Structural insights into the catalytic mechanism of Trypanosoma cruzi GPXI (glutathione peroxidase-like enzyme I)

Current drug therapies against Trypanosoma critzi, the causative agent of Chagas disease, have limited effectiveness and are highly toxic. T. cruzi-specific metabolic pathways that utilize trypanothione for the reduction of peroxides are being explored as potential novel therapeutic targets. In the present study we solved the X-ray crystal structure 4 one of the T. cruzi enzymes involved in peroxide reduction, the glutathione peroxidase-like enzyme TcGPXI (T. critzi glutathione peroxidase-like enzyme I). We also characterized the wild-type, C48G and C96G variants of TcGPXI by NMR spectroscopy and biochemical assays. Our results show that residues Cys(48) and Cys(96) are required for catalytic activity. In solution, the TcGPXI molecule readily forms a Cys(48)-Cys(96) disulfide bridge and the polypeptide segment containing Cys(96) lacks regular secondary structure. NMR spectra of the reduced TcGPXI are indicative of a protein that undergoes widespread conformational exchange on an intermediate time scale. Despite the absence of the disulfide bond, the active site mutant proteins acquired all oxidized-like conformation as judged from their NMR spectra. The protein that was used for crystallization was preoxidized by t-butyl hydroperoxide, however, the electron density maps clearly showed that the active site cysteine residues tire in the reduced thiol form, indicative of X-ray-induced reduction. Our crystallographic and solution studies suggest a level of structural plasticity in TcGPXI consistent with the requirement of the atypical two-cysteine (2-Cys) peroxiredoxin-like mechanism implied by the behaviour of the Cys(48) and Cys(96) mutant proteins.