Probing the specificity of the subclass B3 FEZ-1 metallo-β-lactamase by site-directed mutagenesis

Paola Sandra Mercuri, Isabel García-Sáez, Kris De Vriendt, Iris Thamm, Bart Devreese, Jozef Van Beeumen, Otto Dideberg, Gian Maria Rossolini, Jean Marie Frère, Moreno Galleni

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The subclass B3 FEZ-1 β-lactamase produced by Fluoribacter (Legionella) gormanii is a Zn(II)-containing enzyme that hydrolyzes the β-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. Tyr 156 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.

Original languageEnglish
Pages (from-to)33630-33638
Number of pages9
JournalJournal of Biological Chemistry
Issue number32
Publication statusPublished - Aug 6 2004


ASJC Scopus subject areas

  • Biochemistry

Cite this

Mercuri, P. S., García-Sáez, I., De Vriendt, K., Thamm, I., Devreese, B., Van Beeumen, J., Dideberg, O., Rossolini, G. M., Frère, J. M., & Galleni, M. (2004). Probing the specificity of the subclass B3 FEZ-1 metallo-β-lactamase by site-directed mutagenesis. Journal of Biological Chemistry, 279(32), 33630-33638.