Comparison of Verona Integron-Borne Metallo-β-Lactamase (VIM) Variants Reveals Differences in Stability and Inhibition Profiles

What is it about?

Metallo-β-lactamases (MBLs) are of increasing clinical significance; the development of clinically useful MBL inhibitors is challenged by the rapid evolution of variant MBLs. The Verona integron-borne metallo-β-lactamase (VIM) enzymes are among the most widely distributed MBLs, with >40 VIM variants having been reported. We report on the crystallographic analysis of VIM-5 and comparison of biochemical and biophysical properties of VIM-1, VIM-2, VIM-4, VIM-5, and VIM-38. Recombinant VIM variants were produced and purified, and their secondary structure and thermal stabilities were investigated by circular dichroism analyses. Steady-state kinetic analyses with a representative panel of β-lactam substrates were carried out to compare the catalytic efficiencies of the VIM variants. Furthermore, a set of metalloenzyme inhibitors were screened to compare their effects on the different VIM variants. The results reveal only small variations in the kinetic parameters of the VIM variants but substantial differences in their thermal stabilities and inhibition profiles. Overall, these results support the proposal that protein stability may be a factor in MBL evolution and highlight the importance of screening MBL variants during inhibitor development programs.

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Why is it important?

The increasing problem of antibiotic resistance is a major global public health concern. Today, however, the use of life-saving β-lactam antibiotics is threatened by the production of bacterial β-lactamases. One major challenge in the antibiotic drug discovery field is to identify broad-spectrum bacterial metallo- β-lactamases (MBLs) inhibitors and achieve a breadth of selectivity required for clinical use. In order to meet these criteria, it is important to understand the structural and biochemical properties of MBLs. Verona Integron-borne MBL (VIM) types are amongst the most widely spread MBLs, with 46 VIM reported variants. Accordingly, we report studies on the biochemical and biophysical studies on relevant VIM variants using a combination of different techniques, including kinetic assays, circular dichroism (CD), NMR spectroscopy and X-ray crystallography. VIM variants have been tested against a panel of β-lactam substrates under the same experimental conditions using an absorbance-based assay, previously reported by the group. Remarkably, similar kinetic properties were observed for various variants (VIM-1, VIM-2, VIM-4, VIM-5, and VIM-38). The small differences in their catalytic efficiencies support the proposal that changes in substrate selectivity are not the sole evolutionary driving force for MBLs. Similarly, VIM variants conserved a similar secondary structure as implied by CD analyses; however, clear differences in thermal stability were observed. This finding suggests that protein stability might be an important factor and a driving force in MBL evolution. Inhibition profiles were substantially different with one isoquinoline derivative selectively inhibiting two variants (VIM-5 and VIM-38) more potently than others (VIM-1, VIM-2 and VIM-4). Even though VIM variants differ by a single-amino acid mutation in most cases, their interactions with inhibitors were clearly different. Accordingly, this work highlights the importance of screening various MBL variants at an early stage during inhibitor development programs.