JAC Advance Access originally published online on January 6, 2003
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Journal of Antimicrobial Chemotherapy (2003) 51, 257-266
© 2003 The British Society for Antimicrobial Chemotherapy
On functional and structural heterogeneity of VIM-type metallo-ß-lactamases
1 Dipartimento di Biologia Molecolare, Sezione di Microbiologia, Università di Siena, Policlinico ‘Le Scotte’, Viale Bracci, I-53100 Siena; 3 Dipartimento di Scienze e Tecnologie Biomediche – Cattedra di Biochimica Clinica, Università di L’Aquila, Via Vetoio, Loc. Coppito, I-67100 L’Aquila, Italy; 2 Laboratoire d’Enzymologie & Centre d’Ingénierie des Protéines, Institut de Chimie, Université de Liège, Bat. B6 Allée de la Chimie, Sart Tilman, B-4000 Liège, Belgium
Received 21 January 2002; returned 5 July 2002; revised 1 August 2002; accepted 28 October 2002
The VIM metallo-ß-lactamases are emerging resistance determinants, encoded by mobile genetic elements, that have recently been detected in multidrug-resistant nosocomial isolates of Pseudomonas aeruginosa and other Gram-negative pathogens. In this work a T7-based expression system for overproduction of the VIM-2 enzyme by Escherichia coli was developed, which yielded 
80 mg of protein per litre of culture. The enzyme was mostly released into the medium, from which it was recovered at >99% purity by an initial ammonium sulphate precipitation followed by two chromatography steps, with almost 80% efficiency. Determination of kinetic parameters of VIM-2 under the same experimental conditions previously used for VIM-1 (the first VIM-type enzyme detected in clinical isolates, which is 93% identical to VIM-2) revealed significant differences in Km values and/or turnover rates with several substrates, including penicillins, cephalosporins and carbapenems. Compared with VIM-1, VIM-2 is more susceptible to inactivation by chelators, indicating that the zinc ions of the latter are probably more loosely bound. These data indicated that at least some of the amino acid differences between the two proteins have functional significance. Molecular modelling of the two enzymes identified some amino acid substitutions, including those at positions 223, 224 and 228 (in the BBL numbering), that could be relevant to the changes in catalytic behaviour.
* Corresponding author. Tel: +39-0577-233455; Fax: +39-0577-233325; E-mail: rossolini{at}unisi.it
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