The Journal of antimicrobial chemotherapy · 2007
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.
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Perilli Mariagrazia, Celenza Giuseppe, Fiore Marianna, Segatore Bernardetta, Pellegrini Cristina, Luzzaro Francesco, Rossolini Gian Maria, Amicosante Gianfranco
Аннотация
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.
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.