ingest cdrApp 2017-07-06T13:02:36.911Z f230b17a-68de-497f-ac05-5cb17af9fe4f modifyDatastreamByValue RELS-EXT cdrApp 2017-07-06T13:18:35.663Z Setting exclusive relation modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-01-25T04:44:04.404Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-01-27T05:23:20.000Z modifyDatastreamByValue RELS-EXT fedoraAdmin 2018-02-09T08:41:44.003Z Setting exclusive relation addDatastream MD_TECHNICAL fedoraAdmin 2018-02-09T08:41:55.211Z Adding technical metadata derived by FITS addDatastream MD_FULL_TEXT fedoraAdmin 2018-02-09T08:42:17.576Z Adding full text metadata extracted by Apache Tika modifyDatastreamByValue RELS-EXT fedoraAdmin 2018-02-09T08:42:39.889Z Setting exclusive relation modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-03-14T01:26:07.160Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-05-16T22:48:31.029Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-07-10T23:53:55.254Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-07-17T19:55:18.175Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-08-08T19:22:34.533Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-08-15T16:30:50.928Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-08-16T19:33:51.907Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-09-21T16:59:30.575Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-09-26T20:08:20.339Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-10-11T20:48:06.148Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2019-03-20T14:05:41.298Z Joshua Gober Author Department of Chemistry College of Arts and Sciences RATIONAL ENGINEERING OF CYTOCHROME P450 ENZYMES Cytochrome P450s, which typically catalyze oxidation reactions via an iron-oxo species, have recently been reported to cyclopropanate alkenes in the presence of diazoacetate reagents through formation of an iron carbenoid. Stereoselective enzymes for carbenoid insertion into aryl olefins have been reported; however, engineering selective variants for all of the possible isomers of the reaction remains a challenge. Earlier work with a model P450 (P450 BM3) reported a highly activating mutation of a conserved active site threonine to alanine that resulted in dramatic improvements in enantioselectivity and diastereoselectivity for the model reaction of styrene with ethyl diazoacetate. This work demonstrates that by incorporating this single mutation into a diverse panel of a dozen P450s from various microorganisms, enantioselective and diastereoselective catalysts can be quickly identified for all isomers in the reaction of styrene with ethyl diazoacetate. This work also demonstrates the utility of intermolecular, P450-mediated olefin cyclopropanation for selective, late-stage modification of complex natural products. In this study, a diverse set of engineered P450s were found to catalyze cyclopropanation of dehydroalanines (Dhas), which are commonly found in natural products. P450s involved in the biosynthesis of a pyridine-containing thiopeptide, thiomuracin GZ, were found to catalyze cyclopropanation of thiomuracin derivatives engineered to display Dhas at key residues. This work presents a strategy for chemoselective and stereoselective modification of complex natural products using a rational engineering approach. Spring 2017 2017 Chemistry Biochemistry biocatalysis, cyclopropanation, cytochrome P450, enzyme engineering eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Chemistry Eric Brustad Thesis advisor Marcey Waters Thesis advisor Jeffrey Johnson Thesis advisor Bo Li Thesis advisor Gary Pielak Thesis advisor text Joshua Gober Creator Department of Chemistry College of Arts and Sciences RATIONAL ENGINEERING OF CYTOCHROME P450 ENZYMES Cytochrome P450s, which typically catalyze oxidation reactions via an iron-oxo species, have recently been reported to cyclopropanate alkenes in the presence of diazoacetate reagents through formation of an iron carbenoid. Stereoselective enzymes for carbenoid insertion into aryl olefins have been reported; however, engineering selective variants for all of the possible isomers of the reaction remains a challenge. Earlier work with a model P450 (P450 BM3) reported a highly activating mutation of a conserved active site threonine to alanine that resulted in dramatic improvements in enantioselectivity and diastereoselectivity for the model reaction of