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.

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