title: Resistance to the third-generation cephalosporin ceftazidime by a deacylation-deficient mutant of the TEM β-lactamase by the uncommon covalent-trapping mechanism.
abstract: The Glu166Arg/Met182Thr mutant of Escherichia coli TEM(pTZ19-3) β-lactamase produces a 128-fold increase in the level of resistance to the antibiotic ceftazidime in comparison to that of the parental wild-type enzyme. The single Glu166Arg mutation resulted in a dramatic decrease in both the level of enzyme expression in bacteria and the resistance to penicillins, with a concomitant 4-fold increase in the resistance to ceftazidime, a third-generation cephalosporin. Introduction of the second amino acid substitution, Met182Thr, restored enzyme expression to a level comparable to that of the wild-type enzyme and resulted in an additional 32-fold increase in the minimal inhibitory concentration of ceftazidime to 64 μg/mL. The double mutant formed a stable covalent complex with ceftazidime that remained intact for the entire duration of the monitoring, which exceeded a time period of 40 bacterial generations. Compared to those of the wild-type enzyme, the affinity of the TEM(pTZ19-3) Glu166Arg/Met182Thr mutant for ceftazidime increased by at least 110-fold and the acylation rate constant was augmented by at least 16-fold. The collective experimental data and computer modeling indicate that the deacylation-deficient Glu166Arg/Met182Thr mutant of TEM(pTZ19-3) produces resistance to the third-generation cephalosporin ceftazidime by an uncommon covalent-trapping mechanism. This is the first documentation of such a mechanism by a class A β-lactamase in a manifestation of resistance.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
authors.forename: Nuno T
authors.initials: NT
authors.lastname: Antunes
authors.forename: Hilary
authors.initials: H
authors.lastname: Frase
authors.forename: Marta
authors.initials: M
authors.lastname: Toth
authors.forename: Shahriar
authors.initials: S
authors.lastname: Mobashery
authors.forename: Sergei B
authors.initials: SB
authors.lastname: Vakulenko
mesh_terms: D000215:Acylation / Q000187:drug effects; D002442:Ceftazidime / Q000494:pharmacology*; D003198:Computer Simulation; D024881:Drug Resistance, Bacterial / Q000187:drug effects*; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008954:Models, Biological; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D002442:Ceftazidime; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1