{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/101259"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/101259","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Structural and biochemical studies of enzymes involved in the biosynthesis of value-added products","abstract":"The student, Nektaria Petronikolou, submitted this Dissertation for approval on 2018-02-16 at 18:00.","abstract_html":"The student, Nektaria Petronikolou, submitted this Dissertation for approval on 2018-02-16 at 18:00.","abstract_has_math":false,"creators":["Petronikolou, Nektaria"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biochemistry","degree_department":null,"school":null,"contributors":["Nair, Satish K.","Cronan, John E.","Zhao, Huimin","Jin, Hong"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2018,"date_issued":"2018-09-04T20:46:48Z","date_published":"2018-09-04T20:46:48Z","updated_at":"2026-07-22T22:24:38Z","subjects":["biocatalysis","biofuels","drug resistance"],"languages":["en"],"rights":["Copyright 2018 Nektaria Petronikolou"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/101259","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Nair, Satish K.","Cronan, John E.","Zhao, Huimin","Jin, Hong"]},{"key":"dc:creator","label":"Author","values":["Petronikolou, Nektaria"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2018-09-04T20:46:48Z","2020-09-05T09:15:26Z","2018-03-28","2018-05"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biochemistry"]},{"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":["biocatalysis","biofuels","drug resistance"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2018 Nektaria Petronikolou"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/101259"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["The student, Nektaria Petronikolou, submitted this Dissertation for approval on 2018-02-16 at 18:00.","This Dissertation was approved for publication on 2018-03-28 at 15:31.","DSpace SAF Submission Ingestion Package generated from Vireo submission #12041 on 2018-08-31 at 17:25:10","Made available in DSpace on 2018-09-04T20:46:48Z (GMT). No. of bitstreams: 3 PETRONIKOLOU-DISSERTATION-2018.pdf: 5586351 bytes, checksum: 4ccc9c8c8a66f0350c40658ca9c55631 (MD5) Petronikolou_Dissertation_corrected_final.docx: 14970926 bytes, checksum: 8910cffce5cf54d2aca49ae6894290d7 (MD5) LICENSE.txt: 4218 bytes, checksum: c41f7ffe280f5c8e05272d6c9f960842 (MD5) Previous issue date: 2018-03-28","Embargo set by: Seth Robbins for item 107344 Lift date: 2020-09-04T20:47:38Z Reason: Author requested closed access (OA after 2yrs) in Vireo ETD system","Embargo set by: Seth Robbins for item 107344 Lift date: 2020-09-04T20:50:11Z Reason: Author requested closed access (OA after 2yrs) in Vireo ETD system","Limited Restriction Lifted for Item 107344 on 2020-09-05T09:15:26Z.","Enzymes have been used for decades for the industrial production of high value chemicals such as food additives, antibiotics and other pharmaceutical products. However, in recent years the significant advances in biocatalyst development have led to a rise in the number of commodity chemicals being produced by enzymes instead of extraction from plants or chemical synthesis. Such products include biodiesel and wax esters. For this industrial exploitation of enzymes, it is vital to have an extensive understanding of how the desired enzymes work, including what the enzymes’ substrate preferences and stability are. This knowledge comes from the structural and biochemical characterization of these biocatalysts, and this is the scope of this dissertation; to broaden our knowledge and understanding of enzymes that are involved in the biosynthesis of value-added products, such as biodiesel and drugs. Biodiesel is a mixture of long chain fatty acid alkyl esters (FAAEs), produced mainly by the transesterification of fatty acids derived from vegetable oils and animal fats. In recent years, biodiesel has emerged as a viable resource for utilization in green energy production. However, current production processes utilize methods that are cost ineffective and generate waste byproducts. Recent progress in the field makes use of biocatalysts that are effective in diverting products from primary metabolism to yield fatty acid methyl and ethyl esters in bacterial systems (as well as other commodity chemicals). Two of these enzymes have been the focus of my study: a fatty acid O-methyltransferase (FAMT) from Mycobacterium marinum (MmFAMT), and a bifunctional wax ester synthase/diacylglycerol acyl-transferase (WS/DGAT) from Marinobacter aquaeolei VT8 (Ma-WS/DGAT). In Chapter 1, I present the first kinetic and structural characterization of a mycobacterial fatty acid O-methyltransferase (MmFAMT) previously utilized for the production of biodiesel in E. coli. The structure revealed unexpected similarity with methyltransferases from plant natural product metabolism, and an active-site cavity that can accommodate fatty acids up to 12-14 carbons long. Our results provide the framework for further optimization of an in vivo system that utilizes MmFAMT for production of biodiesel. In Chapter 2, I discuss the structure of a bifunctional wax ester