Synthesis of Mycobacterial Ergothioneine biosynthetic pathway metabolites

Simple item page
dc.contributor.advisorJardine, Anwaren_ZA
dc.contributor.authorKhonde,Lutete Peguyen_ZA
dc.date.accessioned2014-08-22T10:39:40Z
dc.date.available2014-08-22T10:39:40Z
dc.date.issued2013en_ZA
dc.description.abstractMany 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.en_ZA
dc.identifier.apacitation (2013). <i>Synthesis of Mycobacterial Ergothioneine biosynthetic pathway metabolites</i>. (Thesis). University of Cape Town ,Faculty of Science ,Department of Chemistry. Retrieved from http://hdl.handle.net/11427/6675en_ZA
dc.identifier.chicagocitation. <i>"Synthesis of Mycobacterial Ergothioneine biosynthetic pathway metabolites."</i> Thesis., University of Cape Town ,Faculty of Science ,Department of Chemistry, 2013. http://hdl.handle.net/11427/6675en_ZA
dc.identifier.citation 2013. Synthesis of Mycobacterial Ergothioneine biosynthetic pathway metabolites. Thesis. University of Cape Town ,Faculty of Science ,Department of Chemistry. http://hdl.handle.net/11427/6675en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Khonde,Lutete Peguy AB - 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. DA - 2013 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2013 T1 - Synthesis of Mycobacterial Ergothioneine biosynthetic pathway metabolites TI - Synthesis of Mycobacterial Ergothioneine biosynthetic pathway metabolites UR - http://hdl.handle.net/11427/6675 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/6675
dc.identifier.vancouvercitation. Synthesis of Mycobacterial Ergothioneine biosynthetic pathway metabolites. [Thesis]. University of Cape Town ,Faculty of Science ,Department of Chemistry, 2013 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/6675en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDepartment of Chemistryen_ZA
dc.publisher.facultyFaculty of Scienceen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.titleSynthesis of Mycobacterial Ergothioneine biosynthetic pathway metabolitesen_ZA
dc.typeMaster Thesis
dc.type.qualificationlevelMasters
dc.type.qualificationnameMScen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
Files
Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
thesis_sci_2013_khonde_l.pdf
Size:
6.23 MB
Format:
Adobe Portable Document Format
Description:
Download
Collections
Masters