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PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance Graduate School Form 30 Updated 1/15/2015 PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By Megan P. Rogers Entitled INVESTIGATION OF THE EVOLUTIONARY ASPECTS OF THIAMIN DIPHOSPHATE- DEPENDENT DECARBOXYLASES For the degree of Master of Science Is approved by the final examining committee: Michael J. McLeish Chair Eric Long Lei Li To the best of my knowledge and as understood by the student in the Thesis/Dissertation Agreement, Publication Delay, and Certification Disclaimer (Graduate School Form 32), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy of Integrity in Research” and the use of copyright material. Michael J. McLeish Approved by Major Professor(s): Eric Long 4/6/2015 Approved by: Head of the Departmental Graduate Program Date i INVESTIGATION OF THE EVOLUTIONARY ASPECTS OF THIAMIN DIPHOSPHATE- DEPENDENT DECARBOXYLASES A Thesis Submitted to the Faculty of Purdue University by Megan P. Rogers In Partial Fulfillment of the Requirements for the Degree of Master of Science i May 2015 Purdue University Indianapolis, Indiana ii For my family ii iii ACKNOWLEDGEMENTS During my time at IUPUI, there are many people who deserve thanks for helping and guiding me along the way. I would first like to thank my advisor, Dr. Michael J. McLeish for allowing me the opportunity to work under his guidance and for providing support throughout the process. I would also like to thank Dr. Kathleen Marrs for giving me the opportunity to participate in the GK-12 fellowship program and for the ability to present science to students in a unique way. I would like to thank the members of the McLeish group and my fellow peers for their friendship and providing perspective at the most necessary of times. Lastly, I would like to thank my family, especially Will, for the support, patience, and understanding shown to me during this journey. iii iv TABLE OF CONTENTS Page LIST OF TABLES ............................................................................................................................... viii LIST OF FIGURES ................................................................................................................................ ix LIST OF ABBREVIATIONS ............................................................................................................... xii ABSTRACT ........................................................................................................................................... xiv CHAPTER 1. INTRODUCTION.......................................................................................................... 1 1.1 Thiamin Diphosphate .......................................................................................................... 1 1.2 Pyruvate decarboxylase, a modern ThDP-dependent enzyme ........................... 5 1.2.1 The ThDP binding site .............................................................................................. 7 1.2.2 The active site .............................................................................................................. 9 1.2.3 The regulatory site ................................................................................................... 10 1.3 Evolutionary aspects of ThDP-dependent enzymes .............................................. 11 1.4 Research covered in this work ....................................................................................... 15 CHAPTER 2. MATERIALS AND METHODS ............................................................................... 17 2.1 Materials ................................................................................................................................. 17 2.2 Transformations .................................................................................................................. 18 2.2.1 Escherichia coli transformation .......................................................................... 18 2.2.2 Saccharomyces cerevisiae transformation ...................................................... 19 iv 2.3 Construction of expression vectors .............................................................................. 20 2.3.1 Cloning and construction of expression vectors pET17PDC5-His, pET17PDC6-His, and pET17THI3-His ......................................................................... 20 2.3.2 Construction of a zeocin yeast expression vectors ...................................... 20 v Page 2.3.3 Insertion of genes into zeocin yeast expression vector ............................. 22 2.4 Protein expression and purification of His-tagged enzymes ............................. 23 2.4.1 Expression of His-tagged enzymes .................................................................................. 23 2.4.2 General purification of His-tagged proteins ................................................... 23 2.4.3 Purification of phosphonopyruvate decarboxylase and phosphonoacetaldehyde hydrolase ............................................................................. 25 2.5 Protein crystallization ....................................................................................................... 26 2.5.1 Protein expression and purification for crystallization............................. 26 2.5.2 Crystallization of PDC5 ........................................................................................... 26 2.6 Characterization studies ................................................................................................... 27 2.6.1 Kinetic characterization of PDC1 and PDC5 ................................................... 27 2.6.2 Kinetic characterization of the benzoylformate decarboxylase T377L_A460Y variant ........................................................................................................ 27 2.6.3 Steady state analysis of phosphonopyruvate decarboxylase .................. 28 2.6.4 IC50 and Ki determination for difluorophosphonopyruvate ..................... 29 2.6.5 Reaction of difluorophosphonopyruvate with PhPyrDC monitored by circular dichroism (CD) spectroscopy and UV-VIS spectroscopy ......................................................................................................................... 29 2.6.6 Fluoride ion analysis of the reaction of v difluorophosphonopyruvate with PhPyrDC ............................................................. 30 CHAPTER 3. INACTIVATION OF PHOSPHONOPYRUVATE DECARBOXYLASE FROM BACTEROIDES FRAGILIS BY DIFLUOROPHOSPHONOPYRUVATE ..................... 31 3.1 Background ............................................................................................................................ 31 3.2 Results and Discussion ...................................................................................................... 34 3.2.1 Purification and kinetic characterization of PhPyrDC ............................... 34 3.2.2 Design and synthesis of difluorophosphonopyruvate ............................... 36 3.2.3 IC50 and Ki determination for DFPP.................................................................... 37 3.2.4 Dilution studies of PhPyrDC with DFPP........................................................... 39 vi Page 3.2.5 Reaction of DFPP with PhPyrDC monitored by circular dichroism (CD) spectroscopy and UV-Vis spectroscopy ..................................... 39 3.2.6 Fluoride ion analysis of the reaction of DFPP with PhPyrDC .................. 42 3.2.7 Crystallization studies of PhPyrDC .................................................................... 43 3.3 Conclusions and Future Direction ................................................................................ 44 CHAPTER 4. INVESTIGATION AND CHARACTERIZATION OF THE MINOR ISOZYMES OF PYRUVATE DECARBOXYLASE ISOLATED FROM BAKER’S YEAST .... 45 4.1 Background ............................................................................................................................ 45 4.1.1 PDC5 .............................................................................................................................. 46 4.1.2 PDC6 .............................................................................................................................. 48 4.1.3 THI3 ............................................................................................................................... 50 4.2 Results and Discussion ...................................................................................................... 51 4.2.1 Analysis of PDC5 ....................................................................................................... 51 4.2.1.1 Sequence alignments and homology model comparison of PDC5 and PDC1 ............................................................................................................. 51 4.2.1.2 Expression and kinetic characterization of PDC5 ............................. 54 4.2.1.3 Crystallization, X-ray structure and structure analysis for PDC5 .................................................................................................................................. 59 4.2.2 Analysis of PDC6 ....................................................................................................... 62 vi 4.2.2.1 Sequence alignment and homology model comparison of PDC6 and PDC1 ............................................................................................................. 62 4.2.2.2 Expression
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