Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2016 Substrate promiscuity and functional plasticity investigations of class I diterpene synthases Meirong Jia Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biochemistry Commons Recommended Citation Jia, Meirong, "Substrate promiscuity and functional plasticity investigations of class I diterpene synthases" (2016). Graduate Theses and Dissertations. 15941. https://lib.dr.iastate.edu/etd/15941 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]. Substrate promiscuity and functional plasticity investigations of class I diterpene synthases by Meirong Jia A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Biochemistry Program of Study Committee: Reuben J. Peters, Major Professor Richard B. Honzatko Gustavo Maclntosh Scott W. Nelson Zengyi Shao Iowa State University Ames, Iowa 2016 Copyright © Meirong Jia, 2016. All rights reserved. ii TABLE OF CONTENTS Page LIST OF ABBREVIATIONS .................................................................................... iv ACKNOWLEDGEMENTS ....................................................................................... vi ABSTRACT. .............................................................................................................. vii CHAPTER 1. GENERAL INTRODUCTION ....................................................... 1 Biosynthesis of labdane-related diterpenoids ...................................................... 1 Class I diterpene synthases .................................................................................. 2 Promiscuity of class I diterpene synthases........................................................... 3 Plasticity of class I diterpene synthases .............................................................. 4 Evolution of class I diterpene synthases .............................................................. 6 Organization of the dissertation and author contribution .................................... 8 References ............................................................................................................ 9 Figures.................................................................................................................. 12 CHAPTER 2. EXTREME PROMISCUITY OF A BACTERIAL AND A PLANT DITERPENE SYNTHASE ENABLES COMBINATORIAL BIOSYNTHESIS OF LABDANE-RELATED DITERPENES .................................................................... 17 Abstract ................................................................................................................ 17 Introduction .......................................................................................................... 18 Results ............................................................................................................... 22 Discussion and Conclusions ................................................................................ 27 Methods and Materials ......................................................................................... 30 References ............................................................................................................ 34 Figures.................................................................................................................. 40 Supplementary Information ................................................................................. 49 CHAPTER 3. EXTENSIVE PROMISCUITY INVESTIGATIONS OF CLASS I DITERPENE SYNTHASES REVEAL THE STRUCTURE-ACTIVITY RELATIONSHIP OF DITERPENE SUBSTRATES .............................................. 88 Abstract ................................................................................................................ 88 Introduction .......................................................................................................... 90 Results ............................................................................................................... 93 Discussion and Conclusions ................................................................................ 98 Methods and Materials ......................................................................................... 101 References ............................................................................................................ 105 Figures.................................................................................................................. 109 Supplementary Information ................................................................................. 115 iii CHAPTER 4. ONE SINGLE RESIDUE ALTERATION PREDOMINANTLY PREVENTS WATER ATTACK IN SCLAREOL SYNTHASE FROM SALVIA SCLAREA ..................................................................................................... 121 Abstract ................................................................................................................ 121 Introduction .......................................................................................................... 121 Results and Conclusions ...................................................................................... 124 Methods and Materials ......................................................................................... 127 References ............................................................................................................ 130 Figures.................................................................................................................. 132 Supplementary Information ................................................................................. 138 CHAPTER 5. EXTENDING A SINGLE RESIDUE SWITCH FOR ABBREVIATING CATALYSIS IN PLANT ENT-KAURENE SYNTHASES….. 143 Abstract ................................................................................................................ 143 Introduction .......................................................................................................... 144 Methods and Materials ......................................................................................... 146 Results ............................................................................................................... 148 Discussion ............................................................................................................ 151 References ............................................................................................................ 153 Figures.................................................................................................................. 156 Supplementary Information ................................................................................. 160 CHAPTER 6. A PAIR OF RESIDUES THAT INTERACTIVELY AFFECT DITERPENE SYNTHASE PRODUCT OUTCOME ............................................... 163 Abstract ................................................................................................................. 163 Introduction ........................................................................................................... 164 Results ............................................................................................................... 166 Discussion ............................................................................................................ 172 Methods and Materials .......................................................................................... 174 References ............................................................................................................. 176 Figures................................................................................................................... 178 Supplementary Information .................................................................................. 184 CHAPTER 7. CONCLUSIONS AND FUTURE DIRECTIONS............................. 191 References ............................................................................................................ 195 APPENDIX A . DITERPENE SYNTHASE MUTANTS CONSTRUCTED ........... 196 APPENDIX B . ADDITIONAL PROJECTS............................................................. 201 Project A. A Flux-fluctuating Motif in ent-Kaurene Synthases ............................... 201 Project B. Exploring How Water is Added in the Reaction Catalyzed by Abietaenol Synthases ........................................................................................... 202 References ............................................................................................................ 203 Figures.................................................................................................................. 204 iv LIST OF ABBREVIATIONS LRDs labdane-related diterpenoids DTCs diterpene cyclases DTSs diterpene synthases KS ent-kaurene synthase KSL ent-kaurene synthase-like enzyme KgTS terpentetriene synthase from Kitasatospora griseola SsSS sclareol synthase from Salvia sclarea GGPP (E,E,E)-geranylgeranyl diphosphate NNPP (Z,Z,Z)-nerylneryl diphosphate GGPPS GGPP synthase