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

Objectives: Kinetic characterization of TEM-134, a new TEM-type extended-spectrum beta-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 (K-m and k(cat)) 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 (k(cat)/K-m ranging from 5 x 10(3) to 8.0 x 10(5) 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.