Characterisation of glutathione transferase from Arabidopsis thaliana

Glutathione transferases (GSTs) are soluble enzymes with activity towards a wide range of xenobiotic and endogenous compounds. The Arabidopsis genome encoded 54 GSTs, which have been classified into eight classes including Tau and Phi. Members of these Tau and Phi clades are strongly upregulated in response to abiotic stresses such as xenobiotics and biotic stresses including pathogen attack. The most characterised activity of GSTs is the transfer of glutathione to an electrophilic centre to form a polar glutathionylated conjugate. However, increasing number of research demonstrated a non-catalytic activity plants GSTs, especially in the transportation of flavonoids from the cytosol to the vacuole. Despite the wealth of investigations into GSTs, and probably as a result of overlapping substrate specificities, the endogenous roles for the vast majority of these plant GSTs remains unknown.
Here, the binding interaction of camalexin, indole-3-aldehyde, quercetrin and quercetin to GSTF2 in a non-catalytic fashion was observed in three different sites; two identical sites of L1 and one L2 from the X-ray crystallography data. Mutagenesis of the active residues, Q73L, H77A, Y97A and R154A were performed and using isothermal calorimetry (ITC) techniques, lower binding affinities were observed for all mutants towards all ligands except for Y97A and Q73L which showed higher binding affinities with indole-3-aldehyde. This unexpected finding was likely due to the conformational change of the mutant compared to the wild type, as observed in the structure of mutant Y97A.
On elucidating the catalytic activity of GSTs, the structure of GSTU25 in complex with disulphide glutathione was obtained. The GSTU25 has been recently identified to catalyse the denitration of TNT to form 2-glutathionyl- 4,6-dinitrotoluene, a potentially more amenable product for subsequent degradation. This structure complex provides insights of GSTU25 folding upon substrate binding.
The involvement of GSTU25 in the detoxification of TNT was further analysed using CRISPR/Cas9 technology. Subclades of GSTU25, including GSTU24, GSTU21, GSTU19 were knockout with the aim to remove overlapping substrate specificities and to finally reveal TNT-specific phenotypes. Only gstU25 Cas9 segregated Arabidopsis were obtained from the experiment that could potentially be optimised in the future study.