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Exploring the Role of Residue 228 in Substrate and Inhibitor Recognition by VIM Metallo-β-lactamases

DOI: 10.1021/acs.biochem.5b00106 DOI Help

Authors: Maria F. Mojica (Case Western Reserve University) , S. Graciela Mahler (Universidad de la República) , Christopher R. Bethel (Louis Stokes Cleveland Veterans Affairs Medical Center) , Magdalena A. Taracila (Case Western Reserve University) , Magda Kosmopoulou (University of Bristol) , Krisztina M. Papp-wallace (Case Western Reserve University) , Leticia I. Llarrull (Universidad Nacional de Rosario) , Brigid M. Wilson (Louis Stokes Cleveland Veterans Affairs Medical Center) , Steven H. Marshall (Louis Stokes Cleveland Veterans Affairs Medical Center) , Christopher J. Wallace (Louis Stokes Cleveland Veterans Affairs Medical Center) , Maria V. Villegas (CIDEIM) , Michael E. Harris (Case Western Reserve University) , Alejandro J. Vila (Universidad Nacional de Rosario) , James Spencer (University of Bristol) , Robert A. Bonomo (Case Western Reserve University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochemistry , VOL 54 (20) , PAGES 3183 - 3196

State: Published (Approved)
Published: May 2015
Diamond Proposal Number(s): 8922

Abstract: β-Lactamase inhibitors (BLIs) restore the efficacy of otherwise obsolete β-lactams. However, commercially available BLIs are not effective against metallo-β-lactamases (MBLs), which continue to be disseminated globally. One group of the most clinically important MBLs is the VIM family. The discovery of VIM-24, a natural variant of VIM-2, possessing an R228L substitution and a novel phenotype, ompelled us to explore the role of this position and its effects on substrate specificity. We employed mutagenesis, biochemical and biophysical assays, and crystallography. VIM-24 (R228L) confers nhanced resistance to cephems and increases the rate of turnover compared to that of VIM-2 (kcat/KM increased by 6- and 10-fold for ceftazidime and cefepime, respectively). Likely the R → L substitution relieves steric clashes and accommodates the C3N-methyl pyrrolidine group of cephems. Four novel bisthiazolidine (BTZ) inhibitors were next synthesized and tested against these MBLs. These inhibitors inactivated VIM-2 and VIM-24 qually well (Ki* values of 40−640 nM) through a two-step process in which an initial enzyme (E)−inhibitor (I) complex (EI) undergoes a conformational transition to a more stable species, E*I. As both VIM-2 and VIM-24 were inhibited in a similar manner, the crystal structure of a VIM-2−BTZ complex was determined at 1.25 Å and revealed interactions of the inhibitor thiol with the VIM Zn center. Most importantly, BTZs also restored the activity of imipenem against Klebsiella pneumoniae and Pseudomonas aeruginosa in whole cell assays producing VIM-24 and VIM-2, respectively. Our results suggest a role for position 228 in defining the substrate specificity of VIM MBLs and show that BTZ inhibitors are not affected by the R228L substitution.

Subject Areas: Biology and Bio-materials

Instruments: I04-1-Macromolecular Crystallography (fixed wavelength)

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