styrene with ethyl diazoacetate. This work demonstrates that by incorporating this single mutation into a diverse panel of a dozen P450s from various microorganisms, enantioselective and diastereoselective catalysts can be quickly identified for all isomers in the reaction of styrene with ethyl diazoacetate. This work also demonstrates the utility of intermolecular, P450-mediated olefin cyclopropanation for selective, late-stage modification of complex natural products. In this study, a diverse set of engineered P450s were found to catalyze cyclopropanation of dehydroalanines (Dhas), which are commonly found in natural products. P450s involved in the biosynthesis of a pyridine-containing thiopeptide, thiomuracin GZ, were found to catalyze cyclopropanation of thiomuracin derivatives engineered to display Dhas at key residues. This work presents a strategy for chemoselective and stereoselective modification of complex natural products using a rational engineering approach. Spring 2017 2017 Chemistry Biochemistry biocatalysis, cyclopropanation, cytochrome P450, enzyme engineering eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Chemistry Eric Brustad Thesis advisor Marcey Waters Thesis advisor Jeffrey Johnson Thesis advisor Bo Li Thesis advisor Gary Pielak Thesis advisor text Joshua Gober Creator Department of Chemistry College of Arts and Sciences RATIONAL ENGINEERING OF CYTOCHROME P450 ENZYMES Cytochrome P450s, which typically catalyze oxidation reactions via an iron-oxo species, have recently been reported to cyclopropanate alkenes in the presence of diazoacetate reagents through formation of an iron carbenoid. Stereoselective enzymes for carbenoid insertion into aryl olefins have been reported; however, engineering selective variants for all of the possible isomers of the reaction remains a challenge. Earlier work with a model P450 (P450 BM3) reported a highly activating mutation of a conserved active site threonine to alanine that resulted in dramatic improvements in enantioselectivity and diastereoselectivity for the model reaction of styrene with ethyl diazoacetate. This work demonstrates that by incorporating this single mutation into a diverse panel of a dozen P450s from various microorganisms, enantioselective and diastereoselective catalysts can be quickly identified for all isomers in the reaction of styrene with ethyl diazoacetate. This work also demonstrates the utility of intermolecular, P450-mediated olefin cyclopropanation for selective, late-stage modification of complex natural products. In this study, a diverse set of engineered P450s were found to catalyze cyclopropanation of dehydroalanines (Dhas), which are commonly found in natural products. P450s involved in the biosynthesis of a pyridine-containing thiopeptide, thiomuracin GZ, were found to catalyze cyclopropanation of thiomuracin derivatives engineered to display Dhas at key residues. This work presents a strategy for chemoselective and stereoselective modification of complex natural products using a rational engineering approach. Spring 2017 2017 Chemistry Biochemistry biocatalysis, cyclopropanation, cytochrome P450, enzyme engineering eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Chemistry Eric Brustad Thesis advisor Marcey Waters Thesis advisor Jeffrey Johnson Thesis advisor Bo Li Thesis advisor Gary Pielak Thesis advisor text Joshua Gober Creator Department of Chemistry College of Arts and Sciences RATIONAL ENGINEERING OF CYTOCHROME P450 ENZYMES Cytochrome P450s, which typically catalyze oxidation reactions via an iron-oxo species, have recently been reported to cyclopropanate alkenes in the presence of diazoacetate reagents through formation of an iron carbenoid. Stereoselective enzymes for carbenoid insertion into aryl olefins have been reported; however, engineering selective variants for all of the possible isomers of the reaction remains a challenge. Earlier work with a model P450 (P450 BM3) reported a highly activating mutation of a conserved active site threonine to alanine that resulted in dramatic improvements in enantioselectivity and diastereoselectivity for the model reaction of styrene with ethyl diazoacetate. This work demonstrates that by incorporating this single mutation into a diverse panel of a dozen P450s from various microorganisms, enantioselective and diastereoselective catalysts can be quickly identified