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The Pennsylvania State University The Pennsylvania State University The Graduate School Eberly College of Science MECHANISTIC, SPECTROSCOPIC, AND STRUCTURAL CHARACTERIZATION OF TWO NOVEL REACTIONS CATALYZED BY RADICAL S-ADENOSYLMETHIONINE DEPENDENT ENZYMES A Dissertation in Chemistry by Tyler L. Grove 2013 Tyler L. Grove Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2013 The dissertation of Tyler L. Grove was reviewed and approved* by the following: Squire J. Booker Associate Professor of Chemistry and of Biochemistry and Molecular Biology Dissertation Advisor Co-Chair of Committee Joseph Martin Bollinger, Jr. Professor of Chemistry and of Biochemistry and Molecular Biology Co-Chair of Committee Carsten Krebs Associate Professor of Chemistry and of Biochemistry and Molecular Biology Christopher House Associate Professor of Geosciences Barbara J. Garrison Professor of Chemistry Head of the Department of Chemistry *Signatures are on file in the Graduate School iii ABSTRACT Part I: Characterization of Radical SAM-Dependent Methyl Synthases. The bacterial ribosome is the target of about half of all antibiotics currently in use. Antibiotics bind to this huge macromolecular machine, which is composed of both proteins and RNA, and disrupt its function, which is to synthesize proteins required for bacterial survival. Frequently, bacteria develop mechanisms to prevent antibiotics from binding to their ribosomes, which most often involve modifying or changing specific amino acid residues or nucleotides that line the antibiotic binding site. One such modification is the addition of a methyl (–CH3) group to adenosine 2503 (A2503) of 23S ribosomal RNA (rRNA). This nucleotide is absolutely conserved, and is located within domain V of the ribosome which is the catalytic center where peptide bonds are formed. Methylation of carbon 2 of A2503 is catalyzed by the enzyme RlmN. This modification is ubiquitous among prokaryotes, and like most modifications to the ribosome, is believed to be important in optimal functioning of the ribosome. Interestingly, the addition of a methyl group to carbon 8 of A2503 confers resistance to over seven classes of antibiotics, some of which are antibiotics of last resort for patients infected with methicillin resistant Staphylococcus aureus (MRSA) or vancomycin-resistant Enterococcus. This activity is encoded by the product of the cfr gene (Cfr), which has been found in MRSA isolated from hospitalized patients from numerous countries, portending a new and dangerous mechanism for the spread of antibiotic resistance. RlmN and Cfr have been predicted to be evolutionarily related, as well as belong to the radical SAM (RS) superfamily of enzymes. RS enzymes all utilize S- iv adenosylmethionine (SAM), as a precursor to a potently oxidizing 5’-deoxyadenosyl 5’- radical (5’-dA•). The commonality among the reactions catalyzed by these enzymes is that the 5’-dA• initiates catalysis by abstracting a key hydrogen atom from the substrate. Our studies on RlmN and Cfr have elucidated the chemical logic by which these enzymes are capable of installing a methyl group onto an unreactive, sp2-hybridized carbon, revealing a completely new strategy that expands the repertoire of enzyme-catalyzed reactions. To elucidate the mechanistic strategy of RlmN and Cfr, we used high-resolution mass spectrometry in concert with isotopically labeled substrates to show that, unlike most RS enzymes, RlmN and Cfr are capable of activating SAM toward two distinct types of reactivities within a single active site. Interestingly, the first step in each reaction was shown to be the SAM-dependent methylation of a conserved cysteinyl residue, in the process making a methylcysteinyl residue that is subsequently activated by the 5’-dA•. The methylcysteinyl radical then adds to the nucleotide substrate, generating a radical enzyme–substrate crosslink, which we were able to trap in Cfr and characterize by electron paramagnetic resonance and electron-nuclear double resonance spectroscopies. In addition we were able to show that cleave age of this radical enzyme–substrate crosslink is driven by formation of a radical disulfide anion bond between the methyl- carrying Cys residue and another strictly conserved Cys residue, with concomitant release of the product from the protein. In addition, through a collaboration with Professor Amy Rosenzweig at Northwestern University, we were able to determine the crystal structure of RlmN to 2.05 v Å, which allowed the visualization of the methylated cysteinyl residue predicted in our earlier biochemical studies. Part II: Characterization of Radical SAM-Dependent Dehydrogenases The second part of this dissertation was the study of a different and unique class of RS enzymes, the RS dehydrogenases. These