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Investigating the Role of Glutathione Transferases in the Phytodetoxification of Explosives

Tzafestas, Kyriakos (2016) Investigating the Role of Glutathione Transferases in the Phytodetoxification of Explosives. PhD thesis, University of York.

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Abstract

The explosive 2,4,6-trinitrotoluene (TNT) is a major worldwide environmental pollutant. Highly persistent to degradation the presence of this toxic pollutant presents various health and environmental concerns. In the present study the role of two glutathione transferases (GSTs), in the detoxification of TNT is investigated. The Tau class GSTs from Arabidopsis thaliana (Arabidopsis), GST-U24 and GST-U25, were strongly upregulated in response to TNT. Following affinity chromatography purification and characterisation of recombinant forms of both enzymes, three distinct TNT-glutathionyl products were identified. GSTU-25 was able to convert TNT to 2-glutathionyl-4,6-dinitrotoluene, with the concurrent release of nitrite. This conjugate could be chemically weaker than TNT and as a result, potentially more susceptible to biodegradation. To further investigate the detoxification abilities of GST-U24 and GST-U25 in planta 35S-GST-U24 and GST-U25 Arabidopsis lines were generated. These GST overexpressing lines exhibited significantly increased ability to withstand and detoxify TNT with a corresponding reduction in glutathione levels, and displayed higher shoot and root biomass than untransformed plants when grown in the presence of TNT. A Drosophila melanogaster Epsilon class GST (DmGSTE6) was subsequently assessed for its potential for phytoremediation. DmGSTE6 exhibited higher activity than GST-U24 and GST-U25 towards TNT in vitro and produced almost exclusively 2-glutathionyl-4,6-dinitrotoluene. Expressing DmGSTE6 in Arabidopsis resulted in enhanced biomass, when grown on TNT-containing media, when compared to the GST-U24/U25 overexpressing lines, but a similar TNT uptake rate. Finally, to identify key amino residues involved in the catalytic activity of GSTs towards TNT, a site-directed mutagenesis approach was employed. The results highlighted Tyr107 as important to catalytic activity with additional aromatic residues contributing to the stabilisation of aromatic substrates such as TNT. Ultimately the GST-mediated detoxification pathway demonstrated here can be exploited in robust plant species for the phytoremediation of TNT.

Item Type: Thesis (PhD)
Academic Units: The University of York > Biology (York)
Identification Number/EthosID: uk.bl.ethos.686539
Depositing User: Mr Kyriakos Tzafestas
Date Deposited: 24 May 2016 09:44
Last Modified: 08 Sep 2016 13:34
URI: http://etheses.whiterose.ac.uk/id/eprint/13150

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