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Antimicrobial Agents and Chemotherapy, April 2001, p. 1254-1262, Vol. 45, No. 4
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.4.1254-1262.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Biochemical Characterization of the FEZ-1 Metallo-beta -Lactamase of Legionella gormanii ATCC 33297T Produced in Escherichia coli

Paola Sandra Mercuri,1 Fabrice Bouillenne,1 Letizia Boschi,2 Josette Lamotte-Brasseur,1 Gianfranco Amicosante,3 Bart Devreese,4 Jozef van Beeumen,4 Jean-Marie Frère,1 Gian Maria Rossolini,2 and Moreno Galleni1,*

Centre d'Ingénierie des Protéines, Université de Liège, B-4000 Liège,1 and Laboratorium voor Eiwitbiochemie en Eiwitengineering, University of Gent, B-9000 Gent,4 Belgium, and Dipartimento di Biologia Molecolare, Sezione di Microbiologia, Università di Siena, I-53100 Siena, 2 and Dipartimento di Scienze e Tecnologie Biomediche, Università di L'Aquila, I-67100 Coppito, L'Aquila,3 Italy

Received 19 July 2000/Returned for modification 28 October 2000/Accepted 2 January 2001

The blaFEZ-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.

* Corresponding author. Mailing address: Centre d'Ingénierie des Protéines, B6 Sart Tilman, Université de Liège, B-4000 Liège, Belgium. Phone: 32-4-3663419. Fax: 32-4-3663364. E-mail: mgalleni{at}ulg.ac.be.

Antimicrobial Agents and Chemotherapy, April 2001, p. 1254-1262, Vol. 45, No. 4
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.4.1254-1262.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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