Article (Scientific journals)
Purification and biochemical characterization of the VIM-1 metallo-beta-lactamase.
Franceschini, N.; Caravelli, B.; Docquier, Jean-Denis et al.
2000In Antimicrobial Agents and Chemotherapy, 44 (11), p. 3003-7
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Keywords :
Anti-Bacterial Agents/metabolism; Chelating Agents/pharmacology; Escherichia coli/drug effects/enzymology/metabolism; Kinetics; beta-Lactamases/chemistry/isolation & purification/metabolism
Abstract :
[en] 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 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 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/K(m) ratios (>10(6) 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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Franceschini, N.
Caravelli, B.
Docquier, Jean-Denis ;  Université de Liège - ULiège > Département des sciences de la vie > Centre d'ingénierie des protéines
Galleni, Moreno ;  Université de Liège - ULiège
Frère, Jean-Marie ;  Université de Liège > Département des sciences de la vie > Centre d'ingénierie des protéines
Amicosante, G.
Rossolini, G. M.
Language :
English
Title :
Purification and biochemical characterization of the VIM-1 metallo-beta-lactamase.
Publication date :
2000
Journal title :
Antimicrobial Agents and Chemotherapy
ISSN :
0066-4804
eISSN :
1098-6596
Publisher :
American Society for Microbiology, Washington, United States - District of Columbia
Volume :
44
Issue :
11
Pages :
3003-7
Peer reviewed :
Peer Reviewed verified by ORBi
Available on ORBi :
since 01 December 2015

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