presence of free and inhibited enzymes after incubation with clavulanic acid and showed that a cleaved moiety of clavulanic acid leads to the formation of the major inactive complex. The kinetic properties of the N276D mutant could be linked to a salt-bridge interaction of aspartic acid 276 with arginine 244 that alters the electrostatic properties in the substrate binding area.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008826 Microbial Sensitivity Tests Any tests that demonstrate the relative efficacy of different chemotherapeutic agents against specific microorganisms (i.e., bacteria, fungi, viruses). Bacterial Sensitivity Tests,Drug Sensitivity Assay, Microbial,Minimum Inhibitory Concentration,Antibacterial Susceptibility Breakpoint Determination,Antibiogram,Antimicrobial Susceptibility Breakpoint Determination,Bacterial Sensitivity Test,Breakpoint Determination, Antibacterial Susceptibility,Breakpoint Determination, Antimicrobial Susceptibility,Fungal Drug Sensitivity Tests,Fungus Drug Sensitivity Tests,Sensitivity Test, Bacterial,Sensitivity Tests, Bacterial,Test, Bacterial Sensitivity,Tests, Bacterial Sensitivity,Viral Drug Sensitivity Tests,Virus Drug Sensitivity Tests,Antibiograms,Concentration, Minimum Inhibitory,Concentrations, Minimum Inhibitory,Inhibitory Concentration, Minimum,Inhibitory Concentrations, Minimum,Microbial Sensitivity Test,Minimum Inhibitory Concentrations,Sensitivity Test, Microbial,Sensitivity Tests, Microbial,Test, Microbial Sensitivity,Tests, Microbial Sensitivity
D008958 Models, Molecular Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures. Molecular Models,Model, Molecular,Molecular Model
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010957 Plasmids Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS. Episomes,Episome,Plasmid
D002969 Clavulanic Acids Acids, salts, and derivatives of clavulanic acid (C8H9O5N). They consist of those beta-lactam compounds that differ from penicillin in having the sulfur of the thiazolidine ring replaced by an oxygen. They have limited antibacterial action, but block bacterial beta-lactamase irreversibly, so that similar antibiotics are not broken down by the bacterial enzymes and therefore can exert their antibacterial effects. Acids, Clavulanic
D004352 Drug Resistance, Microbial The ability of microorganisms, especially bacteria, to resist or to become tolerant to chemotherapeutic agents, antimicrobial agents, or antibiotics. This resistance may be acquired through gene mutation or foreign DNA in transmissible plasmids (R FACTORS). Antibiotic Resistance,Antibiotic Resistance, Microbial,Antimicrobial Resistance, Drug,Antimicrobial Drug Resistance,Antimicrobial Drug Resistances,Antimicrobial Resistances, Drug,Drug Antimicrobial Resistance,Drug Antimicrobial Resistances,Drug Resistances, Microbial,Resistance, Antibiotic,Resistance, Drug Antimicrobial,Resistances, Drug Antimicrobial
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D001618 beta-Lactamases Enzymes found in many bacteria which catalyze the hydrolysis of the amide bond in the beta-lactam ring. Well known antibiotics destroyed by these enzymes are penicillins and cephalosporins. beta-Lactamase,beta Lactamase,beta Lactamases
D013058 Mass Spectrometry An analytical method used in determining the identity of a chemical based on its mass using mass analyzers/mass spectrometers. Mass Spectroscopy,Spectrometry, Mass,Spectroscopy, Mass,Spectrum Analysis, Mass,Analysis, Mass Spectrum,Mass Spectrum Analysis,Analyses, Mass Spectrum,Mass Spectrum Analyses,Spectrum Analyses, Mass

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