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The Key to the Nornicotine Enantiomeric Composition in Tobacco Leaf University of Kentucky UKnowledge Theses and Dissertations--Plant and Soil Sciences Plant and Soil Sciences 2012 ENANTIOSELECTIVE DEMETHYLATION: THE KEY TO THE NORNICOTINE ENANTIOMERIC COMPOSITION IN TOBACCO LEAF Bin Cai University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Cai, Bin, "ENANTIOSELECTIVE DEMETHYLATION: THE KEY TO THE NORNICOTINE ENANTIOMERIC COMPOSITION IN TOBACCO LEAF" (2012). Theses and Dissertations--Plant and Soil Sciences. 5. https://uknowledge.uky.edu/pss_etds/5 This Doctoral Dissertation is brought to you for free and open access by the Plant and Soil Sciences at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Plant and Soil Sciences by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained and attached hereto needed written permission statements(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine). I hereby grant to The University of Kentucky and its agents the non-exclusive license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless a preapproved embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s dissertation including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Bin Cai, Student Dr. Lowell Bush, Major Professor Dr. Arthur G Hunt, Director of Graduate Studies ENANTIOSELECTIVE DEMETHYLATION: THE KEY TO THE NORNICOTINE ENANTIOMERIC COMPOSITION IN TOBACCO LEAF DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Agriculture at the University of Kentucky By Bin Cai Lexington, Kentucky Director: Dr. Lowell Bush, Professor of Plant Physiology Lexington, Kentucky 2012 Copyright © Bin Cai 2012 ABSTRACT OF DISSERTATION ENANTIOSELECTIVE DEMETHYLATION: THE KEY TO THE NORNICOTINE ENANTIOMERIC COMPOSITION IN TOBACCO LEAF Nicotine and nornicotine are the two main alkaloids that accumulate in Nicotiana tabacum L. (tobacco), and nornicotine is the N-demethylation metabolite of nicotine. Nicotine is synthesized in the root, and probably primarily in the root tip. Both nicotine and nornicotine exist as two isomers that differ from each other by the orientation of H atom at the C-2' position on the pyrrolidine ring. (S)-nicotine is the dominant form in tobacco leaf and the enantiomer fraction of nicotine (EFnic), the fraction of (R)- enantiomer over the total nicotine, is approximately 0.002. Despite considerable efforts to elucidate nicotine and nornicotine related metabolism, a comprehensive understanding of the factors responsible for regulating the variable EF for nornicotine (0.04 to 0.75 ) relative to nicotine has been lacking. The objectives of these investigations were to understand the mechanisms behind the discrepancy. There are three nicotine demethylases reported to be active in tobacco. In vitro recombinant CYP82E4, CYP82E5v2 and CYP82E10 demethylated (R)-nicotine three, ten and ten-fold faster than (S)-nicotine, respectively, and no racemization was observed in either nicotine or nornicotine during demethylation. To confirm these in vitro results, the accumulation and demethylation of nicotine enantiomers throughout the growth cycle and curing process were investigated. Scion stock grafts were used to separate the contributions of roots (source) from leaves (sink) to the final accumulation of nicotine and nornicotine in leaf. The results indicate that nicotine consists of 4% of the R enantiomer (0.04 EFnic) when synthesized. However, (R)-nicotine is selectively demethylated by CYP82E4, CYP82E5 and CYP82E10, resulting in an approximate 0.01 EFnic and 0.60 EFnnic in the root. After most of (R)-nicotine is demethylated in root, nicotine and nornicotine are translocated to leaf, where nicotine is further demethylated. Depending on the CYP82E4 activity, an EFnnic of 0.04 to 0.60 is produced and only 0.2% of the remaining nicotine in the leaf is (R)-configuration. Keywords: (R)-nicotine, nornicotine, cytochrome P450, enantioselectivity, tobacco Bin Cai Student‘s Signature July 13, 2012 Date ENANTIOSELECTIVE DEMETHYLATION: THE KEY TO THE NORNICOTINE ENANTIOMERIC COMPOSITION IN TOBACCO LEAF By Bin Cai Lowell Bush Director of Dissertation Arthur G Hunt Director of Graduate Studies July 13, 2012 ACKNOWLEDGMENTS While an individual work, the following dissertation benefited from the insights and direction of many people. Name Comments Lowell Bush Dissertation chair, mentor, inspiration and guidance thoughout my graduate study Bob Pearce Committee member, valuable suggestions Hongyan Zhu Committee member, valuable suggestions Joe Chappell Committee member, valuable suggestions, provided tools and ideas critical for the experiments in Chpater 3 Sharyn Perry Committee member, valuable suggestions Neil Fannin Lab member, technical support Huihua Ji Lab member, technical support Wenbin Yang Lab member, technical support Anne Jack Lab member, help me design and execute experiments in the field Colin Fisher Lab member, help me design and execute experiments in the field Balazs Siminszky Provided yeast line overexpressing CYP82E4 and CYP82E5v2 enzyme Ralph Dewey Professor at North Carolina State University, provided yeast line overexpressing CYP82E10 enzyme Ramsey Lewis Associate professor at North Carolina State University, provided nicotine demethylase mutant seeds iii List of Contents TITLE .................................................................................................................................. 1 ABSTRACT OF DISSERTATION .................................................................................. III ACKNOWLEDGMENTS ................................................................................................. iii List of Contents .................................................................................................................. iv List of Tables ................................................................................................................... viii List of Figures .................................................................................................................... ix Chapter 1. Literature review ............................................................................................... 1 1.1. Tobacco pyridine alkaloids and derivatives ............................................................. 1 1.2. Biosynthesis of the four main alkaloids in tobacco.................................................. 3 1.2.1. Pyridine, pyrrolidine and piperidine ring formation ......................................... 3 1.2.2. Nicotine, nornicotine, anabasine and anatabine formation ............................... 4 1.3. Nicotine demethylation in tobacco ........................................................................... 7 1.3.1. Nicotine demethylation mechanism .................................................................. 7 1.3.2. Structure and function studies of tobacco nicotine demethylase enzymes ....... 8 1.3.3. Nicotine demethylase specificity .................................................................... 10 1.3.4. Evolution of nicotine demethylases in N. tabacum L. .................................... 12 1.4. Theoretical studies of enzymes responsible for nicotine metabolism .................... 13 1.4.1. Cytochrome P450............................................................................................ 13 1.4.2. Model studies of CYP-catalyzed nicotine demethylation ............................... 15 1.5. Compartmentation and trafficking of nicotine ....................................................... 20 1.5.1. Long distance translocation from roots to leaves ........................................... 20 1.5.2. Cell type .......................................................................................................... 23 1.6. Pharmacological effects of enantiomers of nicotine and TSNAs .......................... 23 1.6.1. Pharmacological effects of nicotine and TSNAs ............................................ 24 iv 1.6.2. Pharmacological effects of the enantiomers of nicotine and TSNAs ............. 24 1.7. Experimental aim of this dissertation ..................................................................... 27 Chapter 2. Variable nornicotine enantiomeric composition caused by nicotine demethylase CYP82E4 in tobacco leaf ............................................................................
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