{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/108217"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/108217","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Investigation and application of substrate tolerant thioether forming enzymes","abstract":"Thioether-containing compounds are involved in different biological functions in both prokaryotic and eukaryotic systems. Nature has evolved a treasure box of intriguing thioether containing natural products. Lanthipeptides contain thioether rings and have demonstrated diverse biological activities. This group of natural products are of great interest due to their diverse ring topology and high substrate tolerance of the biosynthetic enzymes. Amongst them lanthipeptides are one of the most studied ribosomally synthesized and post-translationally modified peptides (RiPPs). In general, a precursor peptide with an N-terminal leader peptide and a C-terminal core peptide is ribosomally synthesized. The core peptides undergoes a series of post-translational modification including dehydration of serines and threonines to the α,β-unsaturated residues 2,3-didehydroalanine (Dha) and (Z)-2,3-didehydrobutyrine (Dhb), respectively. Thioether rings are formed through Michael addition of thiol groups on cysteine residues to theses dehydroamino acids. The removal of the leader peptide yields the mature product, which is exported from the producing cells. The enzymes involved in lanthipeptides biosynthesis are well studied and several platforms have been employed for lanthipeptide engineering using the highly substrate tolerant lanthipeptides synthetases. The potential of lanthipeptide engineering is further demonstrated in chapter 2 using a substrate-tolerant synthetase (ProcM) from Prochlorococcus that dehydrates and cyclizes up to 30 different linear precursor peptides encoded in its genome. A bicyclic lanthipeptide library was constructed, characterized and coupled to a selection system to screen for a protein-protein interaction (PPI) inhibitor. XY3-3 was identified as an inhibitor for the PPI between the HIV p6 protein and the ubiquitin E2 variant (UEV) domain of the human TSG101 protein, which is essential for HIV virus budding from infected cells. A detailed in vitro characterization was performed to identify the binding site of XY3-3 using fluorescence polarization. XY3-3 competes with full length p6 protein for binding to UEV. To optimize XY3-3, two yeast display systems are investigated. Unfortunately, initial data suggested that the displayed peptide formed a disulfide bond which inhibited ProcM modification. Further optimization of yeast display systems or adaption of other display systems may allow the selection of a lanthipeptide scaffold with better binding affinity. While my work illustrated applications for bacterial lanthipeptide synthetases, their mammalian homologs remain mysterious. LanCL proteins, which are homologs of the lanthipeptide cyclase LanC, share similar a structure and conserved zinc binding residues with their bacterial counterparts. Moreover, LanCL proteins bind to glutathione at the putative active site. Recent studies suggested diverse functions of LanCL proteins, yet their enzymatic activity is not well understood. One hypothesis is that LanCL proteins function in a similar way as LanC, by catalyzing glutathionylation of dehydro amino acids to afford the small molecule lanthionine in mammalian systems. To test this hypothesis, in chapter 3, I developed two LC/MS/MS methods to quantify endogenous lanthionine concentrations in both wild type and LanCL knockout mouse brains, where LanCL proteins are highly expressed. Similar levels of about 0.5-2.5 nmol/g tissue were detected for both WT and TKO mouse, suggesting LanCL proteins are not involved in lanthionine synthesis. However, it is still possible that LanCL proteins use glutathione as one of the substrates. Indeed, recent findings in our lab showed LanCL proteins interact with a wide range of kinases and catalyze glutathionylation of Dha/Dhb containing kinase proteins and peptides. In chapter 4, I expanded the substrate scope with non-kinase lanthipeptides, demonstrating LanCL proteins are highly substrate tolerant. To identify LanCL targets, I developed a chemoproteomics method to enrich proteins with LanCL-catalyzed glutathionylation. The initial proteomics data suggest that LanCL protein targets are not limited to kinases and LanCL-catalyzed glutathionylation may function as a general mechanism to eliminate functions of electrophilic Dha/Dhb containing proteins.","abstract_html":"Thioether-containing compounds are involved in different biological functions in both prokaryotic and eukaryotic systems. Nature has evolved a treasure box of intriguing thioether containing natural products. Lanthipeptides contain thioether rings and have demonstrated diverse biological activities. This group of natural products are of great interest due to their diverse ring topology and high substrate tolerance of the biosynthetic enzymes. Amongst them lanthipeptides are one of the most studied ribosomally synthesized and post-translationally modified peptides (RiPPs). In general, a precursor peptide with an N-terminal leader peptide and a C-terminal core peptide