Biochemical characterization of the FEZ-1 metallo-beta-lactamase of Legionella gormanii ATCC 33297T produced in Escherichia coli.
The bla(FEZ-1) gene coding for the metallo-beta-lactamase of Legionella (Fluoribacter) gormanii ATCC 33297T was overexpressed via a T7 expression system in Escherichia coli BL21(DE3)(pLysS). The product was purified to homogeneity in two steps with a yield of 53%. The FEZ-1 metallo-beta-lactamase exhibited a broad-spectrum activity profile, with a preference for cephalosporins such as cephalothin, cefuroxime, and cefotaxime. Monobactams were not hydrolyzed. The beta-lactamase was inhibited by metal chelators. FEZ-1 is a monomeric enzyme with a molecular mass of 29,440 Da which possesses two zinc-binding sites. Its zinc content did not vary in the pH range of 5 to 9, but the presence of zinc ions modified the catalytic efficiency of the enzyme. A model of the FEZ-1 three-dimensional structure was built.
Centre d'Ingénierie des Protéines, Université de Liège, B-4000 Liège, Belgium.
P S
PS
Mercuri
F
F
Bouillenne
L
L
Boschi
J
J
Lamotte-Brasseur
G
G
Amicosante
B
B
Devreese
J
J
van Beeumen
J M
JM
Frère
G M
GM
Rossolini
M
M
Galleni
D000595:Amino Acid Sequence; D001665:Binding Sites; D018550:Cephalosporin Resistance; D002614:Chelating Agents / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007875:Legionella / Q000201:enzymology* / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D015032:Zinc / Q000032:analysis; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D002614:Chelating Agents; C434285:beta-lactamase FEZ-1; D001618:beta-Lactamases; D015032:Zinc