title: Purification and biochemical characterization of the VIM-1 metallo-beta-lactamase.
abstract: 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.
authors.affiliation: Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi L'Aquila, I-67100 Coppito, L'Aquila, Italy.
authors.forename: N
authors.initials: N
authors.lastname: Franceschini
authors.forename: B
authors.initials: B
authors.lastname: Caravelli
authors.forename: J D
authors.initials: JD
authors.lastname: Docquier
authors.forename: M
authors.initials: M
authors.lastname: Galleni
authors.forename: J M
authors.initials: JM
authors.lastname: Frère
authors.forename: G
authors.initials: G
authors.lastname: Amicosante
authors.forename: G M
authors.initials: GM
authors.lastname: Rossolini
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D007700:Kinetics; D001618:beta-Lactamases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D002614:Chelating Agents; C417264:VIM-1 metallo-beta-lactamase; D001618:beta-Lactamases