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

VIM-1 is a new group 3 metallo-beta -lactamase recently detected in carbape nem-resistant nosocomial isolates of Pseudomonas aeruginosa from the Medite rranean area. In this work, VIM-1 was purified from an Escherichia coli str ain carrying the cloned bla(VIM-1) gene by means of an anion-exchange chrom atography step followed by a gel permeation chromatography step. The purifi ed enzyme exhibited a molecular mass of 26 kDa in sodium dodecyl sulfate-po lyacrylamide gel electrophoresis, and an acidic pi of 5.1 in analytical iso electric focusing. Amino-terminal sequencing showed that mature VIM-1 resul ts from the removal of a 26-amino-acid signal peptide from the precursor. V IM-1 hydrolyzes a broad array of beta -lactam compounds, including penicill ins, narrow- to expanded-spectrum cephalosporins, carbapenems, and mechanis m-based serine-beta -lactamase inactivators. Only monobactams escape hydrol ysis. The highest catalytic constant/K-m ratios (>10(6) M-1 . s(-1)) were o bserved with carbenicillin, azlocillin, some cephalosporins (cephaloridine, cephalothin, cefuroxime, cefepime, and cefpirome), imipenem, and biapenem. Kinetic parameters showed remarkable variability with different beta -lact ams and also within the various penam, cephem, and carbapenem compounds, re sulting in no clear preference of the enzyme for any of these beta -lactam subfamilies. Significant differences were observed with some substrates bet ween the kinetic parameters of VIM-1 and those of other metallo-beta -lacta mases. 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 th e inactivation parameters of VIM-1 with different agents.