Exploring the Role of Residue 228 in Substrate and Inhibitor Recognition by VIM Metallo-β-lactamases.
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, compelled 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 enhanced 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 equally 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.
∥Research Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio 44106, United States.
Maria F
MF
Mojica
⊥Laboratorio de Química Farmacéutica, Universidad de la República, Montevideo, Uruguay.
S Graciela
SG
Mahler
∥Research Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio 44106, United States.
Christopher R
CR
Bethel
∥Research Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio 44106, United States.
Magdalena A
MA
Taracila
@School of Cellular and Molecular Medicine, University of Bristol, Bristol, United Kingdom.
Magda
M
Kosmopoulou
∥Research Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio 44106, United States.
Krisztina M
KM
Papp-Wallace
#Instituto de Biología Molecular y Celular de Rosario (IBR), Departamento de Química Biológica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Rosario, Argentina.
Leticia I
LI
Llarrull
∥Research Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio 44106, United States.
Brigid M
BM
Wilson
∥Research Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio 44106, United States.
Steven H
SH
Marshall
∥Research Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio 44106, United States.
Christopher J
CJ
Wallace
∇Centro Internacional de Entrenamiento e Investigaciones Médicas, CIDEIM, Cali, Colombia.
Maria V
MV
Villegas
Michael E
ME
Harris
#Instituto de Biología Molecular y Celular de Rosario (IBR), Departamento de Química Biológica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Rosario, Argentina.
Alejandro J
AJ
Vila
@School of Cellular and Molecular Medicine, University of Bristol, Bristol, United Kingdom.
James
J
Spencer
∥Research Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio 44106, United States.
Robert A
RA
Bonomo
D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000187:drug effects; D015378:Imipenem / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D053778:Thiazolidines / Q000737:chemistry / Q000494:pharmacology*; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D053778:Thiazolidines; D015378:Imipenem; D001618:beta-Lactamases