{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/87954"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/87954","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"On the biosynthesis and discovery of ribosomally synthesized and post-translationally modified peptides","abstract":"Thiazole/oxazole-modified microcins (TOMMs) comprise a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) that all contain thiazole and oxazole heterocycles derived from cysteine, serine, and threonine residues, respectively. The thiazole/oxazole heterocycle is installed over two distinct steps. First, the cyclodehydratase cyclizes an unmodified cysteine, serine, or threonine residue to a thiazoline or oxazoline heterocycle, which can be oxidized by a FMN dependent dehydrogenase to afford a thiazole or oxazole heterocycle. The biosynthesis of a number of TOMMs has been investigated (Chapter 1), yet a number of fundamental questions regarding substrate processing remained. Genome mining efforts revealed an uncharacterized TOMM gene cluster in Bacillus sp. Al Hakam (Balh) that contained substrates and enzymes that ultimately overcame previous limitations. Initial reconstitution of the enzymes revealed cyclization of half of the heterocyclizable residues. This observation inspired a series of experiments to explain the observed selectivity. Using a combination of high-resolution mass spectrometry, site-directed mutagenesis, and kinetics, I found that the location of a heterocyclizable residue and the presence of a preceding glycine largely dictate heterocycle formation, which uniquely proceeds in an overall C- to N-terminal fashion (Chapter 2). While characterizing the cyclodehydratase, it became evident that omission of the dehydrogenase had no effect on cyclodehydratase activity. This provided the opportunity to separate the function of the two enzymes and investigate how the dehydrogenase interacts with the cyclodehydratase and processes substrate (Chapter 3). Many of the tools I developed to study the Balh TOMM synthetase proved useful for structural and biosynthetic studies of other RiPPs (Appendix A).","abstract_html":"Thiazole/oxazole-modified microcins (TOMMs) comprise a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) that all contain thiazole and oxazole heterocycles derived from cysteine, serine, and threonine residues, respectively. The thiazole/oxazole heterocycle is installed over two distinct steps. First, the cyclodehydratase cyclizes an unmodified cysteine, serine, or threonine residue to a thiazoline or oxazoline heterocycle, which can be oxidized by a FMN dependent dehydrogenase to afford a thiazole or oxazole heterocycle. The biosynthesis of a number of TOMMs has been investigated (Chapter 1), yet a number of fundamental questions regarding substrate processing remained. Genome mining efforts revealed an uncharacterized TOMM gene cluster in Bacillus sp. Al Hakam (Balh) that contained substrates and enzymes that ultimately overcame previous limitations. Initial reconstitution of the enzymes revealed cyclization of half of the heterocyclizable residues. This observation inspired a series of experiments to explain the observed selectivity. Using a combination of high-resolution mass spectrometry, site-directed mutagenesis, and kinetics, I found that the location of a heterocyclizable residue and the presence of a preceding glycine largely dictate heterocycle formation, which uniquely proceeds in an overall C- to N-terminal fashion (Chapter 2). While characterizing the cyclodehydratase, it became evident that omission of the dehydrogenase had no effect on cyclodehydratase activity. This provided the opportunity to separate the function of the two enzymes and investigate how the dehydrogenase interacts with the cyclodehydratase and processes substrate (Chapter 3). Many of the tools I developed to study the Balh TOMM synthetase proved useful for structural and biosynthetic studies of other RiPPs (Appendix A).","abstract_has_math":false,"creators":["Melby, Joel Oliver"],"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 M","Bailey, Ryan C.","Hergenrother, Paul J.","Metcalf, William W."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2015,"date_issued":"2015-09-29T20:37:37Z","date_published":"2015-09-29T20:37:37Z","updated_at":"2026-07-22T22:26:31Z","subjects":["Natural Products","Biosynthesis"],"languages":["en"],"rights":["Copyright 2015 Joel Melby"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/87954","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Mitchell, Douglas M","Bailey, Ryan C.","Hergenrother, Paul J.","Metcalf, William W."]