[en] 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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Mercuri, Paola ; Université de Liège > Département des sciences de la vie > Macromolécules biologiques
Garcia-Saez, I.
De Vriendt, K.
Thamm, Iris ; Université de Liège - ULiège > Département des sciences de la vie > Département des sciences de la vie
Devreese, B.
Van Beeumen, J.
Dideberg, O.
Rossolini, G. M.
Frère, Jean-Marie ; Université de Liège - ULiège > Département des sciences de la vie > Département des sciences de la vie
Galleni, Moreno ; Université de Liège - ULiège > Département des sciences de la vie > Macromolécules biologiques
Language :
English
Title :
Probing the specificity of the subclass B3 FEZ-1 metallo-beta-lactamase by site-directed mutagenesis
Publication date :
06 August 2004
Journal title :
Journal of Biological Chemistry
ISSN :
0021-9258
eISSN :
1083-351X
Publisher :
Amer Soc Biochemistry Molecular Biology Inc, Bethesda, United States - Maryland
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