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Antimicrobial Agents and Chemotherapy, November 2000, p. 3003-3007, Vol. 44, No. 11
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Purification and Biochemical Characterization of the VIM-1 Metallo-beta -Lactamase

Nicola Franceschini,1,* Berardo Caravelli,1 Jean-Denis Docquier,2 Moreno Galleni,3 Jean-Marie Frère,3 Gianfranco Amicosante,1 and Gian Maria Rossolini2,*

Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi L'Aquila, I-67100 Coppito, L'Aquila,1 and Dipartimento di Biologia Molecolare, Sez. di Microbiologia, Università di Siena, I-53100 Siena,2 Italy, and Laboratoire d'Enzymologie et Centre d'Ingénierie des Protéines, Institut de Chimie, Université de Liège, Sart Tilman, B-4000 Liège, Belgium3

Received 13 January 2000/Returned for modification 5 May 2000/Accepted 8 August 2000

VIM-1 is a new group 3 metallo-beta -lactamase recently detected in carbapenem-resistant nosocomial isolates of Pseudomonas aeruginosa from the Mediterranean area. In this work, VIM-1 was purified from an Escherichia coli strain carrying the cloned blaVIM-1 gene by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. The purified enzyme exhibited a molecular mass of 26 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an acidic pI of 5.1 in analytical isoelectric focusing. Amino-terminal sequencing showed that mature VIM-1 results from the removal of a 26-amino-acid signal peptide from the precursor. VIM-1 hydrolyzes a broad array of beta -lactam compounds, including penicillins, narrow- to expanded-spectrum cephalosporins, carbapenems, and mechanism-based serine-beta -lactamase inactivators. Only monobactams escape hydrolysis. The highest catalytic constant/Km ratios (>106 M-1 · s-1) were observed with carbenicillin, azlocillin, some cephalosporins (cephaloridine, cephalothin, cefuroxime, cefepime, and cefpirome), imipenem, and biapenem. Kinetic parameters showed remarkable variability with different beta -lactams and also within the various penam, cephem, and carbapenem compounds, resulting in no clear preference of the enzyme for any of these beta -lactam subfamilies. Significant differences were observed with some substrates between the kinetic parameters of VIM-1 and those of other metallo-beta -lactamases. Inactivation assays carried out with various chelating agents (EDTA, 1,10-o-phenanthroline, and pyridine-2,6-dicarboxylic acid) indicated that formation of a ternary enzyme-metal-chelator complex precedes metal removal from the zinc center of the protein and revealed notable differences in the inactivation parameters of VIM-1 with different agents.

* Corresponding author. Mailing address for Nicola Franceschini: Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi de L'Aquila, Via Vetoio, 67100 L'Aquila, Italy. Phone: 39 0862 433456. Fax: 39 0862 433433. E-mail: nicola.franceschini{at}cc.univaq.it.

Antimicrobial Agents and Chemotherapy, November 2000, p. 3003-3007, Vol. 44, No. 11
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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