for all isomers in the reaction of styrene with ethyl diazoacetate. This work also demonstrates the utility of intermolecular, P450-mediated olefin cyclopropanation for selective, late-stage modification of complex natural products. In this study, a diverse set of engineered P450s were found to catalyze cyclopropanation of dehydroalanines (Dhas), which are commonly found in natural products. P450s involved in the biosynthesis of a pyridine-containing thiopeptide, thiomuracin GZ, were found to catalyze cyclopropanation of thiomuracin derivatives engineered to display Dhas at key residues. This work presents a strategy for chemoselective and stereoselective modification of complex natural products using a rational engineering approach. 2017-05 2017 Chemistry Biochemistry biocatalysis, cyclopropanation, cytochrome P450, enzyme engineering eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Chemistry Eric Brustad Thesis advisor Marcey Waters Thesis advisor Jeffrey Johnson Thesis advisor Bo Li Thesis advisor Gary Pielak Thesis advisor text Joshua Gober Creator Department of Chemistry College of Arts and Sciences RATIONAL ENGINEERING OF CYTOCHROME P450 ENZYMES Cytochrome P450s, which typically catalyze oxidation reactions via an iron-oxo species, have recently been reported to cyclopropanate alkenes in the presence of diazoacetate reagents through formation of an iron carbenoid. Stereoselective enzymes for carbenoid insertion into aryl olefins have been reported; however, engineering selective variants for all of the possible isomers of the reaction remains a challenge. Earlier work with a model P450 (P450 BM3) reported a highly activating mutation of a conserved active site threonine to alanine that resulted in dramatic improvements in enantioselectivity and diastereoselectivity for the model reaction of styrene with ethyl diazoacetate. This work demonstrates that by incorporating this single mutation into a diverse panel of a dozen P450s from various microorganisms, enantioselective and diastereoselective catalysts can be quickly identified for all isomers in the reaction of styrene with ethyl diazoacetate. This work also demonstrates the utility of intermolecular, P450-mediated olefin cyclopropanation for selective, late-stage modification of complex natural products. In this study, a diverse set of engineered P450s were found to catalyze cyclopropanation of dehydroalanines (Dhas), which are commonly found in natural products. P450s involved in the biosynthesis of a pyridine-containing thiopeptide, thiomuracin GZ, were found to catalyze cyclopropanation of thiomuracin derivatives engineered to display Dhas at key residues. This work presents a strategy for chemoselective and stereoselective modification of complex natural products using a rational engineering approach. 2017 Chemistry Biochemistry biocatalysis, cyclopropanation, cytochrome P450, enzyme engineering eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Chemistry Eric Brustad Thesis advisor Marcey Waters Thesis advisor Jeffrey Johnson Thesis advisor Bo Li Thesis advisor Gary Pielak Thesis advisor text 2017-05 Joshua Gober Creator Department of Chemistry College of Arts and Sciences RATIONAL ENGINEERING OF CYTOCHROME P450 ENZYMES Cytochrome P450s, which typically catalyze oxidation reactions via an iron-oxo species, have recently been reported to cyclopropanate alkenes in the presence of diazoacetate reagents through formation of an iron carbenoid. Stereoselective enzymes for carbenoid insertion into aryl olefins have been reported; however, engineering selective variants for all of the possible isomers of the reaction remains a challenge. Earlier work with a model P450 (P450 BM3) reported a highly activating mutation of a conserved active site threonine to alanine that resulted in dramatic improvements in enantioselectivity and diastereoselectivity for the model reaction of styrene with ethyl diazoacetate. This work demonstrates that by incorporating this single mutation into a diverse panel of a dozen P450s from various microorganisms, enantioselective and diastereoselective catalysts can be quickly identified for all isomers in the reaction of styrene with ethyl diazoacetate. This work also demonstrates the utility of intermolecular, P450-mediated olefin cyclopropanation for selective, late-stage modification of complex natural products. In this study, a diverse set of engineered P450s were found to catalyze cyclopropanation of dehydroalanines (Dhas), which are commonly found in natural products. P450s involved in the biosynthesis of a pyridine-containing thiopeptide, thiomuracin GZ, were found to catalyze cyclopropanation of thiomuracin derivatives engineered to display Dhas at key residues. This work presents a strategy for chemoselective and stereoselective modification of complex natural products using a rational engineering approach. 