Probing the specificity of the subclass B3 FEZ-1 metallo-beta-lactamase by site-directed mutagenesis.
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.
Mercuri
Paola Sandra
PS
Centre d'Ingénierie des Protéines, B6 Sart Tilman, Université de Liège, B-4000 Liège, Belgium.
García-Sáez
Isabel
I
De Vriendt
Kris
K
Thamm
Iris
I
Devreese
Bart
B
Van Beeumen
Jozef
J
Dideberg
Otto
O
Rossolini
Gian Maria
GM
Frère
Jean-Marie
JM
Galleni
Moreno
M
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*
D002511:Cephalosporins; C434285:beta-lactamase FEZ-1; D001618:beta-Lactamases; D015032:Zinc