enzymes use radical chemistry to effect two-electron oxidations of organic substrates via intermediates containing single unpaired electrons. This unique reaction was predicted to contain multiple iron–sulfur (Fe/S) cluster. All RS enzymes require 1 [4Fe–4S] cluster, which is ligated by cysteines found in the RS motif, CxxxCxxC. The RS cluster interacts with SAM to facilitate its cleavage to the 5’-dA•. The existence and the role of the additional cluster(s) were not as clear. This details the characterization of three enzymes from this class: AtsB, anSMEcpe, and BtrN. BtrN was previously shown to catalyze a key step in the biosynthesis of the antibiotic butirosin B, which is the oxidation of a secondary alcohol located on a deoxysugar to a ketone. AtsB and anSMEcpe, on the other hand, catalyze the oxidation of a seryl or cysteinyl residue on a cognate protein to a formylglycyl (FGly) residue. The FGly residue is found in arylsulfatases, where it plays a unique role as a cofactor catalyzing the hydrolysis of sulfate monoesters. We were able to shown that, in contrast to the majority of radical SAM enzymes, each of these proteins contains two or more [4Fe–4S] clusters that are absolutely required for turnover. In addition, the stoichiometry of the AtsB and anSMEcpe reactions was determined as well as the stereochemical course of their reactions; in both cases the pro-S hydrogen of the substrate is abstracted by the 5’-dA•. In addition, several radical species have been trapped in reactions catalyzed by anSAMcpe which will allow a detail spectroscopic study of the active site. vi To truly understand the role of the auxiliary cluster(s) in these reactions, we developed conditions for crystallizing both BtrN and anSMEcpe in the presence of substrates. In collaboration with Professor Cathy Drennan (MIT), we solved the structure of both anSMEcpe and BtrN to a resolutions of ~1.6 Å. vii TABLE OF CONTENTS List of Abbreviations .......................................................................................................... v List of Figures..................................................................................................................... v List of Tables ...................................................................................................................... vi Acknowledgements ............................................................................................................. vii Chapter 1 ............................................................................................................................ 1 Introduction ......................................................................................................... 1 1.1 Radical SAM enzymes and Human Health. .................................................... 3 1.2 Radical SAM enzymes with multiple iron–sulfur clusters. .............................. 8 1.3 Maturation of complex metallocofactors. ........................................................ 19 1.4 Radical SAM enzymes lacking the canonical CxxxCxxC motif. ..................... 23 1.5 Advances in understanding the reductive cleavage of S- adenosylmethionine. ..................................................................................... 28 1.6 Future directions. ........................................................................................... 30 1.7 References .................................................................................................... 33 Part I Characterization of Radical SAM-Dependent Methyl Synthases. ......................................... 49 Chapter 2 A Radically Different Mechanism for S-adenosylmethionine-dependent Methyltransferases Exhibited by the Antibiotic Resistance Protein Cfr and its Homologue RlmN ....................................................................................................... 50 2.1 Abstract ......................................................................................................... 52 2.2 Introduction. .................................................................................................. 54 2.3 Materials and Methods ................................................................................... 57 2.4 Results ........................................................................................................... 69 2.5 Discussion...................................................................................................... 88 2.6 Acknowledgements ........................................................................................ 90 2.7 References. .................................................................................................... 91 Chapter 3 Cfr and RlmN Contain Only One Binding Site for S-Adenosylmethionine,
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