ENO, REBECCA,FLORENCE,MARY (2019) Structural and Functional Characterisation of Glutathione-S-Transferases
to Combat Multiple Herbicide Resistance in Black Grass (Alopecurus myosuroides). Doctoral thesis, Durham University.
|PDF - Accepted Version|
Black grass (Alopecurus myosuroides) is one of the most common weeds in Western Europe. With the increasing use of herbicides, in response to pressure to produce higher crop yields, incidences of multiple herbicide resistance have been widely reported. Previous work in this area has identified a phi class glutathione-s-transferase, AmGSTF1, as playing a causative role in this multiple herbicide resistance. Two classes of inhibitor have previously been identified: the first CNBF, a multi drug resistance drug; and the second a group of flavone compounds identified from ligand fishing experiments. These have been shown to have activity in vitro inhibiting the action of AmGSTF1 and in vivo against multiple herbicide resistant black grass. However, little was known about their mode of action. This work has focussed on investigating the interactions between these inhibitors and AmGSTF1 using crystallographic, biochemical and complementary biophysical techniques.
Apo AmGSTF1 has successfully been crystallised, with the structure solved to 1.5 Å. In addition a structure has been solved to 2.0 Å with CNBF covalently modifying the Cys120 residue. For both these structures the crystal packing results in loops in the active site region being disordered as well as preventing small molecule binding within the active site. In order to determine the complete structure, a series of mutants were designed to alter crystal packing. The structure of these were determined, and they adopt a different packing arrangement which results in the previously disordered loops being ordered, as well as exposing the binding site. The structure of the F122T mutant was used for in silico modelling to determine the likely binding site for flavonoid ligands.
The mutants were subsequently used for seeding and soaking experiments which allowed for a complete structure of wild type AmGSTF1 to be determined in complex with a glutathione conjugate of CNBF in the active site. This structure gives a significantly better understanding of the mode of action of these inhibitors, in addition to allowing for the possibility of future development of the inhibitors using structure based design.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Faculty and Department:||Faculty of Science > Chemistry, Department of|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||06 Jun 2019 12:43|