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Characterization of the Thioredoxin System of Methanosarcina Mazei

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Characterization of the Thioredoxin System of Methanosarcina Mazei Characterization of the Thioredoxin System of Methanosarcina mazei Usha Loganathan Thesis submitted to the faculty of Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Master of Science In Biological Sciences Committee: Dr. Biswarup Mukhopadhyay, Chair Dr. David L. Popham, Co-Chair Dr. Birgit Scharf December 1, 2014 Blacksburg, Virginia Key words: Methane, fuel, greenhouse gas, Methanogen, Archaea, Methanosarcina mazei, thioredoxin, NADPH thioredoxin reductase, ferredoxin thioredoxin reductase, oxidative stress, redox regulation. Copyright 2014, Usha Loganathan Characterization of the thioredoxin system of Methanosarcina mazei Usha Loganathan ABSTRACT Thioredoxin (Trx) and thioredoxin reductase (TrxR) along with an electron donor form a thioredoxin system. Such systems are widely distributed among the organisms belonging to the three domains of life. It is one of the major disulfide reducing systems, which provides electrons to several enzymes, such as ribonucleotide reductase, methionine sulfoxide reductase and glutathione peroxidase to name a few. It also plays an important role in combating oxidative stress and redox regulation of metabolism. Trx is a small redox protein, about 12 kDa in size, with an active site motif of Cys-X-X-Cys. The reduction of the disulfide in Trx is catalyzed by TrxR. Two types of thioredoxin reductases are known, namely NADPH thioredoxin reductase (NTR) with NADPH as the electron donor and ferredoxin thioredxoin reductase (FTR) which depends on reduced ferredoxin as electron donor. Although NTR is widely distributed in the three domains of life, it is absent in some archaea, whereas FTRs are mostly found in plants, photosynthetic eukaryotes, cyanobacteria, and some archaea. The thioredoxin system has been well studied in plants, mammals, and a few bacteria, but not much is known about the archaeal thioredoxin system. Our laboratory has been studying the thioredoxin systems of methanogenic archaea, and a major focus has been on Methanocaldococcus jannaschii, a deeply rooted archaeon that has two Trxs and one TrxR. My thesis research concerns the thioredoxin system of the late evolving members of the group which are exposed to oxygen more frequently than the deeply rooted members of the group, and have several Trxs and TrxRs. Methanosarcina mazei is one such organism, whose thioredoxin system is composed of one NTR, two FTRs, and five Trx homologs. Characterization of the components of a thioredoxin system sets the basis to further explore its function. I have expressed in Escherichia coli and purified the five Trxs and three TrxRs of M. mazei. I have shown the disulfide reductase activities in MM_Trx1 and MM_Trx5 by their ability to reduce insulin with DTT as the electron donor, and that in MM_Trx3 through the reduction of DTNB by this protein with NADPH as the electron donor, and in the presence of NTR as the enzyme. MM_Trx3 was found to be the only M. mazei thioredoxin to accept electrons through the NTR, and to form a complete Trx - NTR system. The Trx - FTR systems are well studied in plants, and such a system is yet to be defined in archaea. I have proposed a mechanism of action for one of the FTRs. FTR2 harbors a rubredoxin domain, and this unit is the only rubredoxin in this organism. Superoxide reductase, an enzyme that reduces superoxide radical to hydrogen peroxide without forming oxygen, utilizes rubredoxin as the direct electron source and this enzyme is found in certain anaerobes, including Methanosarcina species. Thus, it is possible that FTR2 provides electrons via a Trx to the superoxide reductase of M. mazei. This activity will define FTR2 as a tool in combating oxidative stress in M. mazei. In my thesis research I have laid a foundation to understand a complex thioredoxin system of M. mazei, to find the role of each Trx and TrxR, and to explore their involvement in oxidative stress and redox regulation. iii DEDICATION In memory of my loving husband. To my daughters Uma and Abhi. iv ACKNOWLEDGEMENTS My deepest gratitude and thanks to Dr. Biswarup Mukhopadhyay who has been my advisor, not only through my graduate schooling but also my personal life. The word ‘thanks’ does not do justice for all that he has done for me and my children. He reached out to me when I needed help the most in my life, which I can never forget. He has spent a lot of his valuable time to patiently teach me. He continues to guide me and my children, and I greatly value his opinion and friendship. No amount of words can repay the kindness and help I have received from him. Thank you for believing that I can do it. I thank my committee members Drs. Birgit Scharf and David Popham for serving in my committee, for encouraging and guiding me through my graduate school, and for their valuable time, advice and patience. I thank Dr. Ruth A. Schmitz - Streit at University of Kiel, Germany, for providing the preliminary data on growth of Methanosarcina mazei under oxygen sensitive conditions, and for providing the clones for the NADPH thioredoxin reductase, ferredoxin thioredoxin reductases and one of the thioredoxins. I thank Dr. Bob B. Buchanan of University of California, Berkeley, for the gift of purified spinach ferredoxin NADPH oxidoreductase (FNR). I thank Dr. Jiann-Shin Chen of Virginia Tech, Blacksburg for his gift of purified clostridial ferredoxin and hydrogenase. I thank Virginia Bioinformatics Institute, the Department of Biochemistry, the Department of Biological Sciences, and Virginia Tech for giving me the opportunity to pursue my Masters. I thank my lab members Dr. Endang Purwantini, Dr. Dwi Susanti and Jason Rodriguez who have become my very close friends, and are always there to help, support, guide and give me suggestions. My v special thanks to Dwi who has been mentoring me since the day I joined Biswarup’s lab. I have learned a lot from her over the years. She has helped me get through the rough times of my life. Thank you for cheering me on. Thank you all for being a wonderful friend. I thank my two wonderful daughters, Uma and Abhi, who have stood by me, encouraging and cheering me as I walked through this path. I could not have done it without their support. I thank the community of Virginia Tech and the New River Valley for their kindness and support through the past few years. I have been blessed with the friendship, kindness and support from everyone on earth. I am eternally grateful for all that I have received. I thank you all from the bottom of my heart. vi TABLE OF CONTENTS ABSTRACT ...................................................................................................................................................... ii DEDICATION ................................................................................................................................................. iv ACKNOWLEDGEMENTS ................................................................................................................................. v TABLE OF CONTENTS ................................................................................................................................... vii LIST OF FIGURES ............................................................................................................................................ x LIST OF TABLES ............................................................................................................................................ xiv Chapter 1: LITERATURE REVIEW ................................................................................................................... 1 OXIDATIVE STRESS IN METHANOGENIC ARCHAEA ................................................................................... 1 METHANOGENS .................................................................................................................................... 1 OXIDATIVE STRESS ................................................................................................................................ 3 THIOREDOXIN SYSTEM .......................................................................................................................... 9 RESEARCH INTRODUCTION ................................................................................................................. 13 RESEARCH OBJECTIVES ....................................................................................................................... 14 Chapter 2: MATERIALS AND METHODS ...................................................................................................... 17 Construction of vectors for the expression of M. mazei NTR, FTRs, and Trxs in E. coli .......................... 17 Expression of M. mazei Trxs, NTR, and FTRs in E. coli ............................................................................ 17 Purification of the recombinant proteins ............................................................................................... 18 Western blot ........................................................................................................................................... 20 vii State of oxidation of M. mazei Trxs ........................................................................................................ 20 Oxidation and reduction of M. mazei Trxs .............................................................................................. 21 Analysis of oxidized and re-reduced Trx preparations ..........................................................................
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