The role of glutathione transferases in TNT detoxification

Glutathione transferases (GSTs) are multi-functional enzymes with an important role in plant biochemical mechanisms of xenobiotic detoxification. The GSTs have been extensively studied due to their ability to detoxify herbicides; recently they have been implicated in numerous stress responses, including defence against pathogen attack, oxidative stress, heavy metal toxicity and the detoxification of a wide variety of xenobiotic compounds. GSTs catalyse the conjugation of the tripeptide glutathione (GSH) (γ-Glutamyl-Cysteinyl-Glycine) to a variety of hydrophobic, electrophilic, and usually cytotoxic substrates to form a polar S glutathionylated reaction product. Products of oxidative stress can also be a substrate as GSTs have a role in the protection of tissues against oxidative damage. This project focused on the role of GSTs from Arabidopsis thaliana to detoxify 2,4,6-trinitrotoluene (TNT). Globally, manufacturing, use and storage of TNT has resulted in widespread soil contamination. Current strategies of TNT remediation (incineration, composting and landfilling) are expensive and damaging to the environment. Phytoremediation using the ability of plants to uptake, metabolise and detoxify xenobiotics in situ could present a cost-effective, non-invasive, environmentally friendly alternative. In vitro studies focused on investigating the conjugation activity of purified recombinant GSTs with TNT. In vivo experiments aimed to determine the role of GSTs in TNT detoxification in Arabidopsis plants transformed with 35S-GST constructs in comparison with untransformed, wild type plants. Purified recombinant GSTU24 and GSTU25 directly conjugated TNT to form a range of glutathionylated products. GSTU25 was found to produce 2 glutathionyl-4,6-dinitrotoluene, formed by nucleophilic substitution of a nitro group by glutathione, previously identified in the conjugation reaction of mammalian GSTs with TNT. The data on two other compounds suggest the substances are C glutathionylated 4-hydroxyaminodinitrotoluene and C-glutathionylated 2 hydroxyaminodinitrotoluene with the glutathione attached via the methyl group of TNT. The effect of pH and temperature on product formation and enzyme activity was also studied. Experiments using transgenic lines overexpressing GSTU24 and GSTU25 showed better tolerance of the overexpressing lines compared to the wild type plants, as characterised by enhanced root growth in TNT containing medium and faster TNT removal from the growth medium. In summary, this work presents biochemical data on the first identified glutathionylated TNT conjugates produced by plant GSTs in vitro and in the plants. It also shows that overexpressing detoxification enzymes in the plant results in increased transformation of TNT, improving the ability of plants to tolerate the pollutant, and could lead to the identification and breeding of native plant species with active detoxification systems suitable for the phytoremediation of contaminated sites.