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A Dissertation Entitled Structural and Enzymatic Studies of Essential A Dissertation entitled Structural and Enzymatic Studies of Essential Enzymes in Mycobacterium tuberculosis by Jared J. Lindenberger Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Chemistry _________________________________________ Dr. Donald R. Ronning, Committee Chair _________________________________________ Dr. James T. Slama, Committee Member _________________________________________ Dr. Steven J. Sucheck, Committee Member _________________________________________ Dr. John J. Bellizzi, Committee Member _________________________________________ Dr. Patricia R. Komuniecki, Dean College of Graduate Studies The University of Toledo August 2015 Copyright 2015, Jared J. Lindenberger This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Structural and Enzymatic Studies of Essential Enzymes in Mycobacterium tuberculosis by Jared J. Lindenberger Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Chemistry The University of Toledo August 2015 Tuberculosis (TB) continues to be a global health threat. The World Health Organization estimates that nearly 8.6 million new TB cases were reported, and 1.3 million people succumbed to the disease in 2013. The persistence of TB globally is due in part to the hardiness of the bacterium Mycobacterium tuberculosis (M. tb), the etiological agent of TB, and the ability of M. tb to enter a dormant phase that complicates treatment. Typical therapeutic regimens to treat M. tb infections require at least 6 months of anti-tubercular drugs and patient non-compliance during this treatment period is thought to contribute to the selection of antibiotic resistant strains. In 2012, 1 in 5 cases of TB were multiply drug resistant (MDR-TB), while nearly 1 in 10 of these MDR-TB cases was also extensively drug resistant (XDR-TB). Totally drug resistant TB (TDR-TB), which is untreatable by current TB drugs, has also emerged. With current therapies becoming ineffective and the high TB burden continuing worldwide, the need for new drug targets and new therapeutics is of paramount importance. iii The first part of this work describes the biochemical and structural characterization of GlgE, a maltosyltransferase from M. tb. Knock-out of GlgE causes rapid death of the bacterium, which is the result of a toxic build-up of the substrate maltose-1-phosphate. This novel method of killing makes GlgE an intriguing drug target for the design of novel inhibitors to kill M. tb. Here we report the design of a fluorescent based assay to monitor GlgE activity. This was utilized to characterize the kinetics of the enzyme, measure and characterize mutant forms of the enzyme, and screen a compound library for potential inhibitors. Additionally, we report the crystal structures of the M. tb GlgE in a binary complex with maltose, and a ternary complex with maltose and a maltooligosaccharide, maltohexaose. The second part of this work utilized the structural data obtained from the M. tb GlgE to mutate a homologous GlgE from Streptomyces coelicolor to produce an enzyme that crystallizes to higher resolutions than the M. tb GlgE, while having an identical active site. This S. co GlgEI-V279S was used to structurally characterize interactions between the enzyme and four different inhibitor compounds. We also report the design of a novel coupled assay to monitor GlgE activity. This assay was fast, reproducible, and highly amenable to high-throughput screening applications. The next part of this work looks at an essential enzyme in the biosynthesis of trehalose in Mycobacterium tuberculosis. TPP2, a trehalose-6-phosphate phosphatase, is essential for the viability of the organism. It has been speculated that cellular death is the result of the build-up of trehalose-6-phosphate, the substrate. This method of killing makes it an interesting target for the design of inhibitors. Here we report the design of a novel, coupled assay to monitor the activity of TPP2. This assay was also highly amenable to high- iv throughput screening applications. We also attempted to crystallize and solve the structure of the TPP2 enzyme. However, structural information has remained elusive to this date. Crystallization experiments are ongoing, with several new conditions and crystals awaiting diffraction experiments. The final chapter of this work looks at the structural and biochemical characterization of a mycothiol-S-transferase from M. tb (MtMST). MtMST is responsible for maintaining redox homeostasis in the bacterium, detoxifying xenobiotics and electrophilic toxins, and has been implicated in the neutralization of some of the common anti-tubercular drugs used in the treatment of TB. Here we report the first structure of the MtMST. Additionally; we developed an assay to monitor MtMST activity, and utilized this to determine the ability of MtMST to modify antibiotics in vitro. v Acknowledgements First and foremost, I would like to thank my boss, Dr. Donald Ronning. He has been instrumental towards my success in the lab and I will forever be grateful for the opportunities, and knowledge I have obtained in his lab. I would also like to thank my committee members, Drs. Sucheck, Bellizzi, and Slama, for helping me, and giving me guidance through the various projects. Next, I need to thank all my lab members who have come and gone throughout my time in the lab. This includes, but is not limited to: Dan, Vidhi, Lorenza, Lucile, Erica, Chris, Mike, Cecile, Paniz, all the undergraduates who have been in the lab, and if I missed someone, thank you too. Everyone has been helpful and made every day in the lab enjoyable. I especially need to thank both Sri and Sandeep for helping make the GlgE project successful. I also need to thank all the friends I have made these five years. I love you all, and appreciate everything. Lastly, and most importantly, I want to thank my family for all their love and support. To my mom and pops for their constant love and support over the years, and for all the free meals you’ve supplied, and to my brothers, Japey and Biggins, for always keeping it real with me and never missing an opportunity to make fun of each other. vi Table of Contents Abstract .................................................................................................................................... iii Acknowledgements .................................................................................................................. vi Table of Contents .................................................................................................................... vii List of Tables ......................................................................................................................... xiv Table of Figures ...................................................................................................................... xv List of Equations .................................................................................................................... xix List of Abbreviations .............................................................................................................. xx List of Symbols .................................................................................................................... xxiii 1 Chapter 1 ....................................................................................................................... 24 A primer on Mycobacterium tuberculosis .............................................................................. 24 1.1 Background on Tuberculosis and Mycobacterium tuberculosis ................................... 24 1.1.1 The Tuberculosis Burden ....................................................................................... 24 1.2 Characterization of Mycobacterium tuberculosis ......................................................... 25 1.2.1 The pathogenic Mycobacterium ............................................................................. 25 1.2.2 Description of Mycobacterium tuberculosis .......................................................... 25 1.2.2 Infection and pathogenesis of the disease .............................................................. 26 1.2.3 Treatments .............................................................................................................. 27 vii 1.2.4 Challenge of drug resistance and co-infection with HIV ....................................... 29 1.3 Overview and significance of the different projects ..................................................... 31 1.3.1 GlgE ....................................................................................................................... 31 1.3.2 TPP2 ....................................................................................................................... 32 1.3.3 Mycothiol-S-transferase ......................................................................................... 32 2 Chapter 2 ....................................................................................................................... 34 Kinetic and structural characterization of the Mycobacterium tuberculosis maltosyltransferase, GlgE ....................................................................................................... 34 2.1 Background ..................................................................................................................
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