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An Investigation of Acetobacter Aceti N5-Carboxyaminoimidazole Ribonucleotide Mutase and Its Pure-Purk Operon Charles Constantine Washington University in St

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An Investigation of Acetobacter Aceti N5-Carboxyaminoimidazole Ribonucleotide Mutase and Its Pure-Purk Operon Charles Constantine Washington University in St Washington University in St. Louis Washington University Open Scholarship All Theses and Dissertations (ETDs) January 2009 An Investigation of Acetobacter aceti N5-Carboxyaminoimidazole Ribonucleotide Mutase and Its purE-purK Operon Charles Constantine Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/etd Recommended Citation Constantine, Charles, "An Investigation of Acetobacter aceti N5-Carboxyaminoimidazole Ribonucleotide Mutase and Its purE-purK Operon" (2009). All Theses and Dissertations (ETDs). 74. https://openscholarship.wustl.edu/etd/74 This Dissertation is brought to you for free and open access by Washington University Open Scholarship. It has been accepted for inclusion in All Theses and Dissertations (ETDs) by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY Department of Chemistry Dissertation Examination Committee T. Joseph Kappock, Co-Chair Kevin D. Moeller, Co-Chair Robert Blankenship Michael L. Gross Dewey J. Holten Joseph M. Jez An Investigation of Acetobacter aceti N5-Carboxyaminoimidazole Ribonucleotide Mutase and Its purE-purK Operon by Charles Zoltan Constantine A dissertation presented to the Graduate School of Arts and Sciences of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy August 2009 Saint Louis, Missouri ABSTRACT OF THE DISSERTATION An Investigation of Acetobacter aceti N5-Carboxyaminoimidazole Ribonucleotide Mutase and Its purE-purK Operon by Charles Zoltan Constantine Doctor of Philosophy in Chemistry Washington University in St. Louis T. Joseph Kappock, Co-Chairperson Kevin D. Moeller, Co-Chairperson Acetobacter aceti oxidizes ethanol to acetic acid. While the membrane- permeable acetic acid is toxic to many bacteria, A. aceti survives exposure to acetic acid by tolerating cytoplasmic acidification. The ability to tolerate an acidic cytoplasm suggests that proteins from A. aceti are unusually suited to function in an acidic environment. The ability to tolerate an acidic cytoplasm raises additional questions about biosynthetic pathways that employ acid-labile intermediates. To examine how A. aceti metabolism may have adapted to function under acidic conditions, a biosynthetic conversion involving an acid-labile metabolite has been selected for study. The enzyme N5-carboxyaminoimidazole ribonucleotide mutase (PurE) catalyzes the reversible intramolecular transfer of the carboxylate of N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) to the C-4 position of the imidazole ring to form 4- ii carboxyaminoimidazole ribonucleotide (CAIR). A series of mutants was made to explore the role of two conserved histidines. A pH-rate comparison of AaPurE and the active mutant AaPurE-H59D was used to identify His59 as the key active site acid/base residue. The thermostability of Escherichia coli PurE (EcPurE) over a range of pH was also assessed and compared to AaPurE. AaPurE was found to be significantly more thermostable than EcPurE over the entire pH range surveyed. Comparison of the pH- rate profiles constructed for AaPurE with recently reported pH-rate profiles for EcPurE indicate that the two do not differ significantly, indicating there has been no adaptive change in enzyme mechanism. Also reported is a summary and analysis of a number of crystal structures that have been determined for AaPurE, which suggests a strategy by which proteins may have become resistant to acid-mediated inactivation. Initial functional complementation studies using the purE auxotroph PC0135 suggested that AaPurE may require AaPurK to function. We constructed a set of stable E. coli deletion strains and insertion strains that replace the chromosomal copies of purEEc or purKEc with their counterparts from A. aceti. Functional complementation experiments suggest that a third protein, located upstream of purEAa and conserved in Rhodospirillales, may be involved in the proper functioning of AaPurE. The nonenzymatic decarboxylation of CAIR and the corresponding ribonucleoside (CAIR-s) were examined as models for the PurE reaction. The decarboxylation of CAIR-s was not acid-catalyzed in the pH range examined, and did not accelerate in lower-polarity solvents. iii ACKNOWLEDGEMENTS I would like to thank my advisor Professor T. Joseph Kappock for his invaluable guidance over the years. Experiencing his unique scientific insight has been the highlight of my education. I would like to thank my Ph. D. Advisory Committee members Professors Michael L. Gross and Kevin D. Moeller for their insight in the classroom and during the course of my research. I thank Professors Joseph Jez, Dewey Holten and Robert Blankenship for serving on my dissertation defense committee. Thanks to the members of the Kappock group, past and present, Dr. Julie Francois, Dr. Hong Jiang, Elwood Mullins, and John Hung, and especially Dr. Courtney Starks for their aid and friendship. I would also like to thank members of the Gross group, Dr. David Hambly, Dr. Justin Sperry and Sandy Kerfoot for their aid and accessibility over the years. I would especially like to thank Dr. Ed Hiss; words cannot describe what an asset he is. No list of acknowledgements would be complete without thanking those who got me where I am today. Thank you to my family for their support and encouragement over the years. Thank you to my father for answering the never ending string of whys I provided as a child. Thank you to the amazing teachers and professors I had before coming to Washington University, Cristina Geiger, Gayle Stout, and Dr. Harold Hoops. Thank you to those who are no longer with us: to my Uncle John Constantine for his support in the strange new city of St. Louis, and to my high school chemistry teacher Nancy Middleton; I now realize what a remarkable teacher you were. iv Finally I would like to thank my friends. To Keith Taylor III and Patrick Lynn Clayton, thank you for being friends that I could count on through thick and thin at a moment’s notice. To Dr. Elizabeth Elliott, Brian Barnes, Megan Daschbach, Matt VanDuzor and Dr. Dayna Tucker, without your friendship in graduate school, this would have been a far less enjoyable experience. v TABLE OF CONTENTS Abstract of the Dissertation.................................................................................ii Acknowledgments...............................................................................................iv Table of Contents................................................................................................vi List of Figures......................................................................................................viii List of Tables.......................................................................................................xii List of Schemes...................................................................................................xiv Chapter 1. Introduction 1.1 Perspective............................................................................................. 2 1.2 Purpose.................................................................................................. 6 1.3 References............................................................................................. 8 Chapter 2. Non-enzymatic decarboxylation of 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) and 4-carboxy-5-aminoimidazole ribonucleoside (CAIR-s). 2.1 Introduction……………………………………………………….................................. 11 2.2 Methods and Materials.......................................................................... 19 2.3 Results.................................................................................................... 22 2.4 Discussion............................................................................................... 28 2.5 Future Directions.................................................................................... 38 2.6 References.............................................................................................. 39 Chapter 3. N5-Carboxyaminoimidozole Ribonucleotide Mutase from Acetobacter Aceti 3.1 Introduction........................................................................................... 43 3.2 Methods and Materials.......................................................................... 46 3.3 Results.................................................................................................... 53 3.4 Discussion............................................................................................... 68 3.5 Future Directions.................................................................................... 85 3.6 References.............................................................................................. 86 Chapter 4. The purE-purK operon of A. aceti 4.1 Introduction........................................................................................... 90 4.2 Methods and Materials.......................................................................... 95 4.3 Results.................................................................................................... 133 vi 4.4 Discussion............................................................................................... 160 4.5 Future Directions.................................................................................... 183 4.6 References.............................................................................................
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