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Title: Structural insight into the catalytic mechanism of the unique glutathione-dependent peroxidase enzymes of Trypanosoma cruzi
Author: Patel, Shreenal Harishchandra
ISNI:       0000 0004 2673 8476
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2007
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Kinetoplastid parasites cause a variety of diseases including African sleeping sickness (Trypanosoma brucei) and Chagas disease (Trypanosoma cruzi). The invading parasite is exposed to reactive oxygen species (ROS) generated from the host immune response. In trypanosomes, the glutathione/glutathione reductase antioxidant system (present in the host), is replaced by the analogous trypanosome-specific trypanothione/trypanothione reductase antioxidant system. The differences in the unique oxidative defence system of the parasite from its host, provides potential drug targets. The parasites, like their mammalian hosts, possess glutathione-dependent peroxidase enzymes (GPX), which have a conserved active site cysteine (analogous to selenocysteine in the mammalian hosts). Research now considers these enzymes to fall into a mechanistic family similar to peroxiredoxin enzymes. Peroxiredoxins are a large family of proteins, whereby members fall into the subgroups according to the number of active site cysteine residues present and their distribution (1-Cys or 2Cys typical/atypical). It was recently found that T. brucei glutathione peroxidase enzyme (TbPxIII) follows a mechanism analogous to atypical 2-Cys peroxiredoxin mechanism, supporting a similar mechanism in the homologue T. cruzi glutathione peroxidase I (TcGPXI). The aim of my thesis work was to gain structural information on TcGPXI, to provide insight into the mechanism of this enzyme. Constructs of TcGPXI, TcGPXII, TbPxI and TbPxII were cloned, expressed and purified to produce sufficient protein for structural studies. TcGPXI was investigated by NMR spectroscopy, which facilitated 96% backbone sequence assignments and secondary structure prediction for oxidised wild type TcGPXI. An intra-molecular disulphide bond was observed between the conserved catalytic cysteine and a second cysteine within the protein sequence. To investigate this, cysteine mutants were created to break the disulphide bond and investigated with NMR techniques. Additionally, sequence-specific backbone assignments were obtained for reduced wild type TcGPXI. Established kinetic assays were used to assess catalytic activity. The mutant enzymes exhibited no activity, suggesting that both cysteines involved in the intramolecular disulphide bond, are required for catalysis. Protein from constructs of TcGPXII. TbPxI, TbPxII and oxidised wild type TcGPXI (including mutant forms) were subjected to crystallisation screens. Diffraction data for oxidised wild type TcGPXI was collected, at a resolution of 2.3 A. Molecular replacement was used to solve the crystal structure of TcGPXI. The work presented in this thesis will show that TcGPXI follows a new mechanistic family of enzymes, similar to that of the atypical 2-Cys peroxiredoxin family. The crystal structure reveals the possibility of a disulphide bond in the oxidized state, associated with the unravelling of a helix otherwise present in the thioredoxin fold.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available