ORBi: Mallo Susana - A Tripeptide Deletion in the Class C beta-Lactamase FOX-4 Enzyme Impairs Cefoxitin Hydrolysis and Slightly Increases Susceptibility to beta-Lactamase Inhibitors

Reference : A Tripeptide Deletion in the Class C beta-Lactamase FOX-4 Enzyme Impairs Cefoxitin Hydro...


	Document type :	Scientific journals : Article
	Discipline(s) :	Life sciences : Biochemistry, biophysics & molecular biology
	To cite this reference:	http://hdl.handle.net/2268/40435

Title :	A Tripeptide Deletion in the Class C beta-Lactamase FOX-4 Enzyme Impairs Cefoxitin Hydrolysis and Slightly Increases Susceptibility to beta-Lactamase Inhibitors
Language :	English
Author, co-author :	Mallo, Susana [> >]
	Pérez-Llarena, Francisco J. [> >]
	Kerff, Frédéric [Université de Liège - ULg > > Centre d'ingénierie des protéines >]
	Fernández-Moreira, Esteban [> >]
	Soares, Nelson C. [> >]
	Galleni, Moreno [Université de Liège - ULg > Département des sciences de la vie > Macromolécules biologiques >]
	Bou, German [> >]
Publication date :	Jun-2010
Journal title :	Journal of Antimicrobial Chemotherapy
Publisher :	Oxford University Press
Volume :	65
Issue/season :	6
Pages :	1187-94
Audience :	International
ISSN :	0305-7453
e-ISSN :	1460-2091
City :	London
Country :	United Kingdom
Abstract :	[en] OBJECTIVES: A natural variant of the AmpC enzyme from Escherichia coli HKY28 with a tripeptide deletion (Gly-286/Ser-287/Asp-288) was recently described. The isolate produced an inhibitor-sensitive AmpC beta-lactamase variant that also conferred higher than usual levels of resistance to ceftazidime in the E. coli host. To demonstrate whether this is true in other class C beta-lactamase enzymes, we deleted the equivalent tripeptide in the FOX-4 plasmid-mediated class C beta-lactamase. METHODS: By site-directed mutagenesis, we deleted the tripeptide Gly-306/Asn-307/Ser-308 of FOX-4, thus generating FOX-4(DeltaGNS). The enzymes (FOX-4 wild-type and DeltaGNS) were purified and kinetic parameters (kcat, Km, kcat/Km) as well as IC50 values of several beta-lactams were assessed. Modelling studies were also performed. RESULTS: FOX-4(DeltaGNS) did not increase the catalytic efficiency towards ceftazidime, although it conferred a slight increase in the susceptibility to beta-lactamase inhibitors. There was also a noteworthy decrease in the cefoxitin MIC with the FOX-4(DeltaGNS) mutant (from 512 to 16 mg/L) as well as a 10-fold decrease in kcat/Km towards imipenem, which revealed specific structural features. CONCLUSIONS: Although deletions in the R2-loop are able to extend the substrate spectrum of class C enzymes, the present results do not confirm this hypothesis in FOX-4. The FOX-4 R2 site would already be wide enough to accommodate antibiotic molecules, and thus any amino acid replacement or deletion at this location would not affect the hydrolytic efficiency towards beta-lactams and would have a less drastic effect on the susceptibility to beta-lactamase inhibitors.
Research units :	Centre d'Ingénierie des Protéines - CIP
Permalink :	http://hdl.handle.net/2268/40435
	also: http://hdl.handle.net/2268/74940

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