RlmN and Cfr catalyze S-adenosylmethionine (SAM)-dependent methylation of adenosine 2503 (A2503) of 23S rRNA of the bacterial ribosome at C2 and C8, respectively. C2 methylation is found throughout bacteria, and is believed to aid in the efficiency of peptidyltransfer. By contrast, C8 methylation is an activity acquired by certain pathogenic bacteria that confers upon them resistance to over seven classes of antibiotics that target the large subunit of the bacterial ribosome. C2 and C8 are electrophilic sp2-hybridized carbons, which renders them unreactive toward the catalytic strategy used by almost all other known SAM-dependent methylases. In fact, we have shown that these reactions take place via radical mechanisms, involving i) initial transfer of a methyl group from SAM to a conserved cysteinyl residue via a standard nucleophilic displacement mechanism;ii) abstraction of a hydrogen atom from the resulting methylcysteinyl residue by a 5'-deoxyadenosyl 5'-radical (5'-dA) derived from radical fragmentation of a second SAM molecule;iii) addition of the methylcysteinyl radical intermediate to C2 or C8 of the nucleotide substrate;and iv) resolution of the resulting protein-nucleic cross-link by disulfide-bond formation. We will characterize this reaction further using a variety of kinetic, spectroscopic, and biochemical techniques, and provide biochemical and/or structural evidence for each of the postulated intermediates in the reaction.

Public Health Relevance

RlmN and Cfr are radical S-adenosylmethionine-dependent proteins that catalyze methylation of 23S rRNA of the bacterial ribosome by similar mechanisms involving radical intermediates. Methylation by Cfr confers resistance to over seven classes of antibiotics. This proposal is aimed at elucidating the mechanistic details of these unusual reactions, with a future goal of strategically inhibiting the Cfr protein as a means of protecting our current arsenal of antibacterial agents.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-BCMB-P (02))
Program Officer
Anderson, Vernon
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Pennsylvania State University
Schools of Arts and Sciences
University Park
United States
Zip Code
Badding, Edward D; Grove, Tyler L; Gadsby, Lauren K et al. (2017) Rerouting the Pathway for the Biosynthesis of the Side Ring System of Nosiheptide: The Roles of NosI, NosJ, and NosK. J Am Chem Soc 139:5896-5905
Blaszczyk, Anthony J; Wang, Bo; Silakov, Alexey et al. (2017) Efficient methylation of C2 in l-tryptophan by the cobalamin-dependent radical S-adenosylmethionine methylase TsrM requires an unmodified N1 amine. J Biol Chem 292:15456-15467
Blaszczyk, Anthony J; Wang, Roy X; Booker, Squire J (2017) TsrM as a Model for Purifying and Characterizing Cobalamin-Dependent Radