Objectives: The aim of this article is biochemical and kinetic characterization of CTX-M-43, a natural Asp-240 -> Gly mutant of CTX-M-44 (ex Toho-1), from a clinical isolate of Acinetobacter baumannii isolated in a Bolivian hospital. Methods: Steady-state kinetic parameters (K(m) and k(cat)) were determined for a large pattern of substrates. Analysis of inactivators and transient inactivators was performed to determine the efficiency of acylation (k(+2)/K) and the deacylation constant (k(+3)). Molecular modelling of Michaelis complex of ceftazidime, cefotaxime and ceftibuten, obtained from molecular mechanics calculations, was carried out. Results: CTX-M-43 showed a general increase in affinity towards all cephalosporins tested, with respect to CTX-M-44. Carbapenems acted as inactivators with a good acylation efficiency for meropenem and ertapenem and significant deacylation constant for imipenem. MICs of imipenem obtained at a higher bacterial inoculum of recombinant Escherichia coli were increased. Conclusions: Kinetic data and molecular modelling of Michaelis complex confirmed that Asp-240 -> Gly allows a better accommodation of the bulky C7 beta aminothiazol-oxyimino substituent, resulting in a general increase in the enzyme affinity towards oxyimino cephalosporins. The ascertained potentialities of CTX-M-type enzymes, supported by the kinetic data and the behaviour of the recombinant E. coli at different bacterial inocula towards carbapenems, make a possible evolution of those enzymes towards a carbapenemase activity plausible.

Natural D240G Toho1 mutant conferring resistance to ceftazidime: biochemical characterization of CTX-M-43

CELENZA, GIUSEPPE;ASCHI, MASSIMILIANO;PELLEGRINI C;AMICOSANTE, Gianfranco;PERILLI, MARIAGRAZIA
2008

Abstract

Objectives: The aim of this article is biochemical and kinetic characterization of CTX-M-43, a natural Asp-240 -> Gly mutant of CTX-M-44 (ex Toho-1), from a clinical isolate of Acinetobacter baumannii isolated in a Bolivian hospital. Methods: Steady-state kinetic parameters (K(m) and k(cat)) were determined for a large pattern of substrates. Analysis of inactivators and transient inactivators was performed to determine the efficiency of acylation (k(+2)/K) and the deacylation constant (k(+3)). Molecular modelling of Michaelis complex of ceftazidime, cefotaxime and ceftibuten, obtained from molecular mechanics calculations, was carried out. Results: CTX-M-43 showed a general increase in affinity towards all cephalosporins tested, with respect to CTX-M-44. Carbapenems acted as inactivators with a good acylation efficiency for meropenem and ertapenem and significant deacylation constant for imipenem. MICs of imipenem obtained at a higher bacterial inoculum of recombinant Escherichia coli were increased. Conclusions: Kinetic data and molecular modelling of Michaelis complex confirmed that Asp-240 -> Gly allows a better accommodation of the bulky C7 beta aminothiazol-oxyimino substituent, resulting in a general increase in the enzyme affinity towards oxyimino cephalosporins. The ascertained potentialities of CTX-M-type enzymes, supported by the kinetic data and the behaviour of the recombinant E. coli at different bacterial inocula towards carbapenems, make a possible evolution of those enzymes towards a carbapenemase activity plausible.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11697/1969
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