Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2015 Understanding the BASEs for catalysis in class II diterpene cyclases results in novel product chemistries Kevin Charles Potter Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biochemistry Commons, and the Molecular Biology Commons Recommended Citation Potter, Kevin Charles, "Understanding the BASEs for catalysis in class II diterpene cyclases results in novel product chemistries" (2015). Graduate Theses and Dissertations. 14625. https://lib.dr.iastate.edu/etd/14625 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Understanding the BASEs for catalysis in class II diterpene cyclases results in novel product chemistries by Kevin Charles Potter A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Molecular, Cellular and Developmental Biology Program of Study Committee: Reuben J. Peters Major Professor Michael Shogren-Knaak Mark Hargrove Gustavo MacIntosh Jacqueline V. Shanks Iowa State University Ames, Iowa 2015 Copyright © Kevin Charles Potter, 2015. All rights reserved. ii TABLE OF CONTENTS Page LIST OF ABBREVIATIONS .................................................................................... iv ACKNOWLEDGEMENTS ....................................................................................... v ABSTRACT. .............................................................................................................. vi CHAPTER 1 GENERAL INTRODUCTION ....................................................... 1 Labdane-related diterpenoids ............................................................................... 1 Biosynthesis of labdane-related diterpenoids ....................................................... 2 Evolution of labdane-related diterpenoids ........................................................... 3 Class II diterpene cyclases ................................................................................... 4 Structure of ent-copalyl diphosphate synthase from Arabidopsis thaliana Involved in GA biosynthesis ................................................................................ 6 Organization of the dissertation and author contribution .................................... 7 References ............................................................................................................ 9 Figures.................................................................................................................. 10 CHAPTER 2 NOVEL PRODUCT CHEMISTRY FROM MECHANISTIC ANALYSIS OF ENT-COPALYL DIPHOSPHATE SYNTHASES FROM PLANT HORMONE BIOSYNTHESIS .................................................................................. 13 Abstract ................................................................................................................ 13 Introduction .......................................................................................................... 13 Experimental Section ........................................................................................... 20 References ............................................................................................................ 21 Figures.................................................................................................................. 23 Supplementary Information ................................................................................. 28 CHAPTER 3 AROMATIC REPLACEMENT OF THE CATALYTIC BASE FROM A PLANT ENT-COPALYL DIPHOSPHATE SYNTHASE LEADS TO PRODUCT REARRANGEMENT ........................................................................... 34 Abstract ................................................................................................................ 35 Introduction .......................................................................................................... 37 References ............................................................................................................ 42 Figures.................................................................................................................. 43 Supplementary Information ................................................................................. 48 CHAPTER 4 CONSERVED BASEs FOR THE INITIAL CYCLASE IN GIBBERELLIN BIOSYNTHESIS: FROM BACTERIA TO PLANTS ................... 86 Abstract ................................................................................................................ 86 Introduction .......................................................................................................... 86 Results .................................................................................................................. 90 Discussion ............................................................................................................ 93 iii Experimental Section ........................................................................................... 95 References ............................................................................................................ 97 Figures.................................................................................................................. 100 Supplementary Information ................................................................................. 105 CHAPTER 5 MUTAGENESIS OF THE ORYZA SATIVA SYN-COPALYL DIPHOSPHATE SYNTHASE RESULTS IN NOVEL ENZYME ACTIVITY ....... 107 Abstract ................................................................................................................ 107 Introduction .......................................................................................................... 107 Results .................................................................................................................. 109 Discussion ............................................................................................................ 112 Experimental Section ........................................................................................... 114 References ............................................................................................................ 115 Figures.................................................................................................................. 118 Supplementary Information ................................................................................. 123 CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS.......................... 127 References ............................................................................................................ 130 Figures.................................................................................................................. 131 APPENDIX A DITERPENE CYCLASE MUTANTS CONSTRUCTED ............. 132 APPENDIX B EFFICIENT HETEROCYCLISATION BY (DI)TERPENE SYNTHASES ............................................................................................................ 137 Abstract ................................................................................................................ 137 Introduction .......................................................................................................... 137 References ............................................................................................................ 142 Figures.................................................................................................................. 143 Supplementary Information ................................................................................. 144 APPENDIX C A SINGLE RESIDUE CHANGE LEADS TO HYDROXYLATED PRODUCT FROM THE CLASS II DITERPENE CYCLIZATION CATALYZED BY ABIETADIENE SYNTHASE........................ 150 Abstract ................................................................................................................ 150 Introduction .......................................................................................................... 150 References ......................................................................................................... 155 Figures.................................................................................................................. 156 Supplementary Information ................................................................................. 159 iv LIST OF ABBREVIATIONS CPP copalyl diphosphate CPS copalyl diphosphate synthase DMAPP dimethylallyl pyrophosphate diTPS diterpene synthase diTPC class II diterpene cyclase GA gibberellins GC gas chromatography GC-FID gas chromatography – flame ionization detector GC-MS gas chromatography – mass spectrometry GGPP (E, E, E)-geranylgeranyl diphosphate GGPPS geranylgeranyl diphosphate synthase HPP halimadienyl diphosphate IPP isopentenyl diphosphate KS ent-kaurene synthase KPP kolavenyl diphosphate LRD labdane-related diterpenoids MEP methyl erythritol phosphate MVA mevalonic acid OH hydroxy TPP terpentedienyl diphosphate v ACKNOWLEDGEMENTS These past four years have been a roller coaster ride. There has been many high