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Elucidation of Novel N-Methyltransferases in Tropane and Granatane Alkaloid Biosynthesis

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Elucidation of Novel N-Methyltransferases in Tropane and Granatane Alkaloid Biosynthesis Elucidation of Novel N-methyltransferases in Tropane and Granatane Alkaloid Biosynthesis by Kayla Nicole Anderson, B. S. A Thesis In Chemistry Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCES Approved Dr. John C. D’Auria Chair of Committee Dr. Anthony F. Cozzolino Mark Sheridan Dean of the Graduate School August, 2019 Copyright 2019, Kayla Nicole Anderson Texas Tech University, Kayla Nicole Anderson, August 2019 ACKNOWLEDGMENTS I am grateful for the financial support that Texas Tech University granted to me through the AT&T Chancellor’s fellowship. I am also appreciative for the financial support from Texas Tech University and the National Science Foundation (NMR instrument grant CHE-1048553). This work was partially funded by the National Science Foundation under grant No. (NSF-171423326) to John C. D’Auria. Many thanks and appreciation to Dr. Anthony F. Cozzolino and Shiva Moaven for collaboration in the synthesis of methylated polyamines. I would also like to thank Nikke Tweet for help with the enzymatic synthesis of decarboxylated S-adenosyl-L-methionine. ii Texas Tech University, Kayla Nicole Anderson, August 2019 TABLE OF CONTENTS ACKNOWLEDGMENTS ................................................................................................ ii ABSTRACT .................................................................................................................... viii LIST OF TABLES ........................................................................................................... ix LIST OF FIGURES .......................................................................................................... x LIST OF ABBREVIATIONS ...................................................................................... xxii I. INTRODUCTION ......................................................................................................... 1 Polyamines and Alkaloids in Plants ................................................................................ 1 Pyrrolidine Alkaloids .................................................................................................. 2 Tropane Alkaloids ....................................................................................................... 2 Granatane Alkaloids .................................................................................................... 3 Piperidine Alkaloids .................................................................................................... 4 S-adenosyl-L-methionine N-methyltransferases in Plants .............................................. 5 SABATH methyltransferases ...................................................................................... 5 PEANMT and PavNMT ............................................................................................. 6 PMT and SPDS enzymes ............................................................................................ 6 II. IMPROVED SYNTHESIS OF N-METHYLCADAVERINE ................................. 8 Abstract: .......................................................................................................................... 8 Introduction ..................................................................................................................... 9 Results and Discussion ................................................................................................. 13 Synthesis of N-methylpiperidinium chloride (2·HCl) .............................................. 13 Synthesis of N-methylcadaverine chloride, (1·2 HCl) .............................................. 14 Materials and Methods .................................................................................................. 15 General Methods ....................................................................................................... 15 Synthesis of 5-(benzyl(methyl)amino)pentanenitrile (3) .......................................... 15 Synthesis of N-methylpiperidine hydrochloride (2·HCl).......................................... 16 Synthesis of N1-benzyl-N1-methylpentane-1,5-diamine (4) ..................................... 16 Synthesis of N-methylcadaverine (1·2HCl) .............................................................. 17 Conclusion .................................................................................................................... 17 Supplementary Materials .............................................................................................. 18 Acknowledgments......................................................................................................... 18 Author Contributions .................................................................................................... 18 Conflicts of Interest....................................................................................................... 18 III. ENZYMATIC SYNTHESIS OF DECARBOXYLATED S-ADENOSYL-L- METHIONINE ................................................................................................................ 19 Introduction ................................................................................................................... 19 iii Texas Tech University, Kayla Nicole Anderson, August 2019 Results ........................................................................................................................... 21 Identifying the SAM decarboxylase enzyme in E. coca ........................................... 21 Protein analysis of EcSAMDC ................................................................................. 22 Separation of dcSAM and SAM ............................................................................... 23 Materials and Methods .................................................................................................. 24 Plant material for Erythroxylum coca and Erythroxylum novogranatense............... 24 Total RNA extraction from E. novogranatense ........................................................ 24 cDNA synthesis ........................................................................................................ 25 PCR amplification of candidate genes from cDNA .................................................. 25 E. coli chemical competent transformation .............................................................. 25 E. coli protein expression .......................................................................................... 25 Nickel chelating affinity chromatography ................................................................ 26 Enzyme assay conditions .......................................................................................... 26 SAMDC-assay .......................................................................................................... 26 Derivatization of polyamines .................................................................................... 27 HPLC-UV detection for SAMDC activity ................................................................ 27 Enzymatic synthesis of dcSAM ................................................................................ 27 Dowex 50 H+ chromatography (separation of contaminants from sulfonium compounds) ............................................................................................................... 28 Dowex-1 OH- Chromatography (separation of SAM and dcSAM) ......................... 28 Conclusion .................................................................................................................... 29 Supplemental Material .................................................................................................. 29 Supplemental data and figures can be found in Appendix B. ....................................... 29 Acknowledgments......................................................................................................... 29 Author Contributions .................................................................................................... 29 Conflicts of Interest....................................................................................................... 29 IV. AN ALTERNATE ROUTE IN TROPANE ALKALOID BIOSYNTHESIS IN ERYTHROXYLUM COCA .............................................................................................. 30 Introduction ................................................................................................................... 30 Results ........................................................................................................................... 32 Spermidine N-methyltransferases and spermidine synthases isolated from E. coca 32 Protein analysis of SMT and SPDS candidates in E. coca ....................................... 33 Phylogenetic analysis of EcSPDS1, 2, and 3 ............................................................ 46 Relative abundance and distribution of polyamines in E. coca plants...................... 49 Quantitative PCR of EcSPDS candidates and EcSAMDC ....................................... 52 Synthesis of N-methylspermidine (1) ....................................................................... 62 Discussion ..................................................................................................................... 63 Materials and Methods .................................................................................................. 65 iv Texas Tech University,
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