2017 Chemistry Biochemistry biocatalysis, cyclopropanation, cytochrome P450, enzyme engineering eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Chemistry Eric Brustad Thesis advisor Marcey Waters Thesis advisor Jeffrey Johnson Thesis advisor Bo Li Thesis advisor Gary Pielak Thesis advisor text 2017-05 Joshua Gober Creator Department of Chemistry College of Arts and Sciences RATIONAL ENGINEERING OF CYTOCHROME P450 ENZYMES Cytochrome P450s, which typically catalyze oxidation reactions via an iron-oxo species, have recently been reported to cyclopropanate alkenes in the presence of diazoacetate reagents through formation of an iron carbenoid. Stereoselective enzymes for carbenoid insertion into aryl olefins have been reported; however, engineering selective variants for all of the possible isomers of the reaction remains a challenge. Earlier work with a model P450 (P450 BM3) reported a highly activating mutation of a conserved active site threonine to alanine that resulted in dramatic improvements in enantioselectivity and diastereoselectivity for the model reaction of styrene with ethyl diazoacetate. This work demonstrates that by incorporating this single mutation into a diverse panel of a dozen P450s from various microorganisms, enantioselective and diastereoselective catalysts can be quickly identified for all isomers in the reaction of styrene with ethyl diazoacetate. This work also demonstrates the utility of intermolecular, P450-mediated olefin cyclopropanation for selective, late-stage modification of complex natural products. In this study, a diverse set of engineered P450s were found to catalyze cyclopropanation of dehydroalanines (Dhas), which are commonly found in natural products. P450s involved in the biosynthesis of a pyridine-containing thiopeptide, thiomuracin GZ, were found to catalyze cyclopropanation of thiomuracin derivatives engineered to display Dhas at key residues. This work presents a strategy for chemoselective and stereoselective modification of complex natural products using a rational engineering approach. 2017 Chemistry Biochemistry biocatalysis, cyclopropanation, cytochrome P450, enzyme engineering eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Chemistry Eric Brustad Thesis advisor Marcey Waters Thesis advisor Jeffrey Johnson Thesis advisor Bo Li Thesis advisor Gary Pielak Thesis advisor text 2017-05 Joshua Gober Creator Department of Chemistry College of Arts and Sciences RATIONAL ENGINEERING OF CYTOCHROME P450 ENZYMES Cytochrome P450s, which typically catalyze oxidation reactions via an iron-oxo species, have recently been reported to cyclopropanate alkenes in the presence of diazoacetate reagents through formation of an iron carbenoid. Stereoselective enzymes for carbenoid insertion into aryl olefins have been reported; however, engineering selective variants for all of the possible isomers of the reaction remains a challenge. Earlier work with a model P450 (P450 BM3) reported a highly activating mutation of a conserved active site threonine to alanine that resulted in dramatic improvements in enantioselectivity and diastereoselectivity for the model reaction of styrene with ethyl diazoacetate. This work demonstrates that by incorporating this single mutation into a diverse panel of a dozen P450s from various microorganisms, enantioselective and diastereoselective catalysts can be quickly identified for all isomers in the reaction of styrene with ethyl diazoacetate. This work also demonstrates the utility of intermolecular, P450-mediated olefin cyclopropanation for selective, late-stage modification of complex natural products. In this study, a diverse set of engineered P450s were found to catalyze cyclopropanation of dehydroalanines (Dhas), which are commonly found in natural products. P450s involved in the biosynthesis of a pyridine-containing thiopeptide, thiomuracin GZ, were found to catalyze cyclopropanation of thiomuracin derivatives engineered to display Dhas at key residues. This work presents a strategy for chemoselective and stereoselective modification of complex natural products using a rational engineering approach. 