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

doi:10.1128/AAC.44.11.3003-3007.2000

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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 aeruginosafrom the Mediterranean area. In this work, VIM-1 was purifiedfrom an Escherichia coli strain carrying the cloned bla_VIM-1 geneby means of an anion-exchange chromatography step followed bya gel permeation chromatography step. The purified enzyme exhibiteda molecular mass of 26 kDa in sodium dodecyl sulfate-polyacrylamidegel electrophoresis, and an acidic pI of 5.1 in analytical isoelectricfocusing. Amino-terminal sequencing showed that mature VIM-1 resultsfrom the removal of a 26-amino-acid signal peptide from the precursor.VIM-1 hydrolyzes a broad array of $beta$ -lactam compounds, includingpenicillins, narrow- to expanded-spectrum cephalosporins, carbapenems,and mechanism-based serine- $beta$ -lactamase inactivators. Only monobactamsescape hydrolysis. The highest catalytic constant/K_m ratios (>10⁶ M¹ · s¹) were observed with carbenicillin, azlocillin, some cephalosporins(cephaloridine, cephalothin, cefuroxime, cefepime, and cefpirome),imipenem, and biapenem. Kinetic parameters showed remarkable variabilitywith different $beta$ -lactams and also within the various penam, cephem,and carbapenem compounds, resulting in no clear preference ofthe enzyme for any of these $beta$ -lactam subfamilies. Significantdifferences were observed with some substrates between the kineticparameters of VIM-1 and those of other metallo- $beta$ -lactamases. Inactivationassays carried out with various chelating agents (EDTA, 1,10-o-phenanthroline,and pyridine-2,6-dicarboxylic acid) indicated that formation ofa ternary enzyme-metal-chelator complex precedes metal removalfrom the zinc center of the protein and revealed notable differencesin the inactivation parameters of VIM-1 with differentagents.

^* Corresponding author. Mailing address for Nicola Franceschini: Dipartimento di Scienze e Tecnologie Biomediche, Universitàdegli 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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Purification and Biochemical Characterization of the VIM-1 Metallo--Lactamase

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