Biochemical analysis of TEM-134, a new TEM-type extended-spectrum β-lactamase variant produced in a Citrobacter koseri clinical isolate from an Italian hospital

Mariagrazia Perilli, Giuseppe Celenza, Marianna Fiore, Bernardetta Segatore, Cristina Pellegrini, Francesco Luzzaro, Gian Maria Rossolini, Gianfranco Amicosante

Research output: Contribution to journalArticle

Abstract

Objectives: Kinetic characterization of TEM-134, a new TEM-type extended-spectrum β-lactamase variant isolated from Citrobacter koseri during an Italian nationwide survey. TEM-134 is a natural derivative of TEM-2 with the following substitutions: E104K, R164H and G238S. Methods: Recombinant TEM-134 was purified from Escherichia coli HB101 (pMGP-134) by three chromatographic steps (cation-exchange chromatography, gel permeation and fast chromatofocusing). Steady-state kinetic parameters (Km and kcat) were determined by measuring substrate hydrolysis under initial rate conditions using the Hanes linearization of the Michaelis-Menten equation. Modelling was carried out using the software Modeller (version 9.1). Results: TEM-134 hydrolysed with variable efficiency (kcat/ Km ranging from 5 × 103 to 8.0 × 105 M-1 · s-1) penicillins, narrow-spectrum cephalosporins, cefepime, cefotaxime, ceftazidime and aztreonam, which appeared to be the best substrate. Molecular modelling of the enzyme indicated that the R164H substitution may result in a compromised omega loop in TEM-134 and this may be responsible for its narrower spectrum of activity. Conclusions: Kinetic data and molecular modelling suggested that R164H has a mild detrimental effect on the global activity of the enzyme.

Original languageEnglish
Pages (from-to)877-880
Number of pages4
JournalJournal of Antimicrobial Chemotherapy
Volume60
Issue number4
DOIs
Publication statusPublished - Oct 2007

Keywords

  • Antibiotic resistance
  • Class A
  • Enterobacteriaceae

ASJC Scopus subject areas

  • Pharmacology
  • Microbiology

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