is ribosomally synthesized. The core peptides undergoes a series of post-translational modification including dehydration of serines and threonines to the α,β-unsaturated residues 2,3-didehydroalanine (Dha) and (Z)-2,3-didehydrobutyrine (Dhb), respectively. Thioether rings are formed through Michael addition of thiol groups on cysteine residues to theses dehydroamino acids. The removal of the leader peptide yields the mature product, which is exported from the producing cells. The enzymes involved in lanthipeptides biosynthesis are well studied and several platforms have been employed for lanthipeptide engineering using the highly substrate tolerant lanthipeptides synthetases. The potential of lanthipeptide engineering is further demonstrated in chapter 2 using a substrate-tolerant synthetase (ProcM) from Prochlorococcus that dehydrates and cyclizes up to 30 different linear precursor peptides encoded in its genome. A bicyclic lanthipeptide library was constructed, characterized and coupled to a selection system to screen for a protein-protein interaction (PPI) inhibitor. XY3-3 was identified as an inhibitor for the PPI between the HIV p6 protein and the ubiquitin E2 variant (UEV) domain of the human TSG101 protein, which is essential for HIV virus budding from infected cells. A detailed in vitro characterization was performed to identify the binding site of XY3-3 using fluorescence polarization. XY3-3 competes with full length p6 protein for binding to UEV. To optimize XY3-3, two yeast display systems are investigated. Unfortunately, initial data suggested that the displayed peptide formed a disulfide bond which inhibited ProcM modification. Further optimization of yeast display systems or adaption of other display systems may allow the selection of a lanthipeptide scaffold with better binding affinity. While my work illustrated applications for bacterial lanthipeptide synthetases, their mammalian homologs remain mysterious. LanCL proteins, which are homologs of the lanthipeptide cyclase LanC, share similar a structure and conserved zinc binding residues with their bacterial counterparts. Moreover, LanCL proteins bind to glutathione at the putative active site. Recent studies suggested diverse functions of LanCL proteins, yet their enzymatic activity is not well understood. One hypothesis is that LanCL proteins function in a similar way as LanC, by catalyzing glutathionylation of dehydro amino acids to afford the small molecule lanthionine in mammalian systems. To test this hypothesis, in chapter 3, I developed two LC/MS/MS methods to quantify endogenous lanthionine concentrations in both wild type and LanCL knockout mouse brains, where LanCL proteins are highly expressed. Similar levels of about 0.5-2.5 nmol/g tissue were detected for both WT and TKO mouse, suggesting LanCL proteins are not involved in lanthionine synthesis. However, it is still possible that LanCL proteins use glutathione as one of the substrates. Indeed, recent findings in our lab showed LanCL proteins interact with a wide range of kinases and catalyze glutathionylation of Dha/Dhb containing kinase proteins and peptides. In chapter 4, I expanded the substrate scope with non-kinase lanthipeptides, demonstrating LanCL proteins are highly substrate tolerant. To identify LanCL targets, I developed a chemoproteomics method to enrich proteins with LanCL-catalyzed glutathionylation. The initial proteomics data suggest that LanCL protein targets are not limited to kinases and LanCL-catalyzed glutathionylation may function as a general mechanism to eliminate functions of electrophilic Dha/Dhb containing proteins.","abstract_has_math":false,"creators":["He, Chang"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Chemistry","degree_department":null,"school":null,"contributors":["van der Donk, Wilfred A","Chen, Jie","Sweedler, Jonathan V","Hergenrother, Paul J"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2020,"date_issued":"2020-08-27T00:46:47Z","date_published":"2020-08-27T00:46:47Z","updated_at":"2026-07-22T22:24:48Z","subjects":["Lanthipeptide engineering","Lanthipeptide library","LanC-like protein","glutathionylation"],"languages":["en"],"rights":["Copyright 2020 Chang He"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/108217","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["van der Donk, Wilfred A","Chen, Jie","Sweedler, Jonathan V","Hergenrother, Paul J"]},{"key":"dc:creator","label":"Author","values":["He, Chang"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2020-08-27T00:46:47Z","2022-08-27T00:51:40Z","2020-01-24","2020-05"]},{"key":"dc:type","label":"Dc Type","values":["text","Thesis"]},{"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":["Lanthipeptide engineering","Lanthipeptide library","LanC-like protein","glutathionylation"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2020 Chang He"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/108217"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Thioether-containing compounds are involved in different biological functions in both prokaryotic and eukaryotic systems. Nature has evolved a treasure box of intriguing thioether containing natural products. Lanthipeptides contain thioether rings and have demonstrated diverse