title: Probing the specificity of the subclass B3 FEZ-1 metallo-beta-lactamase by site-directed mutagenesis.
abstract: The subclass B3 FEZ-1 beta-lactamase produced by Fluoribacter (Legionella) gormanii is a Zn(II)-containing enzyme that hydrolyzes the beta-lactam bond in penicillins, cephalosporins, and carbapenems. FEZ-1 has been extensively studied using kinetic, computational modeling and x-ray crystallography. In an effort to probe residues potentially involved in substrate binding and zinc binding, five site-directed mutants of FEZ-1 (H121A, Y156A, S221A, N225A, and Y228A) were prepared and characterized using metal analyses and steady state kinetics. The activity of H121A is dependent on zinc ion concentration. The H121A monozinc form is less active than the dizinc form, which exhibits an activity similar to that of the wild type enzyme. Tyr156 is not essential for binding and hydrolysis of the substrate. Substitution of residues Ser221 and Asn225 modifies the substrate profile by selectively decreasing the activity against carbapenems. The Y228A mutant is inhibited by the product formed upon hydrolysis of cephalosporins. A covalent bond between the side chain of Cys200 and the hydrolyzed cephalosporins leads to the formation of an inactive and stable complex.
authors.affiliation: Centre d'Ingénierie des Protéines, B6 Sart Tilman, Université de Liège, B-4000 Liège, Belgium.
authors.forename: Paola Sandra
authors.initials: PS
authors.lastname: Mercuri
authors.forename: Isabel
authors.initials: I
authors.lastname: García-Sáez
authors.forename: Kris
authors.initials: K
authors.lastname: De Vriendt
authors.forename: Iris
authors.initials: I
authors.lastname: Thamm
authors.forename: Bart
authors.initials: B
authors.lastname: Devreese
authors.forename: Jozef
authors.initials: J
authors.lastname: Van Beeumen
authors.forename: Otto
authors.initials: O
authors.lastname: Dideberg
authors.forename: Gian Maria
authors.initials: GM
authors.lastname: Rossolini
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Frère
authors.forename: Moreno
authors.initials: M
authors.lastname: Galleni
mesh_terms: D001665:Binding Sites; D002511:Cephalosporins / Q000378:metabolism; D003198:Computer Simulation; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006860:Hydrogen Bonding; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D002511:Cephalosporins; C434285:beta-lactamase FEZ-1; D001618:beta-Lactamases; D015032:Zinc