title: Broad antibiotic resistance profile of the subclass B3 metallo-β-lactamase GOB-1, a di-zinc enzyme.
abstract: The metallo-β-lactamase (MBL) GOB-1 was expressed via a T7 expression system in Escherichia coli BL21(DE3). The MBL was purified to homogeneity and shown to exhibit a broad substrate profile, hydrolyzing all the tested β-lactam compounds efficiently. The GOB enzymes are unique among MBLs due to the presence of a glutamine residue at position 116, a zinc-binding residue in all known class B1 and B3 MBL structures. Here we produced and studied the Q116A, Q116N and Q116H mutants. The substrate profiles were similar for each mutant, but with significantly reduced activity compared with that of the wild-type. In contrast to the Q116H enzyme, which bound two zinc ions just like the wild-type, only one zinc ion is present in Q116A and Q116N. These results suggest that the Q116 residue plays a role in the binding of the zinc ion in the QHH site.
authors.affiliation: Centre d'Ingénierie des Protéines, Université de Liège, Liège, Belgium.
authors.forename: Louise E
authors.initials: LE
authors.lastname: Horsfall
authors.forename: Youssef
authors.initials: Y
authors.lastname: Izougarhane
authors.forename: Patricia
authors.initials: P
authors.lastname: Lassaux
authors.forename: Nathalie
authors.initials: N
authors.lastname: Selevsek
authors.forename: Benoit M R
authors.initials: BM
authors.lastname: Liénard
authors.forename: Laurent
authors.initials: L
authors.lastname: Poirel
authors.forename: Michael B
authors.initials: MB
authors.lastname: Kupper
authors.forename: Kurt M
authors.initials: KM
authors.lastname: Hoffmann
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Frère
authors.forename: Moreno
authors.initials: M
authors.lastname: Galleni
authors.forename: Carine
authors.initials: C
authors.lastname: Bebrone
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000201:enzymology; D005973:Glutamine / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*; D047090:beta-Lactams / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D047090:beta-Lactams; D005973:Glutamine; D001618:beta-Lactamases; C558534:beta-lactamase GOB-1, Elizabethkingia meningoseptica; D015032:Zinc