synthase/diacylglycerol acyl-transferase (Ma-WS/DGAT), which is to our knowledge the first structure for any member of this enzyme family to date. Wax esters (WE) are esters of long chain fatty acids and long chain alcohols and have widely been used in cosmetics, lubricants, candles and the food industry. In addition, WE that consist of medium chain fatty acids and ethanol (fatty acid ethyl esters, FAEEs) are biodiesel. Today, there is a strong demand for the development of a large-scale process for the production of low cost WE. The structure may facilitate future engineering efforts aiming at the optimization of this biocatalyst towards production of desired wax esters for industrial use. In addition to biodiesel, value-added chemicals produced by enzymes include bioactive compounds such as chemotherapeutics and antibiotics. In Chapters 3 and 4, I present structural and biochemical data that expand our knowledge for enzymes involved in the biosynthesis of such compounds. In Chapter 3, I discuss the molecular basis for the substrate specificity of loganic acid O-methyltransferase from Catharanthus roseus (CraLAMT), an enzyme that methylates with high stereospecificity an iridoid glycoside to produce loganin. Loganin is an intermediate in the biosynthesis of indole alkaloids in plants - a group of plant natural products with several medicinal uses, and has attracted interest as a bioactive compound itself. Our results may facilitate future engineering efforts to diversify the CraLAMT substrate scaffold towards the design of new potential bioactive compounds. In Chapter 4, I present structural and biochemical data for enzymes that are involved in the biosynthesis of phosphonates. Phosphonates are natural products containing a carbon-phosphorous (C-P) bond. These C-P containing molecules inhibit enzymes which utilize phosphate esters and carboxylic acids by mimicking their substrates. At a time when drug-resistant pathogens pose serious threat to human health, phosphonate natural products provide a pool of potential therapeutic candidates. Two such phosphonate containing compounds are rhizocticin and plumbemycin. Rhizocticin and plumbemycin are di- or tripeptides that contain the same phosphonic amino acid at their C-termini, but differ at their N-termini. This unusual amino acid is (Z)-L-2-amino-5-phosphono-3-pentenoic acid (APPA), and is the active part of rhizocticin and plumbemycin. Upon entering the cell, host peptidases release APPA which subsequently inhibits threonine synthase (ThrC) and kills the cell. Because thrC is an essential gene in bacteria and fungi, but is absent in humans, rhizocticin and plumbemycin are attractive as potential antimicrobials. In Chapter 4, I present data that provide insight into the mechanism of inhibition of ThrC by APPA. In addition, our data shed light on how the producing organisms overcome APPA toxicity and attain resistance.","Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2020-05-01","The student, Nektaria Petronikolou, accepted the attached license on 2018-02-16 at 17:51."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Structural and biochemical studies of enzymes involved in the biosynthesis of value-added products"]}]}],"canonical_facts":{"dc:contributor":["Nair, Satish K.","Cronan, John E.","Zhao, Huimin","Jin, Hong"],"dc:creator":["Petronikolou, Nektaria"],"dc:date":["2018-09-04T20:46:48Z","2020-09-05T09:15:26Z","2018-03-28","2018-05"],"dc:description":["The student, Nektaria Petronikolou, submitted this Dissertation for approval on 2018-02-16 at 18:00.","This Dissertation was approved for publication on 2018-03-28 at 15:31.","DSpace SAF Submission Ingestion Package generated from Vireo submission #12041 on 2018-08-31 at 17:25:10","Made available in DSpace on 2018-09-04T20:46:48Z (GMT). No. of bitstreams: 3 PETRONIKOLOU-DISSERTATION-2018.pdf: 5586351 bytes, checksum: 4ccc9c8c8a66f0350c40658ca9c55631 (MD5) Petronikolou_Dissertation_corrected_final.docx: 14970926 bytes, checksum: 8910cffce5cf54d2aca49ae6894290d7 (MD5) LICENSE.txt: 4218 bytes, checksum: c41f7ffe280f5c8e05272d6c9f960842 (MD5) Previous issue date: 2018-03-28","Embargo set by: Seth Robbins for item 107344 Lift date: 2020-09-04T20:47:38Z Reason: Author requested closed access (OA after 2yrs) in Vireo ETD system","Embargo set by: Seth Robbins for item 107344 Lift date: 2020-09-04T20:50:11Z Reason: Author requested closed access (OA after 2yrs) in Vireo ETD system","Limited Restriction Lifted for Item 107344 on 2020-09-05T09:15:26Z.","Enzymes have been used for decades for the industrial production of high value chemicals such as food additives, antibiotics and other pharmaceutical products. However, in recent years the significant advances in biocatalyst development have led to a rise in the number of commodity chemicals being produced by enzymes instead of extraction from plants or chemical synthesis. Such products include biodiesel and wax esters. For this industrial exploitation of enzymes, it is vital to have an extensive understanding of how the desired enzymes work, including what the enzymes’ substrate preferences and stability are. This knowledge comes from the structural and biochemical