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On functional and structural heterogeneity of VIM-type metallo-beta-lactamases
Jean-Denis Docquier1, Josette Lamotte-Brasseur, Moreno Galleni
1Dipartimento di Biologia Molecolare, Sezione di Microbiologia, Università di Siena, Policlinico 'Le Scotte', Viale Bracci, I-53100 Siena, Italy.
The Journal of Antimicrobial Chemotherapy
|February 4, 2003
Summary
VIM-2 metallo-beta-lactamase, an emerging resistance determinant in Gram-negative pathogens, was successfully overproduced and purified. Differences in kinetic parameters and zinc ion binding compared to VIM-1 highlight functional significance of amino acid variations.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Metallo-beta-lactamases (MBLs) like VIM are critical for bacterial resistance.
- Emergence of VIM-2 in multidrug-resistant Gram-negative pathogens necessitates detailed characterization.
Purpose of the Study:
- To develop an efficient expression system for VIM-2 enzyme production.
- To determine the kinetic parameters and biochemical properties of VIM-2.
- To compare VIM-2 with VIM-1 and understand the functional impact of their amino acid differences.
Main Methods:
- Established a T7-based expression system in Escherichia coli for VIM-2 overproduction.
- Purified VIM-2 using ammonium sulphate precipitation and chromatography.
- Determined kinetic parameters (K(m), turnover rates) for VIM-2 and VIM-1 with various beta-lactam substrates.
- Assessed enzyme susceptibility to chelators and performed molecular modeling.
Main Results:
- Achieved high-yield production (approx. 80 mg/L) of VIM-2, with efficient purification (>99% purity, ~80% yield).
- VIM-2 exhibited distinct kinetic parameters compared to VIM-1 for penicillins, cephalosporins, and carbapenems.
- VIM-2 showed increased susceptibility to chelators, suggesting looser zinc ion binding.
- Molecular modeling identified key amino acid substitutions potentially responsible for altered catalytic behavior.
Conclusions:
- The developed expression system enables robust production of VIM-2 for further study.
- Amino acid variations between VIM-1 and VIM-2 significantly influence enzyme kinetics and substrate specificity.
- Differences in zinc ion binding affinity contribute to the functional divergence between VIM-1 and VIM-2.