biological activities. This group of natural products are of great interest due to their diverse ring topology and high substrate tolerance of the biosynthetic enzymes. Amongst them lanthipeptides are one of the most studied ribosomally synthesized and post-translationally modified peptides (RiPPs). In general, a precursor peptide with an N-terminal leader peptide and a C-terminal core peptide is ribosomally synthesized. The core peptides undergoes a series of post-translational modification including dehydration of serines and threonines to the α,β-unsaturated residues 2,3-didehydroalanine (Dha) and (Z)-2,3-didehydrobutyrine (Dhb), respectively. Thioether rings are formed through Michael addition of thiol groups on cysteine residues to theses dehydroamino acids. The removal of the leader peptide yields the mature product, which is exported from the producing cells. The enzymes involved in lanthipeptides biosynthesis are well studied and several platforms have been employed for lanthipeptide engineering using the highly substrate tolerant lanthipeptides synthetases. The potential of lanthipeptide engineering is further demonstrated in chapter 2 using a substrate-tolerant synthetase (ProcM) from Prochlorococcus that dehydrates and cyclizes up to 30 different linear precursor peptides encoded in its genome. A bicyclic lanthipeptide library was constructed, characterized and coupled to a selection system to screen for a protein-protein interaction (PPI) inhibitor. XY3-3 was identified as an inhibitor for the PPI between the HIV p6 protein and the ubiquitin E2 variant (UEV) domain of the human TSG101 protein, which is essential for HIV virus budding from infected cells. A detailed in vitro characterization was performed to identify the binding site of XY3-3 using fluorescence polarization. XY3-3 competes with full length p6 protein for binding to UEV. To optimize XY3-3, two yeast display systems are investigated. Unfortunately, initial data suggested that the displayed peptide formed a disulfide bond which inhibited ProcM modification. Further optimization of yeast display systems or adaption of other display systems may allow the selection of a lanthipeptide scaffold with better binding affinity. While my work illustrated applications for bacterial lanthipeptide synthetases, their mammalian homologs remain mysterious. LanCL proteins, which are homologs of the lanthipeptide cyclase LanC, share similar a structure and conserved zinc binding residues with their bacterial counterparts. Moreover, LanCL proteins bind to glutathione at the putative active site. Recent studies suggested diverse functions of LanCL proteins, yet their enzymatic activity is not well understood. One hypothesis is that LanCL proteins function in a similar way as LanC, by catalyzing glutathionylation of dehydro amino acids to afford the small molecule lanthionine in mammalian systems. To test this hypothesis, in chapter 3, I developed two LC/MS/MS methods to quantify endogenous lanthionine concentrations in both wild type and LanCL knockout mouse brains, where LanCL proteins are highly expressed. Similar levels of about 0.5-2.5 nmol/g tissue were detected for both WT and TKO mouse, suggesting LanCL proteins are not involved in lanthionine synthesis. However, it is still possible that LanCL proteins use glutathione as one of the substrates. Indeed, recent findings in our lab showed LanCL proteins interact with a wide range of kinases and catalyze glutathionylation of Dha/Dhb containing kinase proteins and peptides. In chapter 4, I expanded the substrate scope with non-kinase lanthipeptides, demonstrating LanCL proteins are highly substrate tolerant. To identify LanCL targets, I developed a chemoproteomics method to enrich proteins with LanCL-catalyzed glutathionylation. The initial proteomics data suggest that LanCL protein targets are not limited to kinases and LanCL-catalyzed glutathionylation may function as a general mechanism to eliminate functions of electrophilic Dha/Dhb containing proteins.","Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2022-05-01","The student, Chang He, accepted the attached license on 2020-01-22 at 10:25.","The student, Chang He, submitted this Dissertation for approval on 2020-01-22 at 10:38.","This Dissertation was approved for publication on 2020-01-24 at 13:53.","DSpace SAF Submission Ingestion Package generated from Vireo submission #14854 on 2020-08-25 at 17:38:25","Made available in DSpace on 2020-08-27T00:46:47Z (GMT). 