Purification and biochemical characterization of the VIM-1 metallo-β-lactamase

N. Franceschini, B. Caravelli, J. D. Docquier, M. Galleni, J. M. Frere, G. Amicosante, G. M. Rossolini

Research output: Contribution to journalArticle

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Abstract

VIM-1 is a new group 3 metallo-β-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 bla(VIM-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 β-lactam compounds, including penicillins, narrow- to expanded-spectrum cephalosporins, carbapenems, and mechanism-based serine-β-lactamase inactivators. Only monobactams escape hydrolysis. The highest catalytic constant/K(m) 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 β-lactams and also within the various penam, cephem, and carbapenem compounds, resulting in no clear preference of the enzyme for any of these β-lactam subfamilies. Significant differences were observed with some substrates between the kinetic parameters of VIM-1 and those of other metallo-β-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.

Original languageEnglish
Pages (from-to)3003-3007
Number of pages5
JournalAntimicrobial Agents and Chemotherapy
Volume44
Issue number11
DOIs
Publication statusPublished - 2000

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Lactams
Carbapenems
cefpirome
Cephalosporins
Chelating Agents
biapenem
Enzymes
Azlocillin
Monobactams
Cephaloridine
Carbenicillin
Cephalothin
Cefuroxime
Imipenem
Coordination Complexes
Isoelectric Focusing
Protein Sorting Signals
Edetic Acid
Sodium Dodecyl Sulfate
Penicillins

ASJC Scopus subject areas

  • Pharmacology (medical)

Cite this

Franceschini, N., Caravelli, B., Docquier, J. D., Galleni, M., Frere, J. M., Amicosante, G., & Rossolini, G. M. (2000). Purification and biochemical characterization of the VIM-1 metallo-β-lactamase. Antimicrobial Agents and Chemotherapy, 44(11), 3003-3007. https://doi.org/10.1128/AAC.44.11.3003-3007.2000

Purification and biochemical characterization of the VIM-1 metallo-β-lactamase. / Franceschini, N.; Caravelli, B.; Docquier, J. D.; Galleni, M.; Frere, J. M.; Amicosante, G.; Rossolini, G. M.

In: Antimicrobial Agents and Chemotherapy, Vol. 44, No. 11, 2000, p. 3003-3007.

Research output: Contribution to journalArticle

Franceschini, N, Caravelli, B, Docquier, JD, Galleni, M, Frere, JM, Amicosante, G & Rossolini, GM 2000, 'Purification and biochemical characterization of the VIM-1 metallo-β-lactamase', Antimicrobial Agents and Chemotherapy, vol. 44, no. 11, pp. 3003-3007. https://doi.org/10.1128/AAC.44.11.3003-3007.2000
Franceschini N, Caravelli B, Docquier JD, Galleni M, Frere JM, Amicosante G et al. Purification and biochemical characterization of the VIM-1 metallo-β-lactamase. Antimicrobial Agents and Chemotherapy. 2000;44(11):3003-3007. https://doi.org/10.1128/AAC.44.11.3003-3007.2000
Franceschini, N. ; Caravelli, B. ; Docquier, J. D. ; Galleni, M. ; Frere, J. M. ; Amicosante, G. ; Rossolini, G. M. / Purification and biochemical characterization of the VIM-1 metallo-β-lactamase. In: Antimicrobial Agents and Chemotherapy. 2000 ; Vol. 44, No. 11. pp. 3003-3007.
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