CONSTRUCTING ARTIFICIAL GENETIC SYSTEMS: A NEW NUCLEOTIDE METABOLISM By MARIKO MATSUURA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2017 © 2017 Mariko Matsuura This dissertation is dedicated to my family: mother Fumiko, father Yuji; brother Junya; sister Nanako; grandparents Isamu and Hisano Totsuka; and Kojiro and Hiroko Matsuura. ACKNOWLEDGMENTS I would like to thank Dr. Steven Benner for the guidance, patience, and the research opportunities he has given me. I am also indebted to all the current and former members and visiting scholars of the Foundation for Applied Molecular Evolution and the Firebird Biomolecular Sciences LLC, especially Dr. Ryan W. Shaw, Ms. Jennifer D. Moses, Dr. Christian B. Winiger, Dr. Dietlind L. Gerloff, Dr. Shuichi Hoshika, Dr. Hyo-Joong Kim, Dr. Myong-Jung Kim, Dr. Myong-Sang Kim, Dr. Nilesh Karalkar, Dr. Nicole A. Leal, Dr. Yoshihiro Furukawa, and Dr. Fei Chen for their guidance, support, and help. Also, I would like to thank my research collaborators Dr. Stefan Lutz (Emory Univ.), Dr. Ashley B. Daugherty (Emory Univ.), Dr. David Baker (U. of Washington), Dr. Per Jr Greisen (U. of Washington), Dr. JoAnne Stubbe (MIT), Dr. Khalil A. Abboud (UF), and Mr. Daisuke Takahashi (UF). I would also like to express appreciation to my current and former committee members, Dr. Steven D. Bruner, Dr. Jamie S. Foster, Dr. Jon D. Stewart, Dr. Weihong Tan, and Dr. Nigel Richards especially my committee chair, Dr. Bruner for his help. Also, Dr. Nicole A. Horenstein and Dr. Kari B. Basso for giving me opportunities and support for my research, Dr. Ben Smith, Ms. Lori Clark, Ms. Lori Ball, Ms. Cassandra Watkins for their administrative assistance, and the Bruner group members for sharing their research talks and discussions with me. I would also like to thank TA supervisors and members for their guidance, and all the students in my classes, who made me a better teacher and scientist. I want to express special gratitude to Dr. Shigeyuki Yokoyama and Dr. Eiko Seki from RIKEN, Japan, who kindly accepts me as a research intern. An additional thank you to the Department of Chemistry, the Foundation for Applied Molecular Evolution, NASA Astrobiology, the National Science Foundation, the Defense Advanced Research Projects 4 Agency, and Templeton World Charity Foundation provided essential educational and financial support, without which my studies would not have been possible. I would also like to thank people who supported me in many ways throughout the Ph.D. program. Firstly, I would like to express my sincere appreciation to my counselors, Dr. Linda A. Lewis and Dr. Jaime Jasser, and all the members in the counseling group. Ms. Megan Williams, Ms. Lauren P. Jadotte, Ms. Lauren McCarthy, and Mr. Robert Ross for their friendship and support for the improvement of my English language skills. I would also like to thank Ms. Stephanie Seguin, Ms. Tiffany Bagby, Ms. Rachel Damiani, and Mr. David Honeycutt for their help with the English language, and all the members in the Gator Toastmasters Club for their help with public speaking. I would like to specifically thank Dr. Eunhui Yoon, Dr. Jules Gliesche, Dr. Shanshan Wang, and Ms. Yuting Wang, for their friendship and D. Marcus Garcia for his love, support and understanding. Lastly, I would like to thank my family, especially my mother Fumiko, for her love and support. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ........................................................................................................... 4 LIST OF TABLES ...................................................................................................................... 9 LIST OF FIGURES .................................................................................................................. 11 LIST OF ABBREVIATIONS.................................................................................................... 14 ABSTRACT ............................................................................................................................. 14 CHAPTER 1 BACKGROUND ............................................................................................................... 17 Designing Systems ............................................................................................................. 17 Design in This Study ................................................................................................... 18 Metabolic and Protein Engineering .............................................................................. 19 Molecules and Analogues ................................................................................................... 19 Nucleotides ................................................................................................................. 19 Artificial Genetic Systems ........................................................................................... 19 Nucleoside/nucleotide Synthesis.................................................................................. 22 Kinases ....................................................................................................................... 24 Nucleoside kinase (NK)........................................................................................ 26 Nucleoside monophosphate kinases (NMPK) ....................................................... 28 Nucleoside diphosphate kinase (NDPK) ............................................................... 30 2 RESEARCH OBJECTIVES ............................................................................................... 32 3 GENERAL METHODS AND PROCEDURES .................................................................. 33 Materials ............................................................................................................................ 33 Nucleobase and Nucleoside/tide Synthesis .................................................................. 33 Oligonucleotides Synthesis .......................................................................................... 33 Standard Methods .............................................................................................................. 33 Cloning/Synthesis of Genes and Construction of Plasmids .......................................... 33 Transformation ............................................................................................................ 34 Enzyme Preparation .................................................................................................... 34 4 ASSAY DEVELOPMENT WITH WILD-TYPE ENZYMES............................................. 35 Materials and Methods ....................................................................................................... 35 Preparation of Enzymes ............................................................................................... 35 Kinase Assays ............................................................................................................. 35 Enzyme-coupled assay ......................................................................................... 35 TLC assay ............................................................................................................ 36 6 Luciferase assay ................................................................................................... 38 Modified 5′ nuclease assay ................................................................................... 39 Results ............................................................................................................................... 42 Assay Development..................................................................................................... 42 Activities of Wild-Type Kinases.................................................................................. 45 Drosophila melanogaster deoxynucleoside kinase (DmdNK) ............................... 46 Guanylate kinases ................................................................................................. 46 E. coli Nucleoside diphosphate kinase (Ndk) ........................................................ 48 Kinetic Parameters ...................................................................................................... 52 Discussion .......................................................................................................................... 54 Assay Comparison ...................................................................................................... 54 Kinase Specificities ..................................................................................................... 58 Ribonucleoside Triphosphate Synthesis ....................................................................... 58 5 KINASE VARIANTS SCREENING ................................................................................. 60 Materials and Methods ....................................................................................................... 60 Enzyme Design and Preparation .................................................................................. 60 D. melanogaster nucleoside kinase (DmdNK) ...................................................... 60 E. coli cytidylate kinase (EcCmk) ......................................................................... 61 E. coli thymidylate kinase (EcTmk) .....................................................................