The Role of Glutathione Transferases in the Detoxification of TNT

Manufacture, use, storage and improper disposal of the explosive 2,4,6-trinitrotoluene (TNT) have led to widespread, global contamination of soil and groundwater. TNT is highly toxic and recalcitrant to degradation resulting in environmental build-up with far reaching ecological and health implications. It is therefore a priority to remove contaminating TNT from the environment. Phytoremediation is a promising solution; suitable plants possess some natural ability to transform TNT, high biomass, deep root systems, requirement for minimal nutrient input and ability to reduce contamination spread by wind or water erosion; making them an attractive remediation system. Key genes involved in the detoxification of TNT by plants have been recently identified by expression studies and the encoded enzymes characterised. This has lead to the thorough investigation of the enzymes in the pathway of TNT detoxification; Phase I transformation includes oxophytodienoate reductases, with uridine diphosphate (UDP) glycosyltransferases (UGTs) playing a role in the Phase II conjugation step. The expression studies identified additional enzymes also likely to be involved in these phases including glutathione transferases (GSTs). GSTs are known to detoxify compounds by conjugation to glutathione (GSH), and like UGTs are Phase II detoxification enzymes. This thesis presents an investigation into whether plant GSTs play a role in the detoxification of TNT. In vitro analysis of recombinant GSTs was performed to elucidate the activity of GSTs towards TNT. Seven GSTs were cloned, expressed and purified from Escherichia coli. TNT assays performed with pure enzyme indicated that at least two of the GSTs were able to transform TNT. Analysis of the reaction product by mass spectrometry showed that TNT was conjugated to glutathione through substitution of a nitro-group, a highly desirable reaction as the removal of a nitro group from TNT is likely to increase the likelihood of subsequent mineralisation of the pollutant. This is the first identification of enzymes capable of this transformation. The two GSTs which exhibited activity towards TNT were overexpressed in Arabidopsis to clarify if the conjugating activity observed in vitro was able to confer increased tolerance to TNT to the transformed plants. Transgenic lines showed no enhanced growth compared to wild type plants on TNT amended media, root lengths appeared slightly shorter while TNT uptake and biomass were reduced. The role of GSTs in the detoxification of TNT remains unresolved however it is likely that GSTs do not play an integral role in TNT detoxification in plants. Nonetheless, the two GSTs characterised in the project are the first examples of plant enzymes which are able to catalyse the removal of nitro groups from TNT. Engineering these GSTs to improve their ability to transform TNT could offer an opportunity for effective environmental remediation.