Biochemical Characterization of Laboratory Mutants of Extended-Spectrum β-Lactamase TEM-60

ABSTRACT Three mutants of the extended-spectrum β-lactamase TEM-60, the P51L, K104E, and S164R mutants, were constructed by site-directed mutagenesis. The kinetic parameters of the mutated enzymes and interactions of inhibitors were significantly different from those of TEM-60, revealing that the L51P mutation plays an important role in enzyme activity and stability in the TEM-60 background.

[1]  P. Bradford.  Extended-Spectrum β-Lactamases in the 21st Century: Characterization, Epidemiology, and Detection of This Important Resistance Threat , 2001, Clinical Microbiology Reviews.

[2]  E. Bruck,et al.  National Committee for Clinical Laboratory Standards. , 1980, Pediatrics.

[3]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[4]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[5]  G. Rossolini,et al.  Characterization of a New Extended-Spectrum β-Lactamase (TEM-87) Isolated in Proteus mirabilis during an Italian Survey , 2002, Antimicrobial Agents and Chemotherapy.

[6]  Mary Jane Ferraro,et al.  Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically : approved standard , 2000 .

[7]  J. Frère,et al.  Use of the chromosomal class A beta-lactamase of Mycobacterium fortuitum D316 to study potentially poor substrates and inhibitory beta-lactam compounds , 1994, Antimicrobial Agents and Chemotherapy.

[8]  J. Frère,et al.  6-beta-Iodopenicillanate as a probe for the classification of beta-lactamases. , 1986, The Biochemical journal.

[9]  J. Knox,et al.  Extended-spectrum and inhibitor-resistant TEM-type beta-lactamases: mutations, specificity, and three-dimensional structure , 1995, Antimicrobial agents and chemotherapy.

[10]  G. Cornaglia,et al.  Ceftazidime and Aztreonam Resistance inProvidencia stuartii: Characterization of a Natural TEM-Derived ExtendedSpectrum β-Lactamase, TEM-60 , 1998, Antimicrobial Agents and Chemotherapy.

[11]  Sergei Vakulenko,et al.  Mutant TEM β-Lactamase Producing Resistance to Ceftazidime, Ampicillins, and β-Lactamase Inhibitors , 2002, Antimicrobial Agents and Chemotherapy.

[12]  G. Jacoby.  EXTENDED-SPECTRUM β-LACTAMASES AND OTHER ENZYMES PROVIDING RESISTANCE TO OXYIMINO-β-LACTAMS , 1997 .

[13]  S. Ho,et al.  Site-directed mutagenesis by overlap extension using the polymerase chain reaction. , 1989, Gene.

[14]  J. Frère,et al.  TEM beta-lactamase mutants hydrolysing third-generation cephalosporins. A kinetic and molecular modelling analysis. , 1994, Journal of molecular biology.