Abstract

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.

Submission Details

ID: WAJXVq7r

Submitter: Paulie Dang

Submission Date: Sept. 26, 2019, 2:40 p.m.

Version: 1

Publication Details

Additional Information

Residues V22 and A23 (BBL standard numbering, PMID 15215079) of the native enzymes have been modified to G22 and P23. VIM-2 and VIM-5 were used as reference structures. VIM-1, VIM-4, and VIM-38 are closely related to VIM-5, for which the study provides a crystal structure. VIM-2 deviates significantly and has been crystallized in a different study (PMID 25411887). The K110A+L183H+A184E+R187S+T239K mutant of VIM-5 (K130A+L224H+A225E+R228S+T308K according to BBL numbering) is VIM-1. The K110A+L183H+A184E+T239K mutant of VIM-5 (K130A+L224H+A225E+T308K according to BBL numbering) is VIM-4. The V247A mutant of VIM-5 (V316A according to BBL numbering) is VIM-38. This residue is close to the C-terminus and has not been resolved in the crystal structure. Inhibition IC50 values are taken from the table instead of the graphs.