Investigation of the Radical SAM RNA Methylases RlmN and Cfr
Open Access
- Author:
- Schwalm, Erica Lynne
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 25, 2018
- Committee Members:
- Squire J Booker, Dissertation Advisor/Co-Advisor
Squire J Booker, Committee Chair/Co-Chair
Amie Kathleen Boal, Committee Member
Carsten Krebs, Committee Member
William O Hancock, Outside Member - Keywords:
- radical SAM
RNA
enzymes
methylation - Abstract:
- Ribosomal RNA (rRNA) is highly post-transcriptionally modified, contributing to the structure and function of the bacterial ribosome in the essential process of protein translation. Methylations represent the most common modification and are typically localized to functional regions. One such modification is the methylation of a specific adenosine residue (A2503 in Escherichia coli rRNA) of domain V of 23S rRNA that resides in the peptidyl transferase center (PTC), the site of the ribosome responsible for the formation of new peptide bonds. RlmN is responsible for the methylation of carbon 2 of A2503, a non-essential modification that contributes to translational fidelity through structural stabilization of the PTC. An evolutionarily related enzyme, Cfr, is capable of methylating the same adenosine residue at carbon 8. The presence of 8-methyladenosine at this position renders bacteria resistant to six classes of clinically relevant antibiotics that target the bacterial ribosome. As members of the radical S-adenosylmethionine (SAM) superfamily of enzymes, RlmN and Cfr contain a [4Fe-4S] cluster cofactor and use SAM to initiate radical chemistry to catalyze difficult chemical transformations. The reactions catalyzed by RlmN and Cfr have been extensively studied in our lab, and the work presented here furthers our understanding of this unique set of enzymes. Using an RlmN variant incapable of producing the final methylated RNA product, a covalent protein-RNA cross-link intermediate of RlmN with a tRNA substrate was trapped and characterized using X-ray crystallography. This structure represents the first structure of a radical SAM methylase in complex with the full substrate, and provides important insight into the mechanism of substrate binding and key catalytic residues. The proposed mechanism for catalysis by RlmN and Cfr involves the formation of a protein-RNA cross-link radical intermediate that has been previously trapped and characterized in the Cfr reaction. Studies using a deuterium labeled rRNA substrate demonstrate a kinetic isotope effect on the decay of the radical intermediate, as well as the formation of the final methylated product. These results support the current proposal for Cfr catalysis and identify the deprotonation of the radical intermediate as a rate-limiting step. A clinical variant of RlmN incapable of catalyzing rRNA methylation in vivo was studied in detail. Results show that the single amino acid insertion slows methyltransfer and prevents reduction of the iron-sulfur cluster by the biological reducing partner. Finally, efforts were made toward the crystallization and structural characterization of Cfr. While no crystal structure was obtained, multiple alternate Cfr protein constructs were designed and characterized.