title: Comparison of Verona Integron-Borne Metallo-β-Lactamase (VIM) Variants Reveals Differences in Stability and Inhibition Profiles.
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.
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom.
authors.forename: Anne
authors.initials: A
authors.lastname: Makena
authors.affiliation: Department of Genetics and Bioengineering, Gümüşhane University, Gümüşhane, Turkey.
authors.forename: Azer Ö
authors.initials: AÖ
authors.lastname: Düzgün
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Brem
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom.
authors.forename: Michael A
authors.initials: MA
authors.lastname: McDonough
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom.
authors.forename: Anna M
authors.initials: AM
authors.lastname: Rydzik
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom.
authors.forename: Martine I
authors.initials: MI
authors.lastname: Abboud
authors.affiliation: Department of Biology, Coruh University, Artvin, Turkey.
authors.forename: Ayşegül
authors.initials: A
authors.lastname: Saral
authors.affiliation: Department of Medical Microbiology, Recep Tayyip Erdoğan University, Rize, Turkey.
authors.forename: Ayşegül Ç
authors.initials: AÇ
authors.lastname: Çiçek
authors.affiliation: Department of Biology, Recep Tayyip Erdoğan University, Rize, Turkey cemal.sandalli@erdogan.edu.tr christopher.schofield@chem.ox.ac.uk.
authors.forename: Cemal
authors.initials: C
authors.lastname: Sandalli
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom cemal.sandalli@erdogan.edu.tr christopher.schofield@chem.ox.ac.uk.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schofield
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D032023:Integrons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D065093:beta-Lactamase Inhibitors / Q000494:pharmacology*; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D065093:beta-Lactamase Inhibitors; C417264:VIM-1 metallo-beta-lactamase; C425108:beta-lactamase bla(vim-2); D001618:beta-Lactamases; C490697:beta-lactamase VIM-5, Pseudomonas aeruginosa; C477893:beta-lactamase bla(VIM-4), Pseudomonas aeruginosa