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Iodotyrosine Deiodinase from Selected Phyla Engineered for Bacterial Expression

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Iodotyrosine Deiodinase from Selected Phyla Engineered for Bacterial Expression ABSTRACT Title of Document: IODOTYROSINE DEIODINASE FROM SELECTED PHYLA ENGINEERED FOR BACTERIAL EXPRESSION Jennifer Marilyn Buss Doctor of Philosophy, 2012 Directed By: Professor Steven E. Rokita Department of Chemistry and Biochemistry Iodide is a well known halogen necessary for development. The majority of iodide processing in biological systems occurs in the thyroid gland. Iodide salvage is essential to thyroid hormone metabolism and metabolic regulation. The DEHAL1 gene product iodotyrosine deiodinase (IYD) is responsible for deiodination of the mono- and diiodotyrosine byproducts of thyroid hormone synthesis (triiodothyronine and thyroxine, T3 and T4, respectively). IYD is a membrane-bound flavoprotein comprised of three domains with the catalytic domain belonging to the NADPH oxidase/flavin reductase structural superfamily. This enzyme required engineering for expression of soluble protein in E. coli and was characterized using CD spectra, kinetic rate constants, binding constants of substrates, and crystal structure. Analysis of the crystal structure of IYD indicates a dimer with an active site comprising of both monomers and orienting the C-I bond of iodotyrosine substrate stacking above the N5 of the flavin mononucleotide (FMN) required for activity. The crystal structure also identifies an active site lid that distinguishes IYD from other proteins in the NADPH oxidase/flavin reductase superfamily. Three amino acids (E153, Y157, and K178) on the active site lid form hydrogen bonding and electrostatic contacts with the zwitterionic portion of the substrate. Mutation to any of these three amino acids significantly decreases substrate-binding affinity and enzymatic activity. Homologous sequences of IYD were identified in other organisms and four sequences as representatives from their phyla were expressed in E. coli. Zebrafish, lancelets, honeybees, and sea anemones each have a protein that acts as a deiodinase. IODOTYROSINE DEIODINASE FROM SELECTED PHYLA ENGINEERED FOR BACTERIAL EXPRESSION By Jennifer Marilyn Buss Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2012 Advisory Committee: Professor Steven E. Rokita, Chair Professor Douglas A. Julin Professor David Fushman Assistant Professor Nicole LaRonde-LeBlanc Professor Steven Mount © Copyright by Jennifer Marilyn Buss 2012 Dedication To my family, for their endless support and encouragement. i Acknowledgements I’d like to begin by thanking my advisor, Dr. Steve Rokita. Thank you for your continued support. Thank you for challenging me to be a better scientist, writer, and thinker. For teaching me to tell a story and a constant reminder to always check the pH. Thank you to my committee Professors Julin, Fushman, LaRonde-LeBlanc, and Mount. I appreciate your input and your expertise. To past members of my committee and other faculty who helped make me a better scientist: Dr. Gerratana, Dr. Cropp, and Dr. Beckett. A special thank you to Dr. Jim Watson. We began as coworkers doing late night experiments and we’ve ended with you as my boss and late night grading. Thank you for keeping it real and putting it all in to perspective. Thank you to all the collaborators that have made my research possible. Dr. Nicole LaRonde-LeBlanc, Dr. Seth Thomas, Dr. Lee Chuenchor for solving crystal structures of our protein that has made my work possible. Dr. Hamza and Dr. Xiao the use of their tissue culture hoods. To Rokita lab members past and present, thank you. To the endless friends I have made. The support that you have provided has been unbelievable. Kathy, words cannot express our time together in school and in the real world. Petrina, Crazy nights adding just a drop more fun. Mike – Go Blue Hens! Patrick, less is more. To the Departmental staff, who have been the moving parts behind closed doors. Thank you, Tia, for keeping everything running smoothly. ii Last but not least, I thank my parents, who have called me Almost Doctor for entirely too many years. The day has come. Thank you for all your encouragement, through the ups and downs, for the best mattress, and the free advice. To my brother and sister, for keeping me laughing, either at them or at myself. iii Table of Contents Dedication ....................................................................................................................... i Acknowledgements ...................................................................................................... ii List of Tables ................................................................................................................ vi List of Figures ............................................................................................................. vii Chapter 1: Introduction .................................................................................................. 1 1.1 Iodide Salvage .................................................................................................. 1 1.2 Cysteines and deiodination ............................................................................... 5 1.3 IYD is a dehalogenase ...................................................................................... 6 1.4 IYD has unique function within its structural superfamily .............................. 7 1.5 Similarity of thyroidal enzymes in model organisms……………………… 11 1.6 Specific Aims ................................................................................................. 12 Chapter 2: Engineering IYD for isolation from E. coli ............................................... 14 2.1 Introduction .................................................................................................... 14 2.2 Experimental Procedures ................................................................................ 16 2.2.1 Materials .................................................................................................. 16 2.2.2 General Methods ...................................................................................... 17 2.2.3 Subcloning of IYD in E. coli expression vectors .................................... 18 2.2.4 Expression in E. coli ................................................................................ 21 2.2.5 Purification of IYD(Δtm)DM .................................................................. 22 2.3 Results and Disucssion ................................................................................... 23 2.3.1 IYD expressed as insoluble protein in E. coli ......................................... 23 2.3.2 IYD(Δtm)DM expressed as soluble protein in E. coli ............................. 26 2.4 Conclusions .................................................................................................... 31 Chapter 3: Characterization of IYD ............................................................................. 33 3.1 Introduction .................................................................................................... 33 3.2 Experimental ................................................................................................... 35 3.2.1 Materials .................................................................................................. 35 3.2.2 Circular Dichroism .................................................................................. 35 3.2.3 Fluorescence quenching .......................................................................... 35 iv 3.2.4 Deiodinase activity .................................................................................. 36 3.2.5 Crystallization of IYD(Δtm)DM ............................................................. 37 3.3 Results and Discussion ................................................................................... 39 3.3.1 CD of flavoprotein iodotyrosine deiodinase ............................................ 39 3.3.2 Kinetics of IYD(Δtm)DM ........................................................................ 42 3.3.3 Equilibrium binding to IYD(Δtm)DM ..................................................... 43 3.3.4 Crystal structure of IYD(∆tm)DM .......................................................... 44 3.4 Conclusions .................................................................................................... 48 Chapter 4: Substrate coordination to the active site lid ............................................... 49 4.1 Introduction .................................................................................................... 49 4.2 Materials and Methods ................................................................................... 53 4.2.1 General Methods ...................................................................................... 53 4.2.2 Site-directed Mutagenesis ........................................................................ 54 4.3 Results and Discussion ................................................................................... 54 4.3.1 Confirmation of soluble and folded protein ............................................ 55 4.3.2 Deiodinase activity of mutants ................................................................ 56 Chapter 5: Expression of homologous IYD ................................................................. 63 5.1 Introduction .................................................................................................... 63 5.2 Experimental ..................................................................................................
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