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Nature has evolved a treasure box of intriguing thioether containing natural products. Lanthipeptides contain thioether rings and have demonstrated diverse biological activities. This group of natural products are of great interest due to their diverse ring topology and high substrate tolerance of the biosynthetic enzymes. Amongst them lanthipeptides are one of the most studied ribosomally synthesized and post-translationally modified peptides (RiPPs). In general, a precursor peptide with an N-terminal leader peptide and a C-terminal core peptide is ribosomally synthesized. The core peptides undergoes a series of post-translational modification including dehydration of serines and threonines to the α,β-unsaturated residues 2,3-didehydroalanine (Dha) and (Z)-2,3-didehydrobutyrine (Dhb), respectively. Thioether rings are formed through Michael addition of thiol groups on cysteine residues to theses dehydroamino acids. The removal of the leader peptide yields the mature product, which is exported from the producing cells. The enzymes involved in lanthipeptides biosynthesis are well studied and several platforms have been employed for lanthipeptide engineering using the highly substrate tolerant lanthipeptides synthetases. The potential of lanthipeptide engineering is further demonstrated in chapter 2 using a substrate-tolerant synthetase (ProcM) from Prochlorococcus that dehydrates and cyclizes up to 30 different linear precursor peptides encoded in its genome. A bicyclic lanthipeptide library was constructed, characterized and coupled to a selection system to screen for a protein-protein interaction (PPI) inhibitor. XY3-3 was identified as an inhibitor for the PPI between the HIV p6 protein and the ubiquitin E2 variant (UEV) domain of the human TSG101 protein, which is essential for HIV virus budding from infected cells. A detailed in vitro characterization was performed to identify the binding site of XY3-3 using fluorescence polarization. XY3-3 competes with full length p6 protein for binding to UEV. To optimize XY3-3, two yeast display systems are investigated. Unfortunately, initial data suggested that the displayed peptide formed a disulfide bond which inhibited ProcM modification. Further optimization of yeast display systems or adaption of other display systems may allow the selection of a lanthipeptide scaffold with better binding affinity. While my work illustrated applications for bacterial lanthipeptide synthetases, their mammalian homologs remain mysterious. LanCL proteins, which are homologs of the lanthipeptide cyclase LanC, share similar a structure and conserved zinc binding residues with their bacterial counterparts. Moreover, LanCL proteins bind to glutathione at the putative active site. Recent studies suggested diverse functions of LanCL proteins, yet their enzymatic activity is not well understood. One hypothesis is that LanCL proteins function in a similar way as LanC, by catalyzing glutathionylation of dehydro amino acids to afford the small molecule lanthionine in mammalian systems. To test this hypothesis, in chapter 3, I developed two LC/MS/MS methods to quantify endogenous lanthionine concentrations in both wild type and LanCL knockout mouse brains, where LanCL proteins are highly expressed. Similar levels of about 0.5-2.5 nmol/g tissue were detected for both WT and TKO mouse, suggesting LanCL proteins are not involved in lanthionine synthesis. However, it is still possible that LanCL proteins use glutathione as one of the substrates. Indeed, recent findings in our lab showed LanCL proteins interact with a wide range of kinases and catalyze glutathionylation of Dha/Dhb containing kinase proteins and peptides. In chapter 4, I expanded the substrate scope with non-kinase lanthipeptides, demonstrating LanCL proteins are highly substrate tolerant. To identify LanCL targets, I developed a chemoproteomics method to enrich proteins with LanCL-catalyzed glutathionylation. The initial proteomics data suggest that LanCL protein targets are not limited to kinases and LanCL-catalyzed glutathionylation may function as a general mechanism to eliminate functions of electrophilic Dha/Dhb containing proteins.","Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2022-05-01","The student, Chang He, accepted the attached license on 2020-01-22 at 10:25.","The student, Chang He, submitted this Dissertation for approval on 2020-01-22 at 10:38.","This Dissertation was approved for publication on 2020-01-24 at 13:53.","DSpace SAF Submission Ingestion Package generated from Vireo submission #14854 on 2020-08-25 at 17:38:25","Made available in DSpace on 2020-08-27T00:46:47Z (GMT). No. of bitstreams: 3 HE-DISSERTATION-2020.pdf: 16009976 bytes, checksum: b77c1f11d5f3f8e3343db9626b378a0a (MD5) LICENSE.txt: 4205 bytes, checksum: 4e76cfc090a983823b631ef4fc664ad1 (MD5) PROQUEST_LICENSE.txt: 4551 bytes, checksum: c2be19e0ce3b3f9c7e78a98070cee850 (MD5) Previous issue date: 2020-01-24","Embargo set by: Seth Robbins for item 115830 Lift date: 2022-08-27T00:46:59Z Reason: Author requested closed access (OA after 2yrs) in Vireo ETD system","Embargo set by: Seth Robbins for item 115830 Lift date: 2022-08-27T00:50:22Z Reason: Author requested closed access (OA after 2yrs) in Vireo ETD system","Embargo set by: Seth Robbins for item 115830 Lift date: 2022-08-27T00:51:40Z Reason: Author requested closed access (OA after 2yrs) in Vireo ETD system","Author requested closed access (OA after 2yrs) in Vireo ETD system","Limited"],"dc:format":["application/pdf"],"dc:identifier":["http://hdl.handle.net/2142/108217"],"dc:language":["en"],"dc:rights":["Copyright 2020 Chang He"],"dc:subject":["Lanthipeptide engineering","Lanthipeptide library","LanC-like protein","glutathionylation"],"dc:title":["Investigation and application of substrate tolerant thioether forming enzymes"],"dc:type":["text","Thesis"],"thesis:degree_discipline":["Chemistry"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:24:48Z"}