2017 Chemistry Biochemistry biocatalysis, cyclopropanation, cytochrome P450, enzyme engineering eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Chemistry Eric Brustad Thesis advisor Marcey Waters Thesis advisor Jeffrey Johnson Thesis advisor Bo Li Thesis advisor Gary J. Pielak Thesis advisor text 2017-05 Joshua Gober Creator Department of Chemistry College of Arts and Sciences RATIONAL ENGINEERING OF CYTOCHROME P450 ENZYMES Cytochrome P450s, which typically catalyze oxidation reactions via an iron-oxo species, have recently been reported to cyclopropanate alkenes in the presence of diazoacetate reagents through formation of an iron carbenoid. Stereoselective enzymes for carbenoid insertion into aryl olefins have been reported; however, engineering selective variants for all of the possible isomers of the reaction remains a challenge. Earlier work with a model P450 (P450 BM3) reported a highly activating mutation of a conserved active site threonine to alanine that resulted in dramatic improvements in enantioselectivity and diastereoselectivity for the model reaction of styrene with ethyl diazoacetate. This work demonstrates that by incorporating this single mutation into a diverse panel of a dozen P450s from various microorganisms, enantioselective and diastereoselective catalysts can be quickly identified for all isomers in the reaction of styrene with ethyl diazoacetate. This work also demonstrates the utility of intermolecular, P450-mediated olefin cyclopropanation for selective, late-stage modification of complex natural products. In this study, a diverse set of engineered P450s were found to catalyze cyclopropanation of dehydroalanines (Dhas), which are commonly found in natural products. P450s involved in the biosynthesis of a pyridine-containing thiopeptide, thiomuracin GZ, were found to catalyze cyclopropanation of thiomuracin derivatives engineered to display Dhas at key residues. This work presents a strategy for chemoselective and stereoselective modification of complex natural products using a rational engineering approach. 2017 Chemistry Biochemistry biocatalysis, cyclopropanation, cytochrome P450, enzyme engineering eng Doctor of Philosophy Dissertation Chemistry Eric Brustad Thesis advisor Marcey Waters Thesis advisor Jeffrey Johnson Thesis advisor Bo Li Thesis advisor Gary J. Pielak Thesis advisor text 2017-05 University of North Carolina at Chapel Hill Degree granting institution Joshua Gober Creator Department of Chemistry College of Arts and Sciences RATIONAL ENGINEERING OF CYTOCHROME P450 ENZYMES Cytochrome P450s, which typically catalyze oxidation reactions via an iron-oxo species, have recently been reported to cyclopropanate alkenes in the presence of diazoacetate reagents through formation of an iron carbenoid. Stereoselective enzymes for carbenoid insertion into aryl olefins have been reported; however, engineering selective variants for all of the possible isomers of the reaction remains a challenge. Earlier work with a model P450 (P450 BM3) reported a highly activating mutation of a conserved active site threonine to alanine that resulted in dramatic improvements in enantioselectivity and diastereoselectivity for the model reaction of styrene with ethyl diazoacetate. This work demonstrates that by incorporating this single mutation into a diverse panel of a dozen P450s from various microorganisms, enantioselective and diastereoselective catalysts can be quickly identified for all isomers in the reaction of styrene with ethyl diazoacetate. This work also demonstrates the utility of intermolecular, P450-mediated olefin cyclopropanation for selective, late-stage modification of complex natural products. In this study, a diverse set of engineered P450s were found to catalyze cyclopropanation of dehydroalanines (Dhas), which are commonly found in natural products. P450s involved in the biosynthesis of a pyridine-containing thiopeptide, thiomuracin GZ, were found to catalyze cyclopropanation of thiomuracin derivatives engineered to display Dhas at key residues. This work presents a strategy for chemoselective and stereoselective modification of complex natural products using a rational engineering approach. 