The aim of this study was to characterize a novel extended-spectrum P-lactamase that belongs to the TEM family, the TEM-149 enzyme, and that was isolated from the urine of two hospitalized patients from different hospitals in southern Italy. The peculiarity of this enzyme was the finding of a valine residue at position 240. The array of amino acid substitutions found in TEM-149 was as follows: E104K, R164S, M182T, and E240V. A reversion of a threonine residue at position 182 was also performed to create a new mutant, TEM-149(T182M), in order to assess the contribution of this substitution on the kinetic profile and the stability of TEM-149. The bla(TEM-149) and bla(TEM-149/T182M) genes were cloned into pBC-SK, and the corresponding enzymes were purified from recombinant Escherichia coli HB101 by the same procedure. Both enzymes hydrolyzed all beta-lactams tested, with a preference for ceftazidime, which was found to be the best substrate. By comparison of the kinetic parameters of the TEM-149 and the TEM-149(T182M) enzymes, a reduction of the catalytic efficiency for the TEM-149(T182M) mutant was observed against all substrates tested except benzylpenicillin, cefotaxime, and aztreonam. Tazobactam, clavulanic acid, and sulbactam were good inhibitors of the TEM-149 P-lactamase.

E240V substitution increases catalytic efficiency toward ceftazidime in a new natural TEM-type extended-spectrum beta-lactamase, TEM-149, from Enterobacter aerogenes and Serratia marcescens clinical isolates

Perilli Mariagrazia;Celenza G;DE SANTIS, FRANCESCA;Pellegrini Cristina;FORCELLA, CHIARA;Amicosante Gianfranco
2008

Abstract

The aim of this study was to characterize a novel extended-spectrum P-lactamase that belongs to the TEM family, the TEM-149 enzyme, and that was isolated from the urine of two hospitalized patients from different hospitals in southern Italy. The peculiarity of this enzyme was the finding of a valine residue at position 240. The array of amino acid substitutions found in TEM-149 was as follows: E104K, R164S, M182T, and E240V. A reversion of a threonine residue at position 182 was also performed to create a new mutant, TEM-149(T182M), in order to assess the contribution of this substitution on the kinetic profile and the stability of TEM-149. The bla(TEM-149) and bla(TEM-149/T182M) genes were cloned into pBC-SK, and the corresponding enzymes were purified from recombinant Escherichia coli HB101 by the same procedure. Both enzymes hydrolyzed all beta-lactams tested, with a preference for ceftazidime, which was found to be the best substrate. By comparison of the kinetic parameters of the TEM-149 and the TEM-149(T182M) enzymes, a reduction of the catalytic efficiency for the TEM-149(T182M) mutant was observed against all substrates tested except benzylpenicillin, cefotaxime, and aztreonam. Tazobactam, clavulanic acid, and sulbactam were good inhibitors of the TEM-149 P-lactamase.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11697/11188
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