Structural and functional studies of C-nucleoside formation enzymes
<p>Throughout the course of human medicinal history, natural products have played a pivotal role in both the promotion of good health and the treatment of diseases. In the 20th century, industrialisation and scientific knowledge saw a boom in active single compounds isolated from nature and us...
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| Format: | Thesis |
| Language: | English |
| Published: |
2023
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| _version_ | 1824459274558898176 |
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| author | Li, W |
| author_facet | Li, W |
| author_sort | Li, W |
| collection | OXFORD |
| description | <p>Throughout the course of human medicinal history, natural products have played a pivotal role in both the promotion of good health and the treatment of diseases. In the 20th century, industrialisation and scientific knowledge saw a boom in active single compounds isolated from nature and used to treat disease directly or after relatively minor chemical modification. However, by the end of the 20th century this approach had fallen out from favour. The difficulty in the <em>de novo</em> design of biologically active chemical scaffolds coupled to revolutions in genomic sequencing and chemical biology, has sparked a renaissance of interest in natural products. Nucleosides are characterized by their structurally privileged backbone, and analogues with antiviral, anti-cancer, and antibacterial activities are well known. Conventional molecules with C-N linkages are labile, meaning breakdown <em>in vivo</em> can be an issue for some therapies. The substitution of C-C glycosidic bonds holds the promise of preserving the drug properties of the parent compound while rendering the compound essentially undegradable.</p>
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<p>The thesis overviews the history of natural product discovery, tracing its evolution from ancient folk traditions to the contemporary application of advanced genetics and chemical modifications for biosynthesis. The introduction chapter details some particularly well-known natural products and their socio-economic impacts. Finally, it emphasizes how structural biology can elucidate the mechanisms of enzyme-catalysis and aid in developing natural product analogies.</p>
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<p>Two chapters focus on the structural, chemical, and biophysical studies of a C-nucleoside formation enzyme, ForT, from the formycin (a C-C nucleoside) biosynthetic pathway. The atomic resolution structures for both binary, ternary, and product complexes are described. Biophysical measurements, computational approaches and kinetics data allow for a detailed description of the mechanism. Chemical synthesis permitted an analysis of the substrates’ scope of the enzyme.</p>
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<p>The thesis builds towards the study the enzyme mechanism using X-ray Free Electron Laser (XFEL). A critical part of this work is the development of photo-activated cage substrates, these will allow synchronization of the catalytic mechanisms. We have successfully designed and synthesized potential photo-caged substrates. Isothermal titration calorimetry (ITC) identified which compounds bound, whilst liquid chromatography-mass spectrometry showed the molecules were inactive. Co-complex crystal structure showed that the caged compounds, which were identified by ITC, did bind in the same orientation as APDA. These structures allow for the rationalization of what molecules can bind at the active site. We established crystallization conditions which yield microcrystals, a necessary preparation for XFEL time-resolved experiments.</p>
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<p>The final experimental chapter describes the structural and functional characterization of a protein related (to ForT) from the methanopterin pathway. This enzyme β-RFAPS synthesizes a C-nucleoside utilizing PRPP and an aromatic compound, 4-aminobenzoic acid. During purification of a modified β-RFAPS enzyme and crystal structural analysis indicated the presence of unidentified molecule at the enzyme’s active site. We collaborated with Prof. Carol Robinson’s group employing native mass spectroscopy and identified the molecule guanosine tetraphosphate (ppGpp). We concluded that ppGpp must be tightly bound, only by treatment with formic acid were we able to remove it. Once ppGpp was removed from the enzyme, the enzyme showed binding affinity of approximately 100 nM. We were able to demonstrate robust initial enzyme kinetic assay.</p> |
| first_indexed | 2025-02-19T04:39:11Z |
| format | Thesis |
| id | oxford-uuid:3352f898-3fb5-4e2f-a947-11e28d6f3822 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2025-02-19T04:39:11Z |
| publishDate | 2023 |
| record_format | dspace |
| spelling | oxford-uuid:3352f898-3fb5-4e2f-a947-11e28d6f38222025-02-17T16:32:51ZStructural and functional studies of C-nucleoside formation enzymesThesishttp://purl.org/coar/resource_type/c_db06uuid:3352f898-3fb5-4e2f-a947-11e28d6f3822Molecular biologyBiochemistryNatural productsBiophysicsEnzyme kineticsEnglishHyrax Deposit2023Li, W<p>Throughout the course of human medicinal history, natural products have played a pivotal role in both the promotion of good health and the treatment of diseases. In the 20th century, industrialisation and scientific knowledge saw a boom in active single compounds isolated from nature and used to treat disease directly or after relatively minor chemical modification. However, by the end of the 20th century this approach had fallen out from favour. The difficulty in the <em>de novo</em> design of biologically active chemical scaffolds coupled to revolutions in genomic sequencing and chemical biology, has sparked a renaissance of interest in natural products. Nucleosides are characterized by their structurally privileged backbone, and analogues with antiviral, anti-cancer, and antibacterial activities are well known. Conventional molecules with C-N linkages are labile, meaning breakdown <em>in vivo</em> can be an issue for some therapies. The substitution of C-C glycosidic bonds holds the promise of preserving the drug properties of the parent compound while rendering the compound essentially undegradable.</p> <br> <p>The thesis overviews the history of natural product discovery, tracing its evolution from ancient folk traditions to the contemporary application of advanced genetics and chemical modifications for biosynthesis. The introduction chapter details some particularly well-known natural products and their socio-economic impacts. Finally, it emphasizes how structural biology can elucidate the mechanisms of enzyme-catalysis and aid in developing natural product analogies.</p> <br> <p>Two chapters focus on the structural, chemical, and biophysical studies of a C-nucleoside formation enzyme, ForT, from the formycin (a C-C nucleoside) biosynthetic pathway. The atomic resolution structures for both binary, ternary, and product complexes are described. Biophysical measurements, computational approaches and kinetics data allow for a detailed description of the mechanism. Chemical synthesis permitted an analysis of the substrates’ scope of the enzyme.</p> <br> <p>The thesis builds towards the study the enzyme mechanism using X-ray Free Electron Laser (XFEL). A critical part of this work is the development of photo-activated cage substrates, these will allow synchronization of the catalytic mechanisms. We have successfully designed and synthesized potential photo-caged substrates. Isothermal titration calorimetry (ITC) identified which compounds bound, whilst liquid chromatography-mass spectrometry showed the molecules were inactive. Co-complex crystal structure showed that the caged compounds, which were identified by ITC, did bind in the same orientation as APDA. These structures allow for the rationalization of what molecules can bind at the active site. We established crystallization conditions which yield microcrystals, a necessary preparation for XFEL time-resolved experiments.</p> <br> <p>The final experimental chapter describes the structural and functional characterization of a protein related (to ForT) from the methanopterin pathway. This enzyme β-RFAPS synthesizes a C-nucleoside utilizing PRPP and an aromatic compound, 4-aminobenzoic acid. During purification of a modified β-RFAPS enzyme and crystal structural analysis indicated the presence of unidentified molecule at the enzyme’s active site. We collaborated with Prof. Carol Robinson’s group employing native mass spectroscopy and identified the molecule guanosine tetraphosphate (ppGpp). We concluded that ppGpp must be tightly bound, only by treatment with formic acid were we able to remove it. Once ppGpp was removed from the enzyme, the enzyme showed binding affinity of approximately 100 nM. We were able to demonstrate robust initial enzyme kinetic assay.</p> |
| spellingShingle | Molecular biology Biochemistry Natural products Biophysics Enzyme kinetics Li, W Structural and functional studies of C-nucleoside formation enzymes |
| title | Structural and functional studies of C-nucleoside formation enzymes |
| title_full | Structural and functional studies of C-nucleoside formation enzymes |
| title_fullStr | Structural and functional studies of C-nucleoside formation enzymes |
| title_full_unstemmed | Structural and functional studies of C-nucleoside formation enzymes |
| title_short | Structural and functional studies of C-nucleoside formation enzymes |
| title_sort | structural and functional studies of c nucleoside formation enzymes |
| topic | Molecular biology Biochemistry Natural products Biophysics Enzyme kinetics |
| work_keys_str_mv | AT liw structuralandfunctionalstudiesofcnucleosideformationenzymes |