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Structure and Mechanism of a Eukaryotic Fmn Adenylyltransferase

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STRUCTURE AND MECHANISM OF A EUKARYOTIC FMN ADENYLYLTRANSFERASE Approved by supervisory committee ____________________________________ Hong Zhang, Ph.D. (Mentor) ____________________________________ Diana Tomchick, Ph.D. (Committee Chair) ____________________________________ Kevin Gardner, Ph.D. ____________________________________ Betsy Goldsmith, Ph.D. This thesis is dedicated to my family. STRUCTURE AND MECHANISM OF A EUKARYOTIC FMN ADENYLYLTRANSFERASE by CARLOS HUERTA JR. DISSERTATION Presented to the Faculty of the Graduate School of Biomedical Sciences The University of Texas Southwestern Medical Center at Dallas In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY The University of Texas Southwestern Medical Center at Dallas Dallas, Texas December, 2009 Copyright by CARLOS HUERTA JR., 2009 All Rights Reserved ACKNOWLEDGEMENTS There are many individuals that contributed to my graduate education and I would like to thank a few of them. First, I would like to honor my mentor, Dr. Hong Zhang. Dr. Zhang’s support and encouragement was essential to the completion of my graduate education. Dr. Zhang’s compassion to X-ray crystallography and biological science was fundamental in transforming me into a structural biologist. I would also like to thank all past and current members of the Zhang laboratory. In particular, I would like to thank Dr. Nian Huang and Dr. Darek Martynowski for discussions in structure refinement and modeling, and Marcelo Raines for teaching me protein purification and crystallization. A special acknowledgement goes to Dr. Dominika Borek for teaching me how to solve my first protein structure and her support through-out my graduate education. My graduate thesis would not be possible without the guidance and understanding of my dissertation committee members Dr. Diana Tomchick, Dr. Kevin Gardner and Dr. Elizabeth Goldsmith. I owe them and Dr. Zhang my deepest gratitude. Finally, I would like to thank my family for all their support and encouragement. Without my mom, Mary Huerta, and my aunt, Jaimi Salas, my path to a graduate education would not exist. I would also like to thank my brother and my sister for encouragement and interest in my research. v STRUCTURE AND MECHANISM OF A EUKARYOTIC FMN ADENYLYLTRANSFERASE Carlos Huerta Jr., Ph.D. The University of Texas Southwestern Medical Center at Dallas, 2009 Supervising Professor: Hong Zhang, Ph.D. ABSTRACT Flavin mononucleotide adenylyltransferase (FMNAT) catalyzes the formation of the essential flavocoenzyme FAD and plays an important role in flavocoenzyme homeostasis regulation. By sequence comparison, bacterial and eukaryotic FMNAT enzymes belong to two different protein superfamilies and apparently utilize different set of active site residues to accomplish the same chemistry. Extensive biochemical studies from endogenous bacterial and mammalian FMNATs using FMN analogs and various cations have suggested that the architectures of the substrate binding and catalytic sites are different. Sequence comparison reveals that eukaryotic FMNAT is related to the PAPS reductase-like family, which belongs to the adenine nucleotide hydrolase-like superfamily. Despite the classification of eukaryotic FMNAT, the residues vi involved in substrate binding and catalysis are not completely known, as eukaryotic FMNAT has no sequence similarity to other known flavin binding proteins. To determine the unique flavin binding site, and to investigate the residues involved in substrate binding and the mechanism of catalysis, we utilized X-ray crystallography and biochemical methods. Here we report the first structural characterization of a eukaryotic FMNAT from a pathogenic yeast Candida glabrata (CgFMNAT). Four crystal structures of CgFMNAT in different complexed forms were determined at resolutions between 1.20-1.95 Å, capturing the enzyme active site states prior to and after catalysis. These structures reveal a novel flavin-binding mode and a unique enzyme-bound FAD conformation. Comparison of the bacterial and eukaryotic FMNAT provides a structural basis for understanding the convergent evolution of the same FMNAT activity from different protein ancestors. The different complexed forms of CgFMNAT allowed a structure-based investigation into the kinetic properties of eukaryotic FMNAT, whereby two “supermutants” were identified from mutagenic analysis. The steady-state kinetics and product inhibition properties of the two “supermutants” provided a basis for understanding the regulatory mechanisms of FAD homeostasis by FMNAT in eukaryotic organisms. vii TABLE OF CONTENTS ACKNOWLEDGEMENT v ABSTRACT vi PRIOR PUBLICATIONS xii LIST OF FIGURES xiii LIST OF TABLES xvii LIST OF ABREVIATIONS xviii CHAPTER 1: General