JAC Advance Access published online on January 6, 2003
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkg067
© 2003 by The British Society for Antimicrobial Chemotherapy
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Original article
1 Dipartimento di Biologia
Molecolare, Sezione di Microbiologia, Università di Siena,
Policlinico ‘Le Scotte',
Viale Bracci, I-53100 Siena, Italy
* Corresponding author. E-mail: rossolini{at}unisi.it.
Received 21 January 2002
; revised 5 July 2002
; accepted 28 October 2002
The VIM metallo-
On functional and structural heterogeneity of VIM-type
metallo-
-lactamases
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
3 Dipartimento
di Scienze e Tecnologie Biomediche - Cattedra di Biochimica
Clinica, Università di L'Aquila, Via Vetoio, Loc.
Coppito, I-67100 L'Aquila, Italy
-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.
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