2017 Chemistry Biochemistry biocatalysis, cyclopropanation, cytochrome P450, enzyme engineering eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Chemistry Eric Brustad Thesis advisor Marcey Waters Thesis advisor Jeffrey Johnson Thesis advisor Bo Li Thesis advisor Gary Pielak Thesis advisor text 2017-05 Joshua Gober Creator Department of Chemistry College of Arts and Sciences RATIONAL ENGINEERING OF CYTOCHROME P450 ENZYMES Cytochrome P450s, which typically catalyze oxidation reactions via an iron-oxo species, have recently been reported to cyclopropanate alkenes in the presence of diazoacetate reagents through formation of an iron carbenoid. Stereoselective enzymes for carbenoid insertion into aryl olefins have been reported; however, engineering selective variants for all of the possible isomers of the reaction remains a challenge. Earlier work with a model P450 (P450 BM3) reported a highly activating mutation of a conserved active site threonine to alanine that resulted in dramatic improvements in enantioselectivity and diastereoselectivity for the model reaction of styrene with ethyl diazoacetate. This work demonstrates that by incorporating this single mutation into a diverse panel of a dozen P450s from various microorganisms, enantioselective and diastereoselective catalysts can be quickly identified for all isomers in the reaction of styrene with ethyl diazoacetate. This work also demonstrates the utility of intermolecular, P450-mediated olefin cyclopropanation for selective, late-stage modification of complex natural products. In this study, a diverse set of engineered P450s were found to catalyze cyclopropanation of dehydroalanines (Dhas), which are commonly found in natural products. P450s involved in the biosynthesis of a pyridine-containing thiopeptide, thiomuracin GZ, were found to catalyze cyclopropanation of thiomuracin derivatives engineered to display Dhas at key residues. This work presents a strategy for chemoselective and stereoselective modification of complex natural products using a rational engineering approach. 2017 Chemistry Biochemistry biocatalysis, cyclopropanation, cytochrome P450, enzyme engineering eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Chemistry Eric Brustad Thesis advisor Marcey Waters Thesis advisor Jeffrey Johnson Thesis advisor Bo Li Thesis advisor Gary Pielak Thesis advisor text 2017-05 Joshua Gober Creator Department of Chemistry College of Arts and Sciences RATIONAL ENGINEERING OF CYTOCHROME P450 ENZYMES Cytochrome P450s, which typically catalyze oxidation reactions via an iron-oxo species, have recently been reported to cyclopropanate alkenes in the presence of diazoacetate reagents through formation of an iron carbenoid. Stereoselective enzymes for carbenoid insertion into aryl olefins have been reported; however, engineering selective variants for all of the possible isomers of the reaction remains a challenge. Earlier work with a model P450 (P450 BM3) reported a highly activating mutation of a conserved active site threonine to alanine that resulted in dramatic improvements in enantioselectivity and diastereoselectivity for the model reaction of styrene with ethyl diazoacetate. This work demonstrates that by incorporating this single mutation into a diverse panel of a dozen P450s from various microorganisms, enantioselective and diastereoselective catalysts can be quickly identified for all isomers in the reaction of styrene with ethyl diazoacetate. This work also demonstrates the utility of intermolecular, P450-mediated olefin cyclopropanation for selective, late-stage modification of complex natural products. In this study, a diverse set of engineered P450s were found to catalyze cyclopropanation of dehydroalanines (Dhas), which are commonly found in natural products. P450s involved in the biosynthesis of a pyridine-containing thiopeptide, thiomuracin GZ, were found to catalyze cyclopropanation of thiomuracin derivatives engineered to display Dhas at key residues. This work presents a strategy for chemoselective and stereoselective modification of complex natural products using a rational engineering approach. 2017 Chemistry Biochemistry biocatalysis; cyclopropanation; cytochrome P450; enzyme engineering eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Chemistry Eric Brustad Thesis advisor Marcey Waters Thesis advisor Jeffrey Johnson Thesis advisor Bo Li Thesis advisor Gary Pielak Thesis advisor text 2017-05 Joshua Gober Creator Department of Chemistry College of Arts and Sciences RATIONAL ENGINEERING OF CYTOCHROME P450 ENZYMES Cytochrome P450s, which typically catalyze oxidation reactions via an iron-oxo species, have recently been reported to cyclopropanate alkenes in the presence of diazoacetate reagents through formation of an iron carbenoid. Stereoselective enzymes for carbenoid insertion into aryl olefins have been reported; however, engineering selective variants for all of the possible isomers of the reaction remains a challenge. Earlier work with a model P450 (P450 BM3) reported a highly activating mutation of a conserved active site threonine to alanine that resulted in dramatic improvements in enantioselectivity and diastereoselectivity for the model reaction of styrene with ethyl diazoacetate. This work demonstrates that by incorporating this single mutation into a diverse panel of a dozen P450s from various microorganisms, enantioselective and diastereoselective catalysts can be quickly identified for all isomers in the reaction of styrene with ethyl diazoacetate. This work also demonstrates the utility of intermolecular, P450-mediated olefin cyclopropanation for selective, late-stage modification of complex natural products. In this study, a diverse set of engineered P450s were found to catalyze cyclopropanation of dehydroalanines (Dhas), which are commonly found in natural products. P450s involved in the biosynthesis of a pyridine-containing thiopeptide, thiomuracin GZ, were found to catalyze cyclopropanation of thiomuracin derivatives engineered to display Dhas at key residues. This work presents a strategy for chemoselective and stereoselective modification of complex natural products using a rational engineering approach. 2017 Chemistry Biochemistry biocatalysis, cyclopropanation, cytochrome P450, enzyme engineering eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Chemistry Eric Brustad Thesis advisor Marcey Waters Thesis advisor Jeffrey Johnson Thesis advisor Bo Li Thesis advisor Gary J. Pielak Thesis advisor text 2017-05 Joshua Gober Creator Department of Chemistry College of Arts and Sciences Cytochrome P450s, which typically catalyze oxidation reactions via an iron-oxo species, have recently been reported to cyclopropanate alkenes in the presence of diazoacetate reagents through formation of an iron carbenoid. Stereoselective enzymes for carbenoid insertion into aryl olefins have been reported; however, engineering selective variants for all of the possible isomers of the reaction remains a challenge. Earlier work with a model P450 (P450 BM3) reported a highly activating mutation of a conserved active site threonine to alanine that resulted in dramatic improvements in enantioselectivity and diastereoselectivity for the model reaction of styrene with ethyl diazoacetate. This work demonstrates that by incorporating this single mutation into a diverse panel of a dozen P450s from various microorganisms, enantioselective and diastereoselective catalysts can be quickly identified for all isomers in the reaction of styrene with ethyl diazoacetate. This work also demonstrates the utility of intermolecular, P450-mediated olefin cyclopropanation for selective, late-stage modification of complex natural products. In this study, a diverse set of engineered P450s were found to catalyze cyclopropanation of dehydroalanines (Dhas), which are commonly found in natural products. P450s involved in the biosynthesis of a pyridine-containing thiopeptide, thiomuracin GZ, were found to catalyze cyclopropanation of thiomuracin derivatives engineered to display Dhas at key residues. This work presents a strategy for chemoselective and stereoselective modification of complex natural products using a rational engineering approach. 2017 Chemistry Biochemistry biocatalysis; cyclopropanation; cytochrome P450; enzyme engineering eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Eric Brustad Thesis advisor Marcey Waters Thesis advisor Jeffrey Johnson Thesis advisor Bo Li Thesis advisor Gary J. Pielak Thesis advisor text 2017-05 Gober_unc_0153D_16975.pdf uuid:6e699591-0ec4-4f73-809a-a6a157b7498e proquest 2019-07-06T00:00:00 2017-04-14T04:24:26Z yes application/pdf 13245059