| dc.contributor |
Studholme, David |
|
| dc.contributor |
Isupov, michail |
|
| dc.contributor |
Van Der Giezen, mark |
|
| dc.creator |
Kontellas, G |
|
| dc.date |
2022-06-13T13:51:09Z |
|
| dc.date |
2022-05-23 |
|
| dc.date |
2022-06-13T13:39:49Z |
|
| dc.date |
2022-06-13T13:51:09Z |
|
| dc.date.accessioned |
2023-02-23T12:14:20Z |
|
| dc.date.available |
2023-02-23T12:14:20Z |
|
| dc.identifier |
ORCID: 0000-0002-7537-3544 (Kontellas, Georgios) |
|
| dc.identifier |
http://hdl.handle.net/10871/129929 |
|
| dc.identifier.uri |
http://localhost:8080/xmlui/handle/CUHPOERS/258531 |
|
| dc.description |
The goal of this project was the investigation of glycolytic enzymes that are vital in the survival and proliferation of adult Fasciola hepatica. These enzymes are triose-phosphate isomerase, phosphoglycerate kinase and cofactor dependent phosphoglycerate mutase. Detailed knowledge about the biochemistry and structure of the above-mentioned fluke enzymes can enable the subsequent development of new and more efficient drugs. The structural comparison of these F. hepatica enzymes with their host mammalian counterparts will elucidate fine structural features that could lead to drugs specifically targeting the trematode enzymes and leaving the host ones unaffected.
In this project, triose-phosphate isomerase from F. hepatica was over-expressed and purified at 95 % of purity, suitable for crystallographic studies. Enzyme kinetics were studied in a range of temperatures (25 – 42 ⁰C) for the substrate glyceraldehyde-3-phosphate. The highest affinity, and correspondingly the lowest Km value, was observed at 25 ⁰C. Optimal pH value for triose-phosphate isomerase was 7.6. The inhibition of triose-phosphate isomerase by the drug triclabendazole is relatively weak according to the performed inhibition assays where the Ki was established. The inhibition mode is potentially non-competitive. Triose-phosphate isomerase was crystallised and the structure was solved at the highest resolution to date of 1.59 Å. Two residues participating in the formation of the dimer interface were found not to be conserved in mammalian hosts. Consequently, the specific dimer interactions formed in F. hepatica triose-phosphate isomerase but are absent in host enzyme, may be playing a role in the binding of triclabendazole to the fluke enzyme.
As the phosphoglycerate kinase published gene model from F. hepatica was incorrect or incomplete, the correct sequence of the gene was predicted. A method for over-expressing the enzyme in milligram quantities was successfully developed. The enzyme was purified at 92 % of purity and kinetic parameters were established over a range of temperatures (25 – 42 ⁰C). Highest activity of phosphoglycerate kinase was observed at 25 ⁰C and the highest affinity according to the established values of Km at different temperatures, was observed at 39 ⁰C. Optimal pH value for phosphoglycerate kinase was 7.6. Clorsulon was shown to be a weak uncompetitive inhibitor of phosphoglycerate kinase and the equilibrium dissociation constant of enzyme-substrate-inhibitor complex (αKi) was established.
Phosphoglycerate mutase from F. hepatica was over-expressed and purified at 93 % of purity, suitable for crystallographic studies. Enzyme kinetics were studied in a range of temperatures (25 – 42 ⁰C) with the highest activity observed at 39 ⁰C and the highest affinity at 42 ⁰C. Optimal pH value for phosphoglycerate mutase was 7.6. The inhibition studies show a relatively weak inhibition of phosphoglycerate mutase by Clorsulon according to the calculated Ki. The inhibition mode of Clorsulon is likely non-competitive. The first crystal structure of phosphoglycerate mutase from F. hepatica was solved at the low resolution of 3.16 Å. The asymmetric unit of the crystal and the size exclusion chromatography experiments revealed that there are four identical chains forming dimers that are part of a tetrameric assembly.
An analysis of the available F. hepatica transcriptome was performed to identify over-expressed non-mammalian proteins during the adult stage that could be important for survival of the parasite. This analysis identified two fluke proteins that share the CAP (cysteine-rich secretory proteins, antigen 5, and pathogenesis related-1 proteins) domain. CAP superfamily proteins in parasitic helminths are thought to be involved in manipulation of host defence responses. Thus, these two proteins could be considered as vaccine targets.
Recent studies have improved the understanding of rhodoquinone synthesis that is absent in mammals. One of the enzymes that is shared among the rhodoquinone and ubiquinone pathways and suggested to be a potential drug target is 4-hydroxybenzoate polyprenyltransferase or COQ2. In this project COQ2 from F. hepatica has been predicted using AlphaFold, a protein structure prediction algorithm with atomic accuracy. Structural comparison of FhCOQ2 with the predicted structure of human COQ2 has revealed two non-conserved residues in the putative active site of the FhCOQ2 enzyme. The two F. hepatica residues might indicate the potentially different substrate specificity of FhCOQ2 utilising tryptophan metabolites for RQ synthesis. |
|
| dc.publisher |
University of Exeter |
|
| dc.publisher |
Biological Sicences |
|
| dc.rights |
2023-12-13 |
|
| dc.rights |
This thesis is embargoed due to subsequent publication parts of it |
|
| dc.rights |
http://www.rioxx.net/licenses/all-rights-reserved |
|
| dc.subject |
Fasciola hepatica |
|
| dc.subject |
glycolysis |
|
| dc.subject |
triose-phosphate isomerase |
|
| dc.subject |
phosphoglycerate kinase |
|
| dc.subject |
phosphoglycerate mutase |
|
| dc.subject |
crystallography |
|
| dc.subject |
transcriptomics |
|
| dc.title |
Glycolytic and unique energy producing enzymes of trematode parasites are potential drug targets: bioinformatics and structural studies |
|
| dc.type |
Thesis or dissertation |
|
| dc.type |
Doctor of Philosophy in Biological Sciences |
|
| dc.type |
Doctoral |
|
| dc.type |
Doctoral Thesis |
|