},{"key":"dc:creator","label":"Author","values":["Melby, Joel Oliver"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2015-09-29T20:37:37Z","2015-08","2015-06-18","2015-8"]},{"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":["Natural Products","Biosynthesis"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2015 Joel Melby"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/87954"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Thiazole/oxazole-modified microcins (TOMMs) comprise a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) that all contain thiazole and oxazole heterocycles derived from cysteine, serine, and threonine residues, respectively. The thiazole/oxazole heterocycle is installed over two distinct steps. First, the cyclodehydratase cyclizes an unmodified cysteine, serine, or threonine residue to a thiazoline or oxazoline heterocycle, which can be oxidized by a FMN dependent dehydrogenase to afford a thiazole or oxazole heterocycle. The biosynthesis of a number of TOMMs has been investigated (Chapter 1), yet a number of fundamental questions regarding substrate processing remained. Genome mining efforts revealed an uncharacterized TOMM gene cluster in Bacillus sp. Al Hakam (Balh) that contained substrates and enzymes that ultimately overcame previous limitations. Initial reconstitution of the enzymes revealed cyclization of half of the heterocyclizable residues. This observation inspired a series of experiments to explain the observed selectivity. Using a combination of high-resolution mass spectrometry, site-directed mutagenesis, and kinetics, I found that the location of a heterocyclizable residue and the presence of a preceding glycine largely dictate heterocycle formation, which uniquely proceeds in an overall C- to N-terminal fashion (Chapter 2). While characterizing the cyclodehydratase, it became evident that omission of the dehydrogenase had no effect on cyclodehydratase activity. This provided the opportunity to separate the function of the two enzymes and investigate how the dehydrogenase interacts with the cyclodehydratase and processes substrate (Chapter 3). Many of the tools I developed to study the Balh TOMM synthetase proved useful for structural and biosynthetic studies of other RiPPs (Appendix A).","Submission original under an indefinite embargo labeled 'Open Access'. The submission was exported from vireo on 2015-09-29 without embargo terms","The student, Joel Melby, accepted the attached license on 2015-06-16 at 19:43.","The student, Joel Melby, submitted this Dissertation for approval on 2015-06-16 at 19:48.","This Dissertation was approved for publication on 2015-06-18 at 08:24.","DSpace SAF Submission Ingestion Package generated from Vireo submission #8254 on 2015-09-29 at 13:21:18","Made available in DSpace on 2015-09-29T20:37:37Z (GMT). No. of bitstreams: 2 MELBY-DISSERTATION-2015.pdf: 75315341 bytes, checksum: 0e25582805f588b5538fa2b910e925aa (MD5) LICENSE.txt: 4207 bytes, checksum: b779928b0265c7c6ad77f06c93b73ef5 (MD5) Previous issue date: 2015-06-18"]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["On the biosynthesis and discovery of ribosomally synthesized and post-translationally modified peptides"]}]}],"canonical_facts":{"dc:contributor":["Mitchell, Douglas M","Bailey, Ryan C.","Hergenrother, Paul J.","Metcalf, William W."],"dc:creator":["Melby, Joel Oliver"],"dc:date":["2015-09-29T20:37:37Z","2015-08","2015-06-18","2015-8"],"dc:description":["Thiazole/oxazole-modified microcins (TOMMs) comprise a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) that all contain thiazole and oxazole heterocycles derived from cysteine, serine, and threonine residues, respectively. The thiazole/oxazole heterocycle is installed over two distinct steps. First, the cyclodehydratase cyclizes an unmodified cysteine, serine, or threonine residue to a thiazoline or oxazoline heterocycle, which can be oxidized by a FMN dependent dehydrogenase to afford a thiazole or oxazole heterocycle. The biosynthesis of a number of TOMMs has been investigated (Chapter 1), yet a number of fundamental questions regarding substrate processing remained. Genome mining efforts revealed an uncharacterized TOMM gene cluster in Bacillus sp. Al Hakam (Balh) that contained substrates and enzymes that ultimately overcame previous limitations. Initial reconstitution of the enzymes revealed cyclization of half of the heterocyclizable residues. This observation inspired a series of experiments to explain the observed selectivity. Using a combination of high-resolution mass spectrometry, site-directed mutagenesis, and kinetics, I found that the location of a heterocyclizable residue and the presence of a preceding glycine largely dictate heterocycle formation, which uniquely proceeds in an overall C- to N-terminal fashion (Chapter 2). While characterizing the cyclodehydratase, it became evident that omission of the dehydrogenase had no effect on cyclodehydratase activity. This provided the opportunity to separate the function of the two enzymes and investigate how the dehydrogenase interacts with the cyclodehydratase and processes substrate (Chapter 3). Many of the tools I developed to study the Balh TOMM synthetase proved useful for structural and biosynthetic studies of other RiPPs (Appendix A).","Submission original under an indefinite embargo labeled 'Open Access'. The submission was exported from vireo on 2015-09-29 without embargo terms","The student, Joel Melby, accepted the attached license on 2015-06-16 at 19:43.","The student, Joel Melby, submitted this Dissertation for approval on 2015-06-16 at 19:48.","This Dissertation was approved for publication on 2015-06-18 at 08:24.","DSpace SAF Submission Ingestion Package generated from Vireo submission #8254 on 2015-09-29 at 13:21:18","Made available in DSpace on 2015-09-29T20:37:37Z (GMT). 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