Impact of Mutations at Arg220 and Thr237 in PER-2 β-Lactamase on Conformation, Activity, and Susceptibility to Inhibitors.
PER-2 accounts for up to 10% of oxyimino-cephalosporin resistance inKlebsiella pneumoniaeandEscherichia coliin Argentina and hydrolyzes both cefotaxime and ceftazidime with high catalytic efficiencies (kcat/Km). Through crystallographic analyses, we recently proposed the existence of a hydrogen bond network connecting Ser70-Gln69-oxyanion water-Thr237-Arg220 that might be important for the activity and inhibition of the enzyme. Mutations at Arg244 in most class A β-lactamases (such as TEM and SHV) reduce susceptibility to mechanism-based inactivators, and Arg220 in PER β-lactamases is equivalent to Arg244. Alterations in the hydrogen bond network of the active site in PER-2, through modifications in key residues such as Arg220 and (to a much lesser extent) Thr237, dramatically impact the overall susceptibility to inactivation, with up to ∼300- and 500-fold reductions in the rate constant of inactivation (kinact)/Kivalues for clavulanic acid and tazobactam, respectively. Hydrolysis on cephalosporins and aztreonam was also affected, although to different extents compared to with wild-type PER-2; for cefepime, only an Arg220Gly mutation resulted in a strong reduction in the catalytic efficiency. Mutations at Arg220 entail modifications in the catalytic activity of PER-2 and probably local perturbations in the protein, but not global conformational changes. Therefore, the apparent structural stability of the mutants suggests that these enzymes could be possibly selectedin vivo.
Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Microbiología, Inmunología y Biotecnología, Cátedra de Microbiología, Buenos Aires, Argentina.|Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.
Melina
M
Ruggiero
Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Química Biológica, Buenos Aires, Argentina.|Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Buenos Aires, Argentina.
Lucrecia
L
Curto
Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Microbiología, Inmunología y Biotecnología, Cátedra de Microbiología, Buenos Aires, Argentina.
Florencia
F
Brunetti
InBioS, Centre d'Ingénierie des Protéines, Université de Liège, Liège, Belgium.
Eric
0000-0001-7372-2133
E
Sauvage
InBioS, Centre d'Ingénierie des Protéines, Université de Liège, Liège, Belgium.
Moreno
M
Galleni
Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Microbiología, Inmunología y Biotecnología, Cátedra de Microbiología, Buenos Aires, Argentina ppower@ffyb.uba.ar ggutkind@ffyb.uba.ar.|Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.
Pablo
0000-0002-7051-9954
P
Power
Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Microbiología, Inmunología y Biotecnología, Cátedra de Microbiología, Buenos Aires, Argentina ppower@ffyb.uba.ar ggutkind@ffyb.uba.ar.|Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.
Gabriel
G
Gutkind
D000077723:Cefepime; D002439:Cefotaxime / Q000494:pharmacology; D002442:Ceftazidime / Q000494:pharmacology; D002511:Cephalosporins / Q000494:pharmacology; D019818:Clavulanic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects; D007711:Klebsiella pneumoniae / Q000187:drug effects; D008826:Microbial Sensitivity Tests; D009154:Mutation / Q000235:genetics; D065093:beta-Lactamase Inhibitors / Q000494:pharmacology*; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D002511:Cephalosporins; D065093:beta-Lactamase Inhibitors; D019818:Clavulanic Acid; D000077723:Cefepime; D002442:Ceftazidime; C071167:beta-lactamase CTX-2; D001618:beta-Lactamases; D002439:Cefotaxime
Arg220; ESBL; Thr237; cefotaximase; ceftazidimase; inhibitor resistant; mechanism-based inhibitor