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Radical-Sam Enzymes with Two Iron-Sulfur Clusters: Cofactor

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RADICAL-SAM ENZYMES WITH TWO IRON-SULFUR CLUSTERS: COFACTOR COMPOSITION AND SPECTROSCOPIC STUDIES OF ESCHERICHIA COLI AND BACILLUS SUBTILIS BIOTIN SYNTHASE, HUMAN MOCS1A, AND THERMOTOGA MARITIMA MIAB by HEATHER LOUISE HERNÁNDEZ (Under the Direction of Michael Kenneth Johnson) ABSTRACT A new class of Fe-S proteins, termed radical-SAM enzymes, catalyzes radical reactions in a variety of biosynthetic processes. These [4Fe-4S]2+,+ cluster-containing enzymes initiate radical enzymatic reactions via reductive cleavage of S-adenosyl-L-methionine (SAM) to yield methionine and an extremely reactive 5'-deoxyadenosyl radical. A growing number of radical-SAM enzymes have recently been discovered to contain a second Fe-S cluster of unknown function, although possible roles include acting as sacrificial S-donor or anchoring and possibly activating the substrate. The combination of analytical and spectroscopic studies, including EPR, Mössbauer, UV-visible absorption/circular dichroism/variable temperature magnetic circular dichroism, and resonance Raman, have been used to investigate the cofactor composition and properties of the two cluster containing radical-SAM enzymes Escherichia coli and Bacillus subtilis biotin synthase (BioB), human MOCS1A, and Thermotoga maritima MiaB. These enzymes are involved in crucial steps in the biosynthesis of biotin and molybdopterin, and in the thiomethylation of tRNA. E. coli and B. subtilis BioB are shown to house a radical-SAM [4Fe-4S] cluster and a [2Fe-2S] cluster in separate binding sites. The function and relevance of the [2Fe-2S] cluster, which has been suggested to be the S-donor to biotin, is addressed. In E. coli BioB, the most active form of the enzyme contains a 1:1 ratio of [2Fe-2S]/[4Fe-4S] clusters and the [2Fe-2S] cluster degrades during turnover. However, Mössbauer studies of E. coli BioB show that [2Fe-2S] cluster degradation is at least an order of magnitude faster than the initial rate of biotin formation and B. subtilis BioB exhibits comparable activity in samples depleted in the [2Fe-2S] cluster. Taken together, these results suggest that a [2Fe-2S] cluster degradation product may be the physiologically relevant S-donor. Both MOCS1A and T. maritima MiaB are characterized and shown to contain a radical-SAM [4Fe-4S] cluster as well as a second [4Fe-4S] cluster. In MOCS1A, the second cluster now appears to play a role in anchoring the 5’- GTP substrate. In T. maritima MiaB, the second cluster does not appear to interact with the tRNA substrate and may function as a S-donor in the thiomethylation reaction. INDEX WORDS: Radical-SAM superfamily, SAM, iron-sulfur cluster, radical generation, biotin synthase, MOCS1A, MiaB, electron paramagnetic resonance, resonance Raman, magnetic circular dichroism, Mössbauer RADICAL-SAM ENZYMES WITH TWO IRON-SULFUR CLUSTERS: COFACTOR COMPOSITION AND SPECTROSCOPIC STUDIES OF ESCHERICHIA COLI AND BACILLUS SUBTILIS BIOTIN SYNTHASE, HUMAN MOCS1A, AND THERMOTOGA MARITIMA MIAB by HEATHER LOUISE HERNÁNDEZ B.S., Michigan State University, 1998 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2006 © 2006 Heather Louise Hernández All Rights Reserved RADICAL-SAM ENZYMES WITH TWO IRON-SULFUR CLUSTERS: COFACTOR COMPOSITION AND SPECTROSCOPIC STUDIES OF ESCHERICHIA COLI AND BACILLUS SUBTILIS BIOTIN SYNTHASE, HUMAN MOCS1A, AND THERMOTOGA MARITIMA MIAB by HEATHER LOUISE HERNÁNDEZ Major Professor: Michael K. Johnson Committee: Michael W. W. Adams Robert A. Scott Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia December 2006 iv DEDICATION To my husband, Noé, our daughter, Nora, and my parents, Leonard and Joanne, for their unwavering love and support. v ACKNOWLEDGEMENTS I would like to thank my advisor, Professor Michael K. Johnson, for his tutelage, direction and time. I greatly appreciated that your door was always open and you were always eager to discuss science or whatever else was on our minds. Thank you for also understanding your students have lives outside of lab (as long as we are producing enough data!). Thank you to my entire advisory committee, Professors Michael W. W. Adams, Marly K. Eidsness, Donald M. Kurtz, Jr. and Robert A. Scott. Our discussions were thought provoking and your time was appreciated. Thanks to all the lab mates that I have had the pleasure of working with during my time in the Johnson lab. Each of you has helped make life in the lab most enjoyable. Mostly, I would like to thank (in alphabetical order) Kala Chandramouli, Michele Mader Cosper, and Elizabeth Mengelt Walters for being such great friends and for providing so much encouragement and support throughout the years. You helped make the not so good times bearable and the good times great. Last, but certainly not least, thank you to my parents, Leonard and Joanne Sackett, and my husband, Noé. Thanks mom and dad for teaching me that the skies not even the limit and that I can accomplish anything that I put my mind to. Nobody could wish for better parents than the two of you. And to my husband, thank you for always being there and always being willing to “do what it takes.” I appreciate you so much. And of course to my beautiful, happy baby, Nora, thank you for being you. Your precious smiles make the rest of the world melt away. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.............................................................................................................v LIST OF TABLES......................................................................................................................... ix LIST OF SCHEMES........................................................................................................................x LIST OF FIGURES ....................................................................................................................... xi CHAPTER 1 Introduction....................................................................................................................1 Structure and Function of Iron-Sulfur Proteins.........................................................1 Radical-SAM Enzymes .............................................................................................6 References ...............................................................................................................21 2 Characterization of the Cofactor Composition of Escherichia coli Biotin Synthase ..54 Abstract ...................................................................................................................56 Introduction .............................................................................................................57 Materials and Methods ............................................................................................61 Results .....................................................................................................................67 Discussion ...............................................................................................................81 References ...............................................................................................................87 3 Role of the [2Fe-2S] Cluster in Recombinant Escherichia coli Biotin Synthase......115 Abstract .................................................................................................................117 Introduction ...........................................................................................................118 vii Materials and Methods ..........................................................................................121 Results ...................................................................................................................123 Discussion .............................................................................................................135 References .............................................................................................................140 4 Cofactor Composition, Spectroscopic Properties and Activity Studies of Bacillus subtilis Biotin Synthase.........................................................................................161 Abstract .................................................................................................................163 Introduction ...........................................................................................................164 Materials and Methods ..........................................................................................167 Results ...................................................................................................................172 Discussion .............................................................................................................183 References .............................................................................................................188 5 Characterization of MOCS1A, an Oxygen-sensitive Iron-Sulfur Protein Involved in Human Molybdenum Cofactor Biosynthesis ........................................................216 Abstract .................................................................................................................218 Introduction ...........................................................................................................219 Experimental Procedures.......................................................................................221
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