Introduction A. Biological Function of Flavin Cofactors 1 B. Biosynthetic Pathway of Flavocofactors 11 1. Biosynthesis of riboflavin 11 2. Biosynthesis of flavocofactors 15 3. Genes of riboflavin biosynthesis 15 4. Genes of flavocofactor biosynthesis 20 5. Riboflavin transporter 23 C. FMNAT Protein Family 24 D. Enzymatic Properties of Bacteria and Eukaryotic FMNATs 26 E. Regulation of FAD Biosynthesis 28 F. Objective of Research 30 CHAPTER 2: Structure Determination of apo-CgFMNAT A. Introduction 32 viii B. Materials 38 C. Methods 1. Cloning, expression and purification of S. cerevisiae 39 and apo-CgFMNAT 2. Crystallization of apo-CgFMNAT 46 3. Data collection, X-ray structure determination and refinement 50 D. Results 1. Quality of apo-CgFMNAT model 55 2. Description of apo-CgFMNAT structure 61 3. Structural comparison to adenine nucleotide 73 hydrolase-like superfamily E. Discussion 77 CHAPTER 3: Structures of CgFMNAT Complexes A. Introduction 81 B. Materials 83 C. Methods 1. Expression and purification 83 2. Crystallization 84 3. Data collection, X-ray structure determination and refinement 89 D. Results 1. Quality of CgFMNAT complex structures 95 ix 2. Comparison of CgFMNAT complex structures 105 3. Substrate binary and ternary complexes − ATP binding site 115 4. Substrate ternary complex – Flavin binding site 123 5. Product ternary complex 127 6. Local conformational changes in different CgFMNAT states 131 7. Comparison of the active site with other 135 adenine nucleotide hydrolase-like proteins 8. Small molecule interaction sites on CgFMNAT surface 144 E. Discussion 1. Novel flavin-binding site in CgFMNAT 150 2. Bacterial and eukaryotic FMNATs – 155 a case of convergent evolution CHAPETER 4: Steady-state kinetics and mutagenic analysis A. Introduction 159 B. Materials 160 C. Methods 1. Mutagenesis, protein expression and purification 161 2. Steady-state kinetics analysis 163 3. Product inhibition analysis 166 D. Results 1. Steady-state kinetic analysis of CgFMNAT 170 x 2. Investigating roles of active residues by mutagenesis 172 3. Product feedback inhibition analysis 176 4. Proposed catalytic mechanism for CgFMNAT 181 E. Discussion 1. Roles of the catalytic residues 185 2. Interaction between ATP and FMN binding site 187 3. Product inhibition of CgFMNAT 189 CHAPTER 5: Conclusion and Future Direction 192 REFERENCES 198 xi PRIOR PUBLICATIONS 1. Yang K, Strauss E, Huerta C, and Zhang H. (2008) “Structural Basis for Substrate Binding and the Catalytic Mechanism of Type III Pantothenate Kinase.” Biochemistry. 47, 1369-1380. 2. Huerta C, Borek D, Machius M, Grishin NV, and Zhang H. (2009) “Structure and Mechanism of a Eukaryotic FMN Adenylyltransferase.” Journal of Molecular Biology. 389, 388-400. xii LIST OF FIGURES Figure 1.1 ............................................................................................................... 3 Figure 1.2 ............................................................................................................... 4 Figure 1.3 ............................................................................................................... 5 Figure 1.4 ............................................................................................................... 6 Figure 1.5 ............................................................................................................... 8 Figure 1.6 ............................................................................................................. 13 Figure 1.7 ............................................................................................................. 16 Figure 1.8 ............................................................................................................. 18 Figure 1.9 ............................................................................................................. 22 Figure 2.1 ............................................................................................................. 34 Figure 2.2 ............................................................................................................. 37 Figure 2.3 ............................................................................................................. 43 Figure 2.4 ............................................................................................................. 44 Figure 2.5 ............................................................................................................. 48 Figure 2.6 ............................................................................................................. 49 Figure 2.7 ............................................................................................................. 57 Figure 2.8 ............................................................................................................
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