Structural studies of herbicide detoxifying enzymes

McEwen, Alastair Graham (2006) Structural studies of herbicide detoxifying enzymes. PhD thesis, University of Glasgow.

Abstract

Glutathione S-transferases (GSTs) [EC 2.5.1.18] are a ubiquitous family of multifunctional enzymes that are widely distributed in nature. GSTs have been identified from bacteria, fungi, insects, cephalopods, fish, amphibians, reptiles, avians, mammals and plants. The major role of GSTs is the conjugation of the tripeptide glutathione (GSH; gamma-L-glutamyl-L-cysteinyl-glycine) to a range of electrophilic substances. Ten GSTs from rice, wheat, petunia and Arabidopsis thaliana were overexpressed in Escherichia coli and purified using an Orange A agarose column, S-hexyl glutathione sepharose column, or a nickel chelation column. Using a variety of crystallization screens, crystals were grown for six of the proteins. The X-ray diffraction data collected for two of these proteins enabled their structure to be solved. The first structure to be solved was that of OsGSTU1-1 in complex with GSH, which is a different subgroup of Tau class GSTs from that of the previously reported TaGSTU4-4. OsGSTUI is similar in sequence to the well characterized ZmGSTUI and ZmGSTU2, safener induced enzymes from maize highly active against the diphenyl ether herbicide fluorodifen. In order to determine the structural basis of OsGSTU1 substrate specificity a number of GSH conjugates were prepared. A conjugate of CDNB with GSH was prepared by enzymatic routes using the enzyme ZmGSTF1. The reaction product of the herbicide fluorodifen with GSH was prepared by a two-step synthetic route by preparing a sulfonamide of the phenyl ring and reacting this with GSH. The structure of OsGSTU1-1 was solved with both of these conjugates bound as well as with that of a conjugate of the chloracetanilide herbicide metolachlor with GSH. The structure with metolachlor-GSH bound was useful in structurally characterizing the mode of binding of chloracetanilide herbicides. The structures with the CDNB conjugate with GSH and the reaction product of fluorodifen with GSH were less useful in determining the mode of binding of diphenyl ether herbicides. Both ligands were similar to each other and were found to be sitting between the two lobes of the active site with the nitro group facing inwards. This suggests that the orientation of the molecule within the active site predicted by Dixon et al., 2003 may be incorrect. The structure of OsGSTU4-4 has also been solved in complex with GSH. This enzyme is similar to TaGSTU4-4 and is highly expressed in rice under a variety of stress conditions. A comparison of the three Tau class GST structures now available, along with enzyme assays against a range of xenobiotics substrates has helped to partially understand the activity of Tau class GSTs in herbicide detoxification. The H-site of Tau class GSTs has two lobes. The previously reported structure of TaGSTU4-4 in complex with S-hexylglutathione has the hexyl chain sitting in lobe 'A', but in the structure with metolachlor-GSH the ligand is sitting in lobe 'B' with the gatekeeper Tyr 116 swung away from the active site and disordered. Conformational changes in plant GSTs were also investigated. A comparison of the structures of apo-form ZmGSTF1-1 with the structures of the enzyme in complex with herbicides showed that a conformation change seemed to occur upon ligand binding. Using UV Difference spectroscopy and circular dichroism a change in the conformation of ZmGSTF1-l was seen upon binding of GSH. However, there were problems with the reproducibility of these results. In the case of AtGSTT1-1, which shows a large difference in conformations between molecules in the apo-form of the crystal structure, both UV difference spectroscopy and circular dichroism showed no spectroscopic changes upon GSH binding.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Adrian Lapthorn
• Keywords: Organic chemistry.
• Colleges/Schools: College of Science and Engineering
• Date of Award: 2006
• Depositing User: Enlighten Team
• Unique ID: glathesis:2006-74075
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 07 Aug 2019 10:45
• Last Modified: 07 Aug 2019 10:51
• URI: https://theses.gla.ac.uk/id/eprint/74075

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