characterization of these biocatalysts, and this is the scope of this dissertation; to broaden our knowledge and understanding of enzymes that are involved in the biosynthesis of value-added products, such as biodiesel and drugs. Biodiesel is a mixture of long chain fatty acid alkyl esters (FAAEs), produced mainly by the transesterification of fatty acids derived from vegetable oils and animal fats. In recent years, biodiesel has emerged as a viable resource for utilization in green energy production. However, current production processes utilize methods that are cost ineffective and generate waste byproducts. Recent progress in the field makes use of biocatalysts that are effective in diverting products from primary metabolism to yield fatty acid methyl and ethyl esters in bacterial systems (as well as other commodity chemicals). Two of these enzymes have been the focus of my study: a fatty acid O-methyltransferase (FAMT) from Mycobacterium marinum (MmFAMT), and a bifunctional wax ester synthase/diacylglycerol acyl-transferase (WS/DGAT) from Marinobacter aquaeolei VT8 (Ma-WS/DGAT). In Chapter 1, I present the first kinetic and structural characterization of a mycobacterial fatty acid O-methyltransferase (MmFAMT) previously utilized for the production of biodiesel in E. coli. The structure revealed unexpected similarity with methyltransferases from plant natural product metabolism, and an active-site cavity that can accommodate fatty acids up to 12-14 carbons long. Our results provide the framework for further optimization of an in vivo system that utilizes MmFAMT for production of biodiesel. In Chapter 2, I discuss the structure of a bifunctional wax ester synthase/diacylglycerol acyl-transferase (Ma-WS/DGAT), which is to our knowledge the first structure for any member of this enzyme family to date. Wax esters (WE) are esters of long chain fatty acids and long chain alcohols and have widely been used in cosmetics, lubricants, candles and the food industry. In addition, WE that consist of medium chain fatty acids and ethanol (fatty acid ethyl esters, FAEEs) are biodiesel. Today, there is a strong demand for the development of a large-scale process for the production of low cost WE. The structure may facilitate future engineering efforts aiming at the optimization of this biocatalyst towards production of desired wax esters for industrial use. In addition to biodiesel, value-added chemicals produced by enzymes include bioactive compounds such as chemotherapeutics and antibiotics. In Chapters 3 and 4, I present structural and biochemical data that expand our knowledge for enzymes involved in the biosynthesis of such compounds. In Chapter 3, I discuss the molecular basis for the substrate specificity of loganic acid O-methyltransferase from Catharanthus roseus (CraLAMT), an enzyme that methylates with high stereospecificity an iridoid glycoside to produce loganin. Loganin is an intermediate in the biosynthesis of indole alkaloids in plants - a group of plant natural products with several medicinal uses, and has attracted interest as a bioactive compound itself. Our results may facilitate future engineering efforts to diversify the CraLAMT substrate scaffold towards the design of new potential bioactive compounds. In Chapter 4, I present structural and biochemical data for enzymes that are involved in the biosynthesis of phosphonates. Phosphonates are natural products containing a carbon-phosphorous (C-P) bond. These C-P containing molecules inhibit enzymes which utilize phosphate esters and carboxylic acids by mimicking their substrates. At a time when drug-resistant pathogens pose serious threat to human health, phosphonate natural products provide a pool of potential therapeutic candidates. Two such phosphonate containing compounds are rhizocticin and plumbemycin. Rhizocticin and plumbemycin are di- or tripeptides that contain the same phosphonic amino acid at their C-termini, but differ at their N-termini. This unusual amino acid is (Z)-L-2-amino-5-phosphono-3-pentenoic acid (APPA), and is the active part of rhizocticin and plumbemycin. Upon entering the cell, host peptidases release APPA which subsequently inhibits threonine synthase (ThrC) and kills the cell. Because thrC is an essential gene in bacteria and fungi, but is absent in humans, rhizocticin and plumbemycin are attractive as potential antimicrobials. In Chapter 4, I present data that provide insight into the mechanism of inhibition of ThrC by APPA. In addition, our data shed light on how the producing organisms overcome APPA toxicity and attain resistance.","Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2020-05-01","The student, Nektaria Petronikolou, accepted the attached license on 2018-02-16 at 17:51."],"dc:format":["application/pdf"],"dc:identifier":["http://hdl.handle.net/2142/101259"],"dc:language":["en"],"dc:rights":["Copyright 2018 Nektaria Petronikolou"],"dc:subject":["biocatalysis","biofuels","drug resistance"],"dc:title":["Structural and biochemical studies of enzymes involved in the biosynthesis of value-added products"],"dc:type":["text"],"thesis:degree_discipline":["Biochemistry"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:24:38Z"}