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Methanobactin: Metal Binding Properties, Physiological Function and Biosynthesis Bipin Sharma Baral Iowa State University

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Methanobactin: Metal Binding Properties, Physiological Function and Biosynthesis Bipin Sharma Baral Iowa State University Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2017 Methanobactin: Metal binding properties, physiological function and biosynthesis Bipin Sharma Baral Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biochemistry Commons Recommended Citation Baral, Bipin Sharma, "Methanobactin: Metal binding properties, physiological function and biosynthesis" (2017). Graduate Theses and Dissertations. 15256. https://lib.dr.iastate.edu/etd/15256 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Methanobactin: Metal binding proterties, physiological functions and biosynthesis by Bipin Sharma Baral A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Biochemistry Program of Study Committee: Alan DiSpirito, Major Professor Amy Andreotti Thomas Bobik Mark Hargrove Gregory Phillips The student author and the program of study committee are solely responsible for the content of this dissertation. The Graduate College will ensure this dissertation is globally accessible and will not permit alterations after a degree is conferred. Iowa State University Ames, Iowa 2017 Copyright © Bipin Sharma Baral, 2017. All rights reserved. ii TABLE OF CONTENTS Page ACKNOWLEDGMENTS ......................................................................................... iv ABSTRACT ............................................................................................................... v CHAPTER 1 GENERAL INTRODUCTION ...................................................... 1 CHAPTER 2 MERCURY BINDING BY METHANOBACTIN FROM MET- CYSTIS STRAIN SB2 .............................................................................................. 29 Abstract ................................................................................................................ 29 Introduction .......................................................................................................... 30 Materials and Methods ......................................................................................... 31 Results .................................................................................................................. 37 Discussion ............................................................................................................ 46 Conclusions .......................................................................................................... 48 Acknowledgements .............................................................................................. 49 References ............................................................................................................ 50 Table .................................................................................................................... 55 Figures.................................................................................................................. 56 Supplemental Information ................................................................................... 60 CHAPTER 3 DETOXIFICATION OF MERCURY BY METHANOBACTIN FROM METHYLOSINUS TRICHOSPORIUM OB3b ............................................ 72 Abstract ................................................................................................................ 72 Introduction .......................................................................................................... 73 Materials and Methods ......................................................................................... 75 Results .................................................................................................................. 80 Discussion ............................................................................................................ 85 References ............................................................................................................ 91 Figures.................................................................................................................. 96 Supplementary Figures ........................................................................................ 101 CHAPTER 4 COMPETITION BETWEEN METALS FOR BINDING TO MET- HANOBACTIN ENABLES EXPRESSION OF SOLUBLE METHANE MONOO- OXYGENASE IN THE PRESENCE OF COPPER .................................................. 103 Abstract ................................................................................................................ 103 Introduction .......................................................................................................... 104 Materials and Methods ......................................................................................... 106 Results .................................................................................................................. 114 Discussion ............................................................................................................ 118 References ............................................................................................................ 123 iii Figure Legends ..................................................................................................... 128 Figures and Table ................................................................................................. 129 Supplemental Information ................................................................................... 132 CHAPTER 5 A TONB-DEPENDENT TRANSPORTER IS RESPONSIBLE FO- FOR METHANOBACTIN UPTAKE BY METHYLOSINUS TRICHOSPORIUM OB3B ......................................................................................................................... 135 Abstract ................................................................................................................ 135 Introduction .......................................................................................................... 136 Materials and Methods ......................................................................................... 138 Results .................................................................................................................. 144 Discussion ............................................................................................................ 146 References ............................................................................................................ 149 Figures.................................................................................................................. 155 CHAPTER 6 AN AMINOTRANSFERASE IS RESPONSIBLE FOR THE DEAMINATION OF THE N-TERMINAL LEUCINE AND REQUIRED FOR FORMATION OF OXAZOLINE RING A IN METHNOBACTIN OF METHYL- OSINUS TRICHOSPORIUM OB3b ......................................................................... 158 Abstract ................................................................................................................ 158 Introduction .......................................................................................................... 159 Materials and Methods ......................................................................................... 161 Results .................................................................................................................. 168 Discussion ............................................................................................................ 173 References ............................................................................................................ 176 Tables and Figures ............................................................................................... 180 Supplementary Information ................................................................................. 185 CHAPTER 7 CONCLUSIONS ............................................................................ 192 iv ACKNOWLEDGEMENTS First of all I would like to thank my major professor Dr Alan A DiSpirito. Without your support and mentoring this work would not have been possible. I would also like to thank my program of study committee members: Dr. Amy Andreotti, Dr. Thomas Bobik, Dr. Mark Hargrove, and Dr.Gregory Phillips for their guidance and advice. I would also like to thank faculty, staff and graduate students in Roy J. Carver Department of Biochemistry, Molecular Biology and Biophysics for help in my research needs as well as making BBMB fun place to come to work. Friends in Ames for being there to share joy and sorrows. Finally, I would like to thank my family for their unwavering support for my education. v ABSTRACT Methanobactins (mbs) are low molecular mass (< 1300 Da) modified peptides secreted by many methanotrophs or methane oxidizing bacteria to sequester copper from the environment. To date, methanobactin has been structurally characterized from six methanotrophs and can be divided into two groups. Group I methanobactins are represented by methanobactins from Methylosinus trichosporium OB3b and Methylosinus sp. LW4. This group is characterized by the presence of two oxazolone rings with adjacent thioamide groups. Two nitrogens
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