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Reducing the Mysteries of Sulfur Metabolism in Mycobacterium Tuberculosis

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Reducing the Mysteries of Sulfur Metabolism in Mycobacterium Tuberculosis Reducing the mysteries of sulfur metabolism in Mycobacterium tuberculosis by Devayani P. Bhave A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemical Biology) in the University of Michigan 2011 Doctoral Committee: Associate Professor Kate S. Carroll, co-Chair Associate Professor Bruce A. Palfey, co-Chair Professor E. Neil G. Marsh Associate Professor Zhaohui Xu Devayani P. Bhave 2011 To my parents, grandparents and brother ii Acknowledgements I would like to express my sincere gratitude to my advisor, Professor Kate S. Carroll. She has truly been an inspirational mentor and facilitated in making my graduate school experience a productive and stimulating one. I have immensely benefitted from her guidance and her rigorous approach to research. I am also thankful to her for setting up challenging goals and for encouraging me to achieve them. I am grateful to my dissertation committee members, Professors Neil Marsh, Zhaohui Xu, Vincent Pecoraro and Bruce Palfey for providing valuable feedback and many helpful suggestions to improve my research. I especially thank Professor Palfey for his flexibility and openness in serving on my committee. I owe appreciation to all our collaborators for many discussions and detailed feedback, which contributed to this project: Professor Carsten Krebs (Pennsylvania State University), Professor Louis Noodleman and Dr. Wenge Han (The Scripps Research Institute), Professor James E. Penner-Hahn and Dr. Samuel PaZicni (University of Michigan). I am also grateful to Professor Stephen Ragsdale, Dr. Ryan KunZ, and all the members of the Ragsdale lab. I would like to acknowledge all the past and present members of the Carroll lab. I was fortunate to work alongside such fun, intelligent and hard-working scientists. In particular, I thank Jiyoung Hong, Stephen Leonard and Candice Paulsen for their camaraderie, encouragement and for iii sharing the honor of being the first batch of graduate students in the Carroll Lab. They have been wonderful friends upon whom I could rely on for advice and support. I am grateful to Jiyoung for sharing the highs and lows of our project with me, to Steve, for his cheerful disposition and kindness, and to Candie for always being jovial and positive. Khalilah Reddie and Young Ho Seo deserve special thanks for always taking the time to discuss ideas or proofread manuscripts. I thank Thu Ha Truong for sharing her enthusiasm and baking delicious cake pops. Thanks to Francisco Garcia for sharing his expertise in mass spectrometry, to Kaysia Ludford for her hard work and Hanumantharao Paritala for helpful discussions. It was always a joy to work with Jesse Song, the most discerning and sincere undergraduate student I have met. I am grateful for the opportunity to learn from the diverse group of scientists in the Chemical Biology Program in Ann Arbor. I am also grateful to the Scripps Florida community for providing a supportive environment during the final year of my doctoral work. The past five years were made more meaningful and enjoyable by a great group of friends: Gayatri-Yogesh-Nimisa, Manasi-Rohit-Reva, Nithyashanti, Preksha, Radhika, Vaishnavi, Lyra, Neha, Amit, Devesh, Shilpa-Munish-Rhea, Tapan, Nagaraj, Saumil, Minal-Harshad, Paul, Gayatri- Victor, Rajeev, Abhijit, Madhulika, Sirisha-Hanu and many more. I thank them all—this journey would be not have been as worthwhile if it were not for them. I am indebted to Vineeta and Dilip Nagarkar, Karen Husby-Coupland, Vijaya Nagesh, Sandhya, Rita, and Subhash Subhedar for their warmth and hospitality through the years. A very special thanks goes out to my teachers from various stages of life: Mrs. E, Matthew, Mrs. D. Biswas, and Professors S. M. Chitale, C. K. Desai, Amitav Mallik, and Subhash Padhye. They iv have provided guidance, motivation and kindness and taught me by example to persevere. I am fortunate for being a part of a large and loving family: my grandparents, uncles, aunts and cousins have all been supportive and always willing to lend a helping hand, especially through the tough times. I am especially grateful to my parents who always inspire me to be a better person, and encourage me to pursue my dreams. This might be the only time I put it down in words but my brother, Devasheesh, deserves a large part of the credit for this work. I also owe thanks to my sister-in-law Dipti for her thoughtfulness and enthusiasm. And I am fortunate to have had the unconditional love of our spirited dog, Dodo—his name never fails to bring a smile or a tear (or both at most times). v Preface This thesis is the compilation of published and unpublished work towards reducing the mysteries of sulfur metabolism in Mycobacterium tuberculosis. In particular, we elucidate mechanistic details of sulfonucleotide reductases (SRs) that catalyze the first committed step of reductive sulfur assimilation en route to the biosynthesis of all sulfur-containing metabolites in plants and bacteria. SRs have no human homolog and represent promising targets for therapeutic intervention. In Chapter 1, we discuss sulfur metabolism enZymes that facilitate mycobacterial survival and introduce adenosine-5'-phosphosulfate reductase (APR) as an iron-sulfur cluster protein essential for the survival of persistent M. tuberculosis. Functions of iron-sulfur clusters in different proteins are summariZed with a view to understanding the role of the cluster in APR. A portion of this discussion is published as a review for which the citation is Bhave, D. P., Muse, W.B., and Carroll, K.S., "Drug Targets in Mycobacterial Sulfur Metabolism," (2007) Infect. Disord. Drug Targets 7; 140-158. Chapter 2 details the molecular determinants that underlie binding and specificity in APR and provides an active site model as a pharmacological roadmap for the rational design of potential inhibitors of APR. This study was published as Hong, J.A., Bhave, D.P., and Carroll, K.S., "Identification of Critical Ligand Binding Determinants in Mycobacterium tuberculosis Adenosine-5'-phosphosulfate Reductase," (2009) J. Med. Chem. 52; 5485-95. vi Chapter 3 focuses on the spectroscopic characteriZation of the [4Fe-4S] cluster in APR from M. tuberculosis. A paramagnetic state of the cluster has been generated for the first time permitting the application of electron paramagnetic resonance and other forms of spectroscopies. Further, an essential role has been identified for active site residue Lys144, whose side chain serves as a link between with the [4Fe-4S] cluster and the substrate. Based on these findings, a role for the cluster in the catalytic mechanism of APR has been discussed. The citation for this article is Bhave, D.P., Hong, J.A., Lee, M., Jiang, W., Krebs, C., and Carroll, K.S., "Spectroscopic Studies on the [4Fe-4S] Cluster in Adenosine 5'-Phosphosulfate Reductase from Mycobacterium tuberculosis," (2010) J. Biol. Chem. 286; 1216-1226. In Chapter 4, we present density functional theory calculations and extended x-ray fine structure spectroscopic analyses that reveals insights into the coordination, geometry and electrostatics of the [4Fe-4S] cluster in APR. Additionally, a comparison between models with and without the tandem cysteine pair coordination of the cluster suggests a role for the unique coordination in facilitating a compact geometric structure and modulating the electrostatics of the cluster. These findings are published as Bhave, D.P., Han, W.-G., PaZicni, S., Penner-Hahn, J. E., Carroll, K.S., and Noodleman, L., "Geometric and Electrostatic Study of the [4Fe-4S] Cluster of Adenosine-5'-Phosphosulfate Reductase from Broken Symmetry Density Functional Calculations and Extended X-ray Absorption Fine Structure Spectroscopy," (2011) Inorg. Chem. 50; 6610- 6625. Chapter 5 examines substrate specificity in the family of SRs and reports that contrary to prevailing view the phosphate-binding loop in SRs has a modest effect on substrate discrimination. Instead, by means of metalloprotein engineering, spectroscopic and kinetic vii analyses, we demonstrate that the iron-sulfur cluster plays a pivotal role in substrate specificity and catalysis. The findings offer new insights into the evolution of this enZyme family, and have broader implications regarding the function of protein-bound iron-sulfur clusters. This work is under review as Bhave, D.P., Hong, J.A., Keller, R.L., M., Krebs, C., and Carroll, K.S., "Iron-Sulfur Cluster Engineering Provides Insight into the Evolution of Substrate Specificity among the Family of Sulfonucleotide Reductases," (2011), manuscript under review in ACS Chem. Bio. Finally, Chapter 6 summariZes the key findings of this thesis and provides a discussion of future directions for understanding mechanistic details of APR and the involvement of the iron-sulfur cluster toward the rational development of inhibitors for APR. viii Table of Contents Dedication ii Acknowledgements iii Preface vi List of Figures xv List of Schemes xvii List of Tables xviii List of Abbreviations xix Abstract xxii Chapter 1. Identification of Drug Targets in Mycobacterial Sulfur Metabolism with a Focus on Adenosine-5'-phosphosulfate Reductase 1.1. Abstract 1 1.1.1. Mycobacterium tuberculosis 2 1.1.2. Overview of TB Infection 3 1.1.3. Sulfur and Mycobacterial Survival 4 1.2. Sulfate Assimilation in Mycobacteria: An Overview 7 1.2.1. Sulfate Import and Activation 8 1.2.2. Sulfotransferases and Sulfation 12 1.3. Oxidative Macrophage Antimicrobial Activity 15 1.4. Sulfate Reduction 17 ix 1.5. Adenosine-5'-Phosphosulfate Reductase 20 1.5.1. Iron-Sulfur Proteins 21 1.6. Outlook 25 1.7. References 26 2. Identification of Critical Ligand Binding Determinants in Mycobacterium tuberculosis Adenosine-5'-phosphosulfate Reductase 2.1. Abstract 35 2.2. Introduction 36 2.3. Results and Discussion 40 2.3.1. Substrate Affinity 40 2.3.2. Affinity of Substrate Fragments 42 2.3.3. Affinity of Substrate Analogs 43 2.3.3.1. β-Nucleotide Substitution 43 2.3.3.2.
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