Scanning Chimeragenesis: The Approach Used to Change Monoxygenase Cytochrome P450 BM3 into ω-Hydroxylase CYP4C7

Persistent Link:
http://hdl.handle.net/10150/195451
Title:
Scanning Chimeragenesis: The Approach Used to Change Monoxygenase Cytochrome P450 BM3 into ω-Hydroxylase CYP4C7
Author:
Chen, Chiung-Kuang
Issue Date:
2007
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
It is believed that the specificity of cytochrome P450 is determined by a specific set of protein fragments that form the Substrate Recognition Site (SRS-1) and are responsible for a particular orientation of the bound substrate relative to the activated oxygen atom. Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium, is known for its high catalytic activity. Wild type BM3 catalyzes the oxidation of medium and long chain fatty acids (C12-18), and of farnesol, but the two form different metabolites, forms 9-hydroxyfarnesol and 10,11- and 2,3-epoxyfarnesol in a ratio of 3:3:2 and 12-hydroxyfarnesol, respectively. CYP102A1 and CYP4C7 share a common substrate, farnesol. Therefore, CYP4C7 has become the target for homologous replacements in CYP102A1. CYP4C7 from Diploptera Punctata (Pacific Beetle Cockroach) only catalyzes farnesol to produce 12-hydroxyfarnesol as its primary metabolite, with no activity towards fatty acids. By using the technique of scanning chimeragenesis, in this work three generations of chimeras have created twenty chimeric proteins. By starting with CYP102A1 as the experimental model and employing sequential rounds of selective mutagenesis, the third generation mutant C(78-82,F87L,328-330) was produced, which catalyzed the 12- and 15-hydroxylation of farnesol as its major products in a 3:1 ratio with a hundred-fold increase in catalytic activity compared to the wild type CYP4C7, and a two-fold increase over CYP102A1. Based on the activity assay results for the chimeric proteins with substrates geranyl-geraniol, 10,11-epoxymethylfarnesoate (JH III), methylfarnesoate, farnesol, geraniol, 3,7-dimethyl-1-octanol, and lauric and palmitic acids, most chimeric proteins showed a change in substrate selectivity and/or regiospecificity. Scanning chimeragenesis can be used as a tool to not only study the relationship between the protein fragments that form the substrate binding site, but also to help elucidate the roles of substrate selectivity and regiospecificity among any two cytochromes P450. Furthermore, this investigation has resulted in the production of highly efficient chimeric enzymes that have previously evaded other methods of sequence modification by mutagenesis or directed evolution and chemical synthesis.
Type:
text; Electronic Dissertation
Degree Name:
PhD
Degree Level:
doctoral
Degree Program:
Chemistry; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Walker, Francis A
Committee Chair:
Walker, Francis A

Full metadata record

DC FieldValue Language
dc.language.isoENen_US
dc.titleScanning Chimeragenesis: The Approach Used to Change Monoxygenase Cytochrome P450 BM3 into ω-Hydroxylase CYP4C7en_US
dc.creatorChen, Chiung-Kuangen_US
dc.contributor.authorChen, Chiung-Kuangen_US
dc.date.issued2007en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractIt is believed that the specificity of cytochrome P450 is determined by a specific set of protein fragments that form the Substrate Recognition Site (SRS-1) and are responsible for a particular orientation of the bound substrate relative to the activated oxygen atom. Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium, is known for its high catalytic activity. Wild type BM3 catalyzes the oxidation of medium and long chain fatty acids (C12-18), and of farnesol, but the two form different metabolites, forms 9-hydroxyfarnesol and 10,11- and 2,3-epoxyfarnesol in a ratio of 3:3:2 and 12-hydroxyfarnesol, respectively. CYP102A1 and CYP4C7 share a common substrate, farnesol. Therefore, CYP4C7 has become the target for homologous replacements in CYP102A1. CYP4C7 from Diploptera Punctata (Pacific Beetle Cockroach) only catalyzes farnesol to produce 12-hydroxyfarnesol as its primary metabolite, with no activity towards fatty acids. By using the technique of scanning chimeragenesis, in this work three generations of chimeras have created twenty chimeric proteins. By starting with CYP102A1 as the experimental model and employing sequential rounds of selective mutagenesis, the third generation mutant C(78-82,F87L,328-330) was produced, which catalyzed the 12- and 15-hydroxylation of farnesol as its major products in a 3:1 ratio with a hundred-fold increase in catalytic activity compared to the wild type CYP4C7, and a two-fold increase over CYP102A1. Based on the activity assay results for the chimeric proteins with substrates geranyl-geraniol, 10,11-epoxymethylfarnesoate (JH III), methylfarnesoate, farnesol, geraniol, 3,7-dimethyl-1-octanol, and lauric and palmitic acids, most chimeric proteins showed a change in substrate selectivity and/or regiospecificity. Scanning chimeragenesis can be used as a tool to not only study the relationship between the protein fragments that form the substrate binding site, but also to help elucidate the roles of substrate selectivity and regiospecificity among any two cytochromes P450. Furthermore, this investigation has resulted in the production of highly efficient chimeric enzymes that have previously evaded other methods of sequence modification by mutagenesis or directed evolution and chemical synthesis.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
thesis.degree.namePhDen_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorWalker, Francis Aen_US
dc.contributor.chairWalker, Francis Aen_US
dc.contributor.committeememberMash, Eugene A.en_US
dc.contributor.committeememberMiranda, Katrina M.en_US
dc.contributor.committeememberOlenyuk, Bogdan Z.en_US
dc.contributor.committeememberZheng, Zhipingen_US
dc.identifier.proquest2378en_US
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