title: Natural D240G Toho-1 mutant conferring resistance to ceftazidime: biochemical characterization of CTX-M-43.
abstract: OBJECTIVE
  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 C7beta 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.
authors.affiliation: Department of Biomedical Sciences and Technologies, University of L'Aquila, L'Aquila, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Celenza
authors.forename: Carla
authors.initials: C
authors.lastname: Luzi
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Aschi
authors.forename: Bernardetta
authors.initials: B
authors.lastname: Segatore
authors.forename: Domenico
authors.initials: D
authors.lastname: Setacci
authors.forename: Cristina
authors.initials: C
authors.lastname: Pellegrini
authors.forename: Chiara
authors.initials: C
authors.lastname: Forcella
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Amicosante
authors.forename: Mariagrazia
authors.initials: M
authors.lastname: Perilli