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Synthesis of Mycobacterial Ergothioneine biosynthetic pathway metabolites
Many gram positive bacteria, such as Mycobacterium tuberculosis, lack the redox protective molecule, glutathione and produce mycothiol (MSH) and ergothioneine (ESH) as their principal low molecular mass thiol instead. Ergothioneine has been known for a while as an anti-oxidant; however its role as...
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| Format: | Thesis |
| Language: | English |
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Department of Chemistry
2014
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| _version_ | 1867613296939499522 |
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| access_status_str | Open Access |
| author | Khonde,Lutete Peguy |
| author2 | Jardine, Anwar |
| author_browse | Jardine, Anwar Khonde,Lutete Peguy |
| author_facet | Jardine, Anwar Khonde,Lutete Peguy |
| author_sort | Khonde,Lutete Peguy |
| collection | Thesis |
| description | Many gram positive bacteria, such as Mycobacterium tuberculosis, lack the redox protective molecule, glutathione and produce mycothiol (MSH) and ergothioneine (ESH) as their principal low molecular mass thiol instead. Ergothioneine has been known for a while as an anti-oxidant; however its role as a protective thiol in Mycobacterium tuberculosis is still undefined. Present knowledge indicates that ergothioneine may play a critical role in the in vivo and in vitro survival of Mycobacteria, hence enzymes involved in ESH synthesis can be considered as potential drug targets. Ergothioneine is synthesized by the sequential action of five enzymes, encoded by the genes egtA, egtB, egtC, egtD and egtE. Three of these enzymes implicated in the ergothioneine synthesis have been expressed and purified. The last step catalyzed by EgtE, a pyridoxal 5-phosphate (PLP)-dependent β-lyase convert the S-(β-amino-β-carboxyethyl) ergothioneine sulfoxide to ergothioneine. This thesis describes the synthesis of ESH pathway enzyme substrates, with the main focus on the synthesis of the EgtE enzyme substrate, ESH biosynthetic precursor, R- and S-(β-amino-β-carboxyethyl) ergothioneine sulfoxide. In order to prove that synthesized compounds are indeed mycobacterium ESH pathway enzyme substrates, crude M. smeg cell free lysate enzyme was used to convert substrates to ESH. Enzymatic transformation was followed by LCMS analysis. One of two synthetic routes studied, required sufficient quantities of ESH and were therefore thoroughly reviewed. An improved synthetic procedure for ESH was obtained. Deuterated hercynine and ESH was prepared, which will serve as a valuable internal standards and probes for ergothioneine metabolic studies. Enantioselective sulfoxidation of EgtE enzyme substrate precursor, hercynyl cysteine sulfide gave the required hercynyl cysteine sulfoxide derivative and in same cases the hercynyl cysteine sulfone. Crude enzyme mediated transformation of substrates indicated that the hercynyl cysteine sulfide undergoes efficient conversion to ESH. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/6675 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:33:54.099Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2014 |
| publishDateRange | 2014 |
| publishDateSort | 2014 |
| publisher | Department of Chemistry |
| publisherStr | Department of Chemistry |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/6675 Synthesis of Mycobacterial Ergothioneine biosynthetic pathway metabolites Khonde,Lutete Peguy Jardine, Anwar Many gram positive bacteria, such as Mycobacterium tuberculosis, lack the redox protective molecule, glutathione and produce mycothiol (MSH) and ergothioneine (ESH) as their principal low molecular mass thiol instead. Ergothioneine has been known for a while as an anti-oxidant; however its role as a protective thiol in Mycobacterium tuberculosis is still undefined. Present knowledge indicates that ergothioneine may play a critical role in the in vivo and in vitro survival of Mycobacteria, hence enzymes involved in ESH synthesis can be considered as potential drug targets. Ergothioneine is synthesized by the sequential action of five enzymes, encoded by the genes egtA, egtB, egtC, egtD and egtE. Three of these enzymes implicated in the ergothioneine synthesis have been expressed and purified. The last step catalyzed by EgtE, a pyridoxal 5-phosphate (PLP)-dependent β-lyase convert the S-(β-amino-β-carboxyethyl) ergothioneine sulfoxide to ergothioneine. This thesis describes the synthesis of ESH pathway enzyme substrates, with the main focus on the synthesis of the EgtE enzyme substrate, ESH biosynthetic precursor, R- and S-(β-amino-β-carboxyethyl) ergothioneine sulfoxide. In order to prove that synthesized compounds are indeed mycobacterium ESH pathway enzyme substrates, crude M. smeg cell free lysate enzyme was used to convert substrates to ESH. Enzymatic transformation was followed by LCMS analysis. One of two synthetic routes studied, required sufficient quantities of ESH and were therefore thoroughly reviewed. An improved synthetic procedure for ESH was obtained. Deuterated hercynine and ESH was prepared, which will serve as a valuable internal standards and probes for ergothioneine metabolic studies. Enantioselective sulfoxidation of EgtE enzyme substrate precursor, hercynyl cysteine sulfide gave the required hercynyl cysteine sulfoxide derivative and in same cases the hercynyl cysteine sulfone. Crude enzyme mediated transformation of substrates indicated that the hercynyl cysteine sulfide undergoes efficient conversion to ESH. 2014-08-22T10:39:40Z 2014-08-22T10:39:40Z 2013 Master Thesis Masters MSc http://hdl.handle.net/11427/6675 eng application/pdf Department of Chemistry Faculty of Science University of Cape Town |
| spellingShingle | Khonde,Lutete Peguy Synthesis of Mycobacterial Ergothioneine biosynthetic pathway metabolites |
| thesis_degree_str | Master's |
| title | Synthesis of Mycobacterial Ergothioneine biosynthetic pathway metabolites |
| title_full | Synthesis of Mycobacterial Ergothioneine biosynthetic pathway metabolites |
| title_fullStr | Synthesis of Mycobacterial Ergothioneine biosynthetic pathway metabolites |
| title_full_unstemmed | Synthesis of Mycobacterial Ergothioneine biosynthetic pathway metabolites |
| title_short | Synthesis of Mycobacterial Ergothioneine biosynthetic pathway metabolites |
| title_sort | synthesis of mycobacterial ergothioneine biosynthetic pathway metabolites |
| url | http://hdl.handle.net/11427/6675 |
| work_keys_str_mv | AT khondelutetepeguy synthesisofmycobacterialergothioneinebiosyntheticpathwaymetabolites |