Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2013 Exploring and understanding the substrate diversity of b-ketoacyl-ACP synthase III enzymes Shivani Garg Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biochemistry Commons Recommended Citation Garg, Shivani, "Exploring and understanding the substrate diversity of b-ketoacyl-ACP synthase III enzymes" (2013). Graduate Theses and Dissertations. 16085. https://lib.dr.iastate.edu/etd/16085 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]. Exploring and understanding the substrate diversity of β-ketoacyl-ACP synthase III enzymes by Shivani Garg 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: Guru Rao, Co-Major Professor Drena Dobbs, Co-Major Professor Basil J. Nikolau Marna D. Yandeau-Nelson Thomas Bobik Edward Yu Iowa State University Ames, Iowa 2013 Copyright © Shivani Garg, 2013. All rights reserved. ii “Two great things you can give your children: one is roots, the other is wings.” – Hodding Carter Dedicated to my grandfather and parents who gave me both roots and wings iii TABLE OF CONTENTS Page DEDICATION ......................................................................................................... ii ACKNOWLEDGEMENTS .................................................................................... v ABSTRACT………………………………. ............................................................. vii CHAPTER I. GENERAL INTRODUCTION ....................................................... 1 Introduction to β-ketoacyl-ACP synthase III ....................................................... 1 Questions explored in the dissertation ................................................................ 4 Dissertation organization .................................................................................... 5 Literature review ................................................................................................. 7 Significance ......................................................................................................... 16 References ......................................................................................................... 17 CHAPTER II. DISSECTING THE STRUCTURAL BASIS OF DIVERSE FUNCTIONALITIES OF E. COLI AND B. SUBTILIS KASIII ENZYMES USING STD NMR BASED LIGAND INTERACTIONS ........................................ 23 Abstract ......................................................................................................... 23 Introduction ......................................................................................................... 24 Materials and methods ........................................................................................ 28 Results ......................................................................................................... 33 Discussion ......................................................................................................... 41 Tables ......................................................................................................... 47 Figures ......................................................................................................... 49 References ......................................................................................................... 55 CHAPTER III. DELINEATING THE STRUCTURE-FUNCTION RELATIONSHIPS OF -KETOACYL-ACP SYNTHASE III BASED ON PHYLOGENETIC AND FUNCTIONAL COMPARISONS ................................... 59 Abstract ......................................................................................................... 59 Introduction ......................................................................................................... 60 Materials and methods ......................................................................................... 63 Results ......................................................................................................... 66 Discussion ......................................................................................................... 73 iv Page Tables ......................................................................................................... 81 Figures ......................................................................................................... 83 References ......................................................................................................... 90 CHAPTER IV. IDENTIFICATION OF KASIII ENZYMES WITH NOVEL SUBSTRATE SPECIFICITIES: DEMONSTRATION OF IN VIVO PRODUCTION OF NOVEL ω-1 HYDROXYLATED FATTY ACIDS USING A NOVEL KASIII ....................................................................................... 94 Abstract ......................................................................................................... 94 Introduction ......................................................................................................... 95 Materials and methods ......................................................................................... 97 Results ......................................................................................................... 104 Discussion ......................................................................................................... 110 Tables ......................................................................................................... 116 Figures ......................................................................................................... 118 References ......................................................................................................... 123 CHAPTER V. CONCLUSIONS............................................................................. 129 Conclusions .......................................................................................................... 129 References ......................................................................................................... 139 APPENDIX. BUSINESS PLAN FOR OMEGACHEA BIORENEWABLES LLC ......................................................................................................... 142 v ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Basil J. Nikolau, who gave me the opportunity to work on this exciting research topic. This work would have been impossible without his constant support and guidance throughout my graduate school years. I am so grateful for the numerous opportunities and enriching experiences that I had in his lab such as mentoring REU students, developing collaborations, undertaking biorenewable chemicals minor and exploring commercial aspects of my research; these experiences along with his confidence in my abilities have helped me grow tremendously as a researcher and as a person. Next, I would like to thank Dr. Marna D. Yandeau-Nelson, my co-advisor, who provided invaluable advice and mentorship contributing greatly to the development of this work. I also want to thank my POS committee members, Dr. Thomas Bobik, Dr. Edward Yu, Dr. Drena Dobbs and Dr. Guru Rao for their valuable suggestions and willingness to help at all times. This research was funded by NSF and CBiRC, and I am grateful for the interdisciplinary research environment of CBiRC that provided unique opportunities to extend my research into areas I had not imagined before. Dr. Peter Keeling, CBiRC Innovation Director, was instrumental in helping me develop a commercialization path for my research. Most cherished are my conversations with him that broadened my vision and enabled me to look at research from an industrial point of view. The constant encouragement from Dr. Brent Shanks, CBiRC Director, motivated me to do better at my research. I am thankful for the collaboration opportunities that CBiRC provided, including a research visit to Dr. Joseph Noel’s lab at the Salk Institute for Biological Studies, which markedly vi enhanced my research and technical skills. I am thankful to Dr. Charles Stewart, research specialist in the Noel lab, for his expert mentorship and for teaching me thermal shift assays and crystallization techniques that brought a new perspective to my research. I want to thank Dr. Bruce Fulton, NMR facility manager, who facilitated saturation transfer difference NMR experiments and helped me analyze the NMR experimental results. I would like to acknowledge the W. M. Keck Metabolomics lab where enzyme activity assays were conducted, and Dr. Ann Perera was helpful in solving technical issues related to instrumentation. I highly appreciate the time and the sincerest efforts that my undergraduate students, Luke Prest, Kim-Xuan Nguyen, Morgan Becker, Joshua Scaralia, Ruben Garcia and Niibari Menegbo, devoted to this research. Amy Arhens and Katie Blair, former and current MCDB program coordinators, were always accessible to answer my questions regarding administrative and program completion requirements. I deeply appreciate their timely help and advice whenever I needed it. I wish to thank