Open JDG Dissertation March2020

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Open JDG Dissertation March2020 The Pennsylvania State University The Graduate School BIOCHEMICAL CHARACTERIZATION OF PHYLOGENETICALLY DIVERSE HOMOLOGS OF THE ANTIBIOTIC RESISTANCE PROTEIN CFR AND RELATED GENOME NEIGHBORS A Dissertation in Chemistry by James David Gumkowski Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2020 The dissertation of James David Gumkowski was reviewed and approved by the following: Amie K. Boal Associate Professor of Chemistry Associate Professor of Biochemistry and Molecular Biology Dissertation Advisor Chair of Committee Squire J. Booker Howard Hughes Medical Investigator Professor of Chemistry Professor of Biochemistry and Molecular Biology Eberly Distinguished Chair in Science Carsten Krebs Professor of Chemistry Professor of Biochemistry and Molecular Biology Kenneth Keiler Professor of Biochemistry and Molecular Biology Associate Department Head for Graduate Education Philip Bevilacqua Department Head, Chemistry Distinguished Professor of Chemistry Distinguished Professor of Biochemistry and Molecular Biology ii ABSTRACT Radical S-adenosylmethionine (SAM) enzymes are one of the largest protein superfamilies identified to date, with more than 500,000 annotated members. While the functions of these enzymes vary widely, one of the most challenging chemical transformations that radical SAM enzymes are known to perform is methylation of unactivated carbon or phosphorous centers. The enzymes that catalyze these difficult reactions are known as the Class A-D radical SAM methyltransferases. The best characterized of the radical SAM methyltransferases are Class A, composed of RlmN and Cfr. RlmN is a housekeeping enzyme found in many bacteria that methylates rRNA and some tRNAs and is thought to promote translational fidelity. In contrast, Cfr is found in relatively few bacteria, largely Firmicutes, and its modification of rRNA has been shown to confer antibiotic resistance to agents which target the exit tunnel of the peptidyl transferase center. It accomplishes this by appending a methyl group to the C8 position of A2503 (Escherichia coli numbering). cfr is of particular concern presently because it has been found on transposable elements and is appearing with increasing frequency in clinical settings. The original aim of this work was to structurally characterize a member of the Cfr family, a goal which required finding sequences which diverge from model system Staphylococcus aureus Cfr. In chapter 2, four phylogenetically diverse Cfr homologs were isolated and characterized. Though none proved amenable to structural studies, biophysical and spectroscopic analyses revealed that all four homologs contained the radical SAM [4Fe-4S] cluster and were able to perform C8 methylation of a 155mer substrate mimic with varying efficiencies. These results suggest that Cfr-like activity iii associated with drug resistance can be found in a diverse set of organisms, some of which are human pathogens. I also showed that the identity of the iron-sulfur cluster reductant can have a significant impact on activity. The small molecule reductant dithionite yielded more dimethylated product compared to a flavodoxin protein-based reducing system. This work underscores the importance of studying Cfr activity with native reductants, because reaction outcome can be influenced by the identity of the reducing system and the rate at which it operates relative to RNA product release. In this dissertation I show that a clostridial Cfr homolog is an active rRNA methylase in vitro, a surprising observation given the lack of activity in other homologs from the clostridial clade. Additionally, the Mössbauer spectrum of Clostridioides difficile Cfr demonstrated the presence of a mononuclear iron cofactor in the C-terminal domain. In chapter 3, I use truncation variants to verify the biophysical and functional properties of this domain. When isolated separately, the domain binds a single iron (II) ion with rubredoxin-like properties. Addition of this domain in trans to a clostridial Cfr variant containing only the radical SAM domain did not affect activity. However, mismetallation of the domain or mutation of a predicted Cys ligand nearly eliminates methylation of a 155mer RNA substrate, suggesting an essential role for iron when the domain is present. Co-elution of the domain with RNA could also indicate a function related to RNA substrate binding. These results raise the possibility that the limited activity demonstrated by some clostridial Cfrs could be due to a lack of additional protein factors available in the native host organisms but missing when the RNA methylase is overexpressed by itself in a heterologous host. iv The C-terminal domain identified on Clostridioides difficile Cfr is homologous to members of a family of small, uncharacterized proteins containing two pairs of CXXC motifs named Cys-rich KTR. In chapter 4, I isolate a standalone KTR from Staphylococcus aureus and demonstrate that it, like the C-terminal domain of the clostridial Cfr, coordinates a single iron and binds RNA. This result suggests that the members of this family could share a requirement for iron and that they may all function in RNA-binding or electron transfer. Bioinformatic analysis of the clostridial Cfrs revealed that all conserve either a Cys-rich KTR or a Lsa family ABC-F type ribosomal protection protein. This result suggests links between RNA methylases, ribosomal protection proteins, and a new family of metalloprotein, cys-rich KTRs, all of which may be necessary for efficient RNA methylation activity and drug resistance in these systems. The work in this dissertation expands the understanding of Cfr activity from diverse phylogenetic sources, especially those from clostridial organisms. It also provides a basis for further investigation of the connection between Cfr, Lsa, and KTR function in clostridial pathogens and commensal organisms. Structure determination of a Cfr remains a top priority because it would allow for rational inhibitor design. Structural characterization of a member of the newly discovered metalloprotein family Cys-rich KTR would also validate many of the findings in this dissertation and give further insight into the genomic proximity to antibiotic resistance determinants. v TABLES OF CONTENTS LIST OF FIGURES.......................................................................................................... vii LIST OF TABLES............................................................................................................. xi ACKNOWLEDGEMENTS............................................................................................. xiii Chapter 1: Introduction....................................................................................................... 1 Project statement........................................................................................................... 2 RNA modification and its importance in ribosome function........................................ 3 Radical SAM enzymes.................................................................................................. 4 Radical SAM methylases............................................................................................ 10 Mechanistic characterization of RlmN and Cfr.......................................................... 15 Project objectives........................................................................................................ 25 References................................................................................................................... 27 Chapter 2: Phylogenetic and biochemical analyses of four radical S-adenosylmethionine Cfr homologs of diverse origin......................................................................................... 33 Abstract....................................................................................................................... 34 Introduction................................................................................................................. 35 Materials and Methods................................................................................................ 40 Results......................................................................................................................... 50 Discussion................................................................................................................... 81 References................................................................................................................... 91 Chapter 3: Biochemical and spectroscopic characterization of a C-terminal rubredoxin- like domain in Clostridioides difficile Cfr........................................................................ 95 Abstract....................................................................................................................... 96 Introduction................................................................................................................. 97 Materials and Methods................................................................................................ 99 Results....................................................................................................................... 108 Discussion................................................................................................................. 123 References................................................................................................................
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