{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/73109"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/73109","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Revealing the molecular details of azoline formation in ribosomal natural product biosynthesis","abstract":"The thiazole/oxazole-modified microcins (TOMMs) are a recently grouped class of ribosomally synthesized and posttranslationally modified peptides. Encoded by many bacteria and archaea, these natural products occupy a large chemical and functional space and are linked by the presence of the eponymous azole moieties. Previous work has demonstrated that an evolutionarily conserved, heterotrimeric enzyme complex (TOMM synthetase) is responsible for azole biogenesis. The TOMM synthetase transforms select serine, threonine and cysteine residues in the core peptide into azole heterocycles via a two-step cyclodehydration-dehydrogenation reaction. The installation of the azol(in)e heterocycles endows the peptide with rigidity and, in all characterized TOMM natural products, is required for biological activity. Despite being reconstituted nearly two decades ago, many questions remain regarding the role of each protein in the TOMM synthetase and the mechanism of cyclodehydration. Previous attempts to elucidate the molecular details of azole formation were impeded by the poor stability/solubility of reconstituted TOMM synthetases. In this thesis I report the characterization of the biosynthetic machinery from two novel TOMM cluster from Bacillus sp. Al Hakam that do not suffer from these issues. Using these robust synthetase complexes, I demonstrate that the cyclodehydration reaction proceeds via a phosphorylated hemi-orthoamide intermediate (chapter 2) and provide definitive roles from each of the proteins in the cyclodehydratase complex (chapter 4 and 5). My findings identify an unprecedented role for ATP and provide the first function to the previously uncharacterized YcaO superfamily of proteins. Furthermore, in order to complete my mechanistic investigations of the TOMM synthetase, I developed a novel strategy for generating peptides with site-specific carbonyl oxygen isotope labels (chapter 3). I also demonstrate that this strategy can be expanded to the site-specific installation of thioamides.","abstract_html":"The thiazole/oxazole-modified microcins (TOMMs) are a recently grouped class of ribosomally synthesized and posttranslationally modified peptides. Encoded by many bacteria and archaea, these natural products occupy a large chemical and functional space and are linked by the presence of the eponymous azole moieties. Previous work has demonstrated that an evolutionarily conserved, heterotrimeric enzyme complex (TOMM synthetase) is responsible for azole biogenesis. The TOMM synthetase transforms select serine, threonine and cysteine residues in the core peptide into azole heterocycles via a two-step cyclodehydration-dehydrogenation reaction. The installation of the azol(in)e heterocycles endows the peptide with rigidity and, in all characterized TOMM natural products, is required for biological activity. Despite being reconstituted nearly two decades ago, many questions remain regarding the role of each protein in the TOMM synthetase and the mechanism of cyclodehydration. Previous attempts to elucidate the molecular details of azole formation were impeded by the poor stability/solubility of reconstituted TOMM synthetases. In this thesis I report the characterization of the biosynthetic machinery from two novel TOMM cluster from Bacillus sp. Al Hakam that do not suffer from these issues. Using these robust synthetase complexes, I demonstrate that the cyclodehydration reaction proceeds via a phosphorylated hemi-orthoamide intermediate (chapter 2) and provide definitive roles from each of the proteins in the cyclodehydratase complex (chapter 4 and 5). My findings identify an unprecedented role for ATP and provide the first function to the previously uncharacterized YcaO superfamily of proteins. Furthermore, in order to complete my mechanistic investigations of the TOMM synthetase, I developed a novel strategy for generating peptides with site-specific carbonyl oxygen isotope labels (chapter 3). I also demonstrate that this strategy can be expanded to the site-specific installation of thioamides.","abstract_has_math":false,"creators":["Dunbar, Kyle"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Chemistry","degree_department":null,"school":null,"contributors":["Mitchell, Douglas A.","Mitchell, Douglas A","van der Donk, Wilfred A.","Nair, Satish K.","Gerlt, John A."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2015,"date_issued":"2015-01-21T19:59:36Z","date_published":"2015-01-21T19:59:36Z","updated_at":"2026-07-22T22:26:07Z","subjects":["Azoline Biosynthesis","ribosomally synthesized and post-translationally modified peptide (RiPP) Biosynthesis","thiazole/oxazole-modified microcin (TOMM) Biosynthesis","Cyclodehydratase","YcaO"],"languages":["en"],"rights":["Copyright 2014 Kyle L. Dunbar"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/73109","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Mitchell, Douglas A.","Mitchell, Douglas A","van der Donk, Wilfred A.","Nair, Satish K.","Gerlt, John A."]},{"key":"dc:creator","label":"Author","values":["Dunbar, Kyle"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2015-01-21T19:59:36Z","2017-01-22T10:15:29Z","2014-12","2015-01-21"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Chemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Azoline Biosynthesis","ribosomally synthesized and post-translationally modified peptide (RiPP) Biosynthesis","thiazole/oxazole-modified microcin (TOMM) Biosynthesis","Cyclodehydratase","YcaO"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2014 Kyle L. Dunbar"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/73109"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["The thiazole/oxazole-modified microcins (TOMMs) are a recently grouped class of ribosomally synthesized and posttranslationally modified peptides. Encoded by many bacteria and archaea, these natural products occupy a large chemical and functional space and are linked by the presence of the eponymous azole moieties. Previous work has demonstrated that an evolutionarily conserved, heterotrimeric enzyme complex (TOMM synthetase) is responsible for azole biogenesis. The TOMM synthetase transforms select serine, threonine and cysteine residues in the core peptide into azole heterocycles via a two-step cyclodehydration-dehydrogenation reaction. The installation of the azol(in)e heterocycles endows the peptide with rigidity and, in all characterized TOMM natural products, is required for biological activity. Despite being reconstituted nearly two decades ago, many questions remain regarding the role of each protein in the TOMM synthetase and the mechanism of cyclodehydration. Previous attempts to elucidate the molecular details of azole formation were impeded by the poor stability/solubility of reconstituted TOMM synthetases. In this thesis I report the characterization of the biosynthetic machinery from two novel TOMM cluster from Bacillus sp. Al Hakam that do not suffer from these issues. Using these robust synthetase complexes, I demonstrate that the cyclodehydration reaction proceeds via a phosphorylated hemi-orthoamide intermediate (chapter 2) and provide definitive roles from each of the proteins in the cyclodehydratase complex (chapter 4 and 5). My findings identify an unprecedented role for ATP and provide the first function to the previously uncharacterized YcaO superfamily of proteins. Furthermore, in order to complete my mechanistic investigations of the TOMM synthetase, I developed a novel strategy for generating peptides with site-specific carbonyl oxygen isotope labels (chapter 3). I also demonstrate that this strategy can be expanded to the site-specific installation of thioamides.","Item withdrawn by Mark Zulauf (zulauf@illinois.edu) on 2014-09-26T19:09:28Z Item was in collections: University of Illinois Theses & Dissertations (ID: 1) No. of bitstreams: 2 Dunbar_Kyle.docx: 42379813 bytes, checksum: 0b55fd49f5f7bc2347181863fbd94a7b (MD5) Dunbar_Kyle.pdf: 44811773 bytes, checksum: 68ffd712ea43a76b2e6df3d1eb7aa42d (MD5)","Made available in DSpace on 2015-01-21T19:59:36Z (GMT). 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Encoded by many bacteria and archaea, these natural products occupy a large chemical and functional space and are linked by the presence of the eponymous azole moieties. Previous work has demonstrated that an evolutionarily conserved, heterotrimeric enzyme complex (TOMM synthetase) is responsible for azole biogenesis. The TOMM synthetase transforms select serine, threonine and cysteine residues in the core peptide into azole heterocycles via a two-step cyclodehydration-dehydrogenation reaction. The installation of the azol(in)e heterocycles endows the peptide with rigidity and, in all characterized TOMM natural products, is required for biological activity. Despite being reconstituted nearly two decades ago, many questions remain regarding the role of each protein in the TOMM synthetase and the mechanism of cyclodehydration. Previous attempts to elucidate the molecular details of azole formation were impeded by the poor stability/solubility of reconstituted TOMM synthetases. In this thesis I report the characterization of the biosynthetic machinery from two novel TOMM cluster from Bacillus sp. Al Hakam that do not suffer from these issues. Using these robust synthetase complexes, I demonstrate that the cyclodehydration reaction proceeds via a phosphorylated hemi-orthoamide intermediate (chapter 2) and provide definitive roles from each of the proteins in the cyclodehydratase complex (chapter 4 and 5). My findings identify an unprecedented role for ATP and provide the first function to the previously uncharacterized YcaO superfamily of proteins. Furthermore, in order to complete my mechanistic investigations of the TOMM synthetase, I developed a novel strategy for generating peptides with site-specific carbonyl oxygen isotope labels (chapter 3). I also demonstrate that this strategy can be expanded to the site-specific installation of thioamides.","Item withdrawn by Mark Zulauf (zulauf@illinois.edu) on 2014-09-26T19:09:28Z Item was in collections: University of Illinois Theses & Dissertations (ID: 1) No. of bitstreams: 2 Dunbar_Kyle.docx: 42379813 bytes, checksum: 0b55fd49f5f7bc2347181863fbd94a7b (MD5) Dunbar_Kyle.pdf: 44811773 bytes, checksum: 68ffd712ea43a76b2e6df3d1eb7aa42d (MD5)","Made available in DSpace on 2015-01-21T19:59:36Z (GMT). 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Dunbar"],"dc:subject":["Azoline Biosynthesis","ribosomally synthesized and post-translationally modified peptide (RiPP) Biosynthesis","thiazole/oxazole-modified microcin (TOMM) Biosynthesis","Cyclodehydratase","YcaO"],"dc:title":["Revealing the molecular details of azoline formation in ribosomal natural product biosynthesis"],"dc:type":["text"],"thesis:degree_discipline":["Chemistry"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:26:07Z"}