Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2019 Regulation of acetyl-CoA metabolism in plants: Role of acetyl-CoA synthetase and sirtuin proteins Naazneen Sofeo Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biochemistry Commons Recommended Citation Sofeo, Naazneen, "Regulation of acetyl-CoA metabolism in plants: Role of acetyl-CoA synthetase and sirtuin proteins" (2019). Graduate Theses and Dissertations. 17322. https://lib.dr.iastate.edu/etd/17322 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]. Regulation of acetyl-CoA metabolism in plants: Role of acetyl-CoA synthetase and sirtuin proteins by Naazneen Sofeo 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: Basil J Nikolau, Co-major Professor Marna D. Yandeau-Nelson, Co-major Professor Richard B Honzatko Scott W Nelson Zengyi Shao The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this dissertation. The Graduate College will ensure this dissertation is globally accessible and will not permit alterations after a degree is conferred. Iowa State University Ames, Iowa 2019 Copyright © Naazneen Sofeo, 2019. All rights reserved. ii TABLE OF CONTENTS ACKNOWLEDGMENTS ................................................................................................. iv ABSTRACT ........................................................................................................................ v CHAPTER 1. INTRODUCTION AND DISSERTATION ORGANIZATION ................ 1 General Introduction ...................................................................................................... 1 Significance ................................................................................................................... 4 Dissertation Organization .............................................................................................. 6 References...................................................................................................................... 8 CHAPTER 2. ALTERING THE SUBSTRATE SPECIFICITY OF ACETYL-COA SYNTHETASE BY RATIONAL MUTAGENESIS OF THE CARBOXYLATE BINDING POCKET ......................................................................................................... 12 Abstract ........................................................................................................................ 13 Introduction.................................................................................................................. 14 Results & Discussion ................................................................................................... 16 Conclusion ................................................................................................................... 26 Methods ....................................................................................................................... 27 Abbreviations ............................................................................................................... 31 Author Contributions ................................................................................................... 31 Acknowledment ........................................................................................................... 31 References.................................................................................................................... 32 Figures ......................................................................................................................... 36 Tables ........................................................................................................................... 43 Supporting Information ............................................................................................... 45 CHAPTER 3. UNDERSTANDING EFFECTS OF ACETYLATION IN REGULATING ARABIDOPSIS ACETYL-COA SYNTHETASE ................................ 47 Abstract ........................................................................................................................ 47 Introduction.................................................................................................................. 48 Material & Methods ..................................................................................................... 49 Results.......................................................................................................................... 54 Discussion .................................................................................................................... 57 References.................................................................................................................... 58 Figures ......................................................................................................................... 62 CHAPTER 4. SIRTUIN-MEDIATED REGULATION OF PLANT ACETYLOME AND METABOLOME ..................................................................................................... 70 Abstract ........................................................................................................................ 70 Introduction.................................................................................................................. 70 Results.......................................................................................................................... 72 Discussion .................................................................................................................... 78 Methods ....................................................................................................................... 80 iii References.................................................................................................................... 91 Figures ......................................................................................................................... 96 Tables ......................................................................................................................... 104 Supplemental Information ......................................................................................... 104 CHAPTER 5. GENERAL CONCLUSION .................................................................... 105 Conclusions................................................................................................................ 105 References.................................................................................................................. 108 APPENDIX A. USING A STRATEGY THAT EXPANDS THE GENETIC CODE TO GENERATE IN VIVO ACETYLATED LYS-622 MODIFICATION OF THE ARABIDOPSIS ACETYL-COA SYNTHETASE ......................................................... 110 Introduction................................................................................................................ 110 Methods ..................................................................................................................... 111 Results and Discussion .............................................................................................. 114 References.................................................................................................................. 116 Figures ....................................................................................................................... 118 Tables ......................................................................................................................... 124 APPENDIX B. SUBCELLULAR LOCALIZATION OF THE SRT1 PROTEIN IN ARABIDOPSIS THALIANA ........................................................................................... 125 Introduction................................................................................................................ 125 Methods ..................................................................................................................... 125 Results and Conclusion.............................................................................................. 126 References.................................................................................................................. 127 Figures ....................................................................................................................... 128 Tables ......................................................................................................................... 133 iv ACKNOWLEDGEMENTS I would like to first thank my major professor, Dr. Basil J Nikolau for giving me an opportunity to be a part of his research group. It has been a true privilege and joy to work in this group. I also would like to thank my co-major professor, Dr. Marna D. Yandeau-Nelson for her valuable support, guidance and encouragement throughout. I would like to thank Dr. Richard B Honzatko, Dr. Scott W. Nelson, and Dr. Zengyi Shao, for serving as my valuable Program of Study Committee members.