Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2012 Insights into the catalytic mechanisms of the protein enzymes, PFKFB3 and VldE, using x-ray crystallography Michael Christopher Cavalier Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Recommended Citation Cavalier, Michael Christopher, "Insights into the catalytic mechanisms of the protein enzymes, PFKFB3 and VldE, using x-ray crystallography" (2012). LSU Doctoral Dissertations. 3945. https://digitalcommons.lsu.edu/gradschool_dissertations/3945 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. INSIGHTS INTO THE CATALYTIC MECHANISMS OF THE PROTEIN ENZYMES, PFKFB3 AND VLDE, USING X-RAY CRYSTALLOGRAPHY A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Biological Sciences by Michael Christopher Cavalier B.S., Louisiana State University, 2006 December 2012 ACKNOWLEDGMENTS I would like to begin by thanking my major professor, Dr. Yong-Hwan Lee, for taking me into his laboratory and providing me with the opportunity to learn the skill of protein crystallography. I thank Dr. Marcia Newcomer, Dr. Grover Waldrop, and Dr. Bing-Hao Luo for joining my graduate committee and for all the advice they have offered. I thank Dr. Henry Bellamy and Dr. David Neau for contributing their expertise in synchrotron data collection. I thank Dr. Erin Hawkins for her guidance during my teaching duties and for her continuing encouragement. I thank my fellow lab members; Blake Crochet, Dr. Jeong-Do Kim, Dr. Young- Sun Yim, Dr. Song-Gun Kim, and Minsuh Seo for the valuable discussions and for the contributions they have made to my education. I also offer a special thanks to my parents, Bonnie and Nickie, and to my brother, Matthew, for their unrelenting support during this long trying process. I also thank my fiancée, Heather, whose patient love enabled me to complete this work. This would not be possible without them. ii TABLE OF CONTENTS ACKNOWLEDGMENTS .............................................................................................................. ii LIST OF TABLES ......................................................................................................................... vi LIST OF FIGURES ...................................................................................................................... vii ABSTRACT ................................................................................................................................... ix CHAPTER 1: REVIEW OF LITERATURE, THE BIFUNCTIONAL ENZYME .........................1 Carbohydrate Metabolism ....................................................................................................1 History of Metabolism .............................................................................................1 Glycolysis ................................................................................................................1 Gluconeogenesis ......................................................................................................4 The Discovery of Fructose-2,6-Bisphosphate and the Bifunctional Enzyme ..........5 Two Functional Folds ..........................................................................................................8 Opposing Reactions .................................................................................................8 Histidine Phosphatases.............................................................................................8 P-loop Kinases .......................................................................................................11 The Tissue Specific Isoforms of the Bifunctional Enzyme ...............................................14 Conserved Structure ...............................................................................................14 Liver .......................................................................................................................18 Heart .......................................................................................................................19 Testis ......................................................................................................................19 Inducible ................................................................................................................20 PFKFB3 and the Warburg Effect .......................................................................................20 The Glycolytic Phenotype of Cancer .....................................................................20 PFKFB3 and Cancer ..............................................................................................21 The Hypoxia-Inducible Transcription Factor HIF-1..............................................22 PFKFB3 as a Therapeutic Target ...........................................................................23 CHAPTER 2: MOLECULAR BASIS OF THE FRUCTOSE-2,6- BISPHOSPHATASE REACTION OF PFKFB3: TRANSITION STATE AND THE C-TERMINAL FUNCTION ..................................................25 Introduction ........................................................................................................................25 Results ................................................................................................................................29 Overall Structure ....................................................................................................29 PFKFB3-P•F-6-P Structure ....................................................................................29 PFKFB3•AlF4 Structure .........................................................................................30 PFKFB3•PPi Structure ...........................................................................................33 Comparison of Bisphosphatase Active Sites .........................................................33 Degradation of Fructose-2,6-Bisphosphate ...........................................................36 Discussion ..........................................................................................................................40 Materials and Methods .......................................................................................................42 Preparation and Crystallization of PFKFB3 ..........................................................42 iii Data Collection and Processing .............................................................................43 Structure Determination and Refinement ..............................................................43 CHAPTER 3: REVIEW OF LITERATURE, VLDE ....................................................................46 Diversification of Natural Products ...................................................................................46 Natural Products Offer Unique Scaffolding ..........................................................46 Diversification of the Erythromycin Scaffold by Genetic Manipulation...............47 Diversification of the Avermectin Scaffold by Alternative Precursors .................50 Diversification of the Antitumor Indolocarbazoles by Recombinant Enzymes ....51 The Aminocyclitol class of Natural Products ....................................................................52 The Variety of Aminocyclitols ..............................................................................52 The Aminoglycosides ............................................................................................53 The Five-Membered Ring Aminocyclitols ............................................................56 The C7N Aminocyclitols ........................................................................................58 Validamycin A ...................................................................................................................61 Early Uses of Validamycin A ................................................................................61 Discovery of the Validamycin A Gene Cluster .....................................................62 Biosynthetic Pathway of Validamycin A ...............................................................62 Glycosyltransferases ..........................................................................................................66 A Large and Diverse Class of Enzymes ................................................................66 GT-A and GT-B Topologies ..................................................................................67 Inverting Glycosyltransferases ...............................................................................69 Retaining Glycosyltransferases ..............................................................................72 Oxocarbenium Ion-Like Transition State ..............................................................74 CHAPTER 4: MECHANISTIC INSIGHTS INTO VALIDOXYLAMINE A 7'-PHOSPHATE SYNTHESIS BY VLDE USING THE STRUCTURE OF THE ENTIRE PRODUCT COMPLEX ..................................75 Introduction ........................................................................................................................75