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.
OBJECTIVE
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.
Department of Sciences and Biomedical Technologies, University of L'Aquila, L'Aquila, Italy.
Mariagrazia
M
Perilli
Giuseppe
G
Celenza
Marianna
M
Fiore
Bernardetta
B
Segatore
Cristina
C
Pellegrini
Francesco
F
Luzzaro
Gian Maria
GM
Rossolini
Gianfranco
G
Amicosante
D019943:Amino Acid Substitution / Q000235:genetics; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D041041:Citrobacter koseri / Q000201:enzymology* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004756:Enterobacteriaceae Infections / Q000382:microbiology; D004926:Escherichia coli / Q000235:genetics; D006761:Hospitals; D007525:Isoelectric Focusing; D007558:Italy; D007700:Kinetics; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D047090:beta-Lactams / Q000378:metabolism
D011994:Recombinant Proteins; D047090:beta-Lactams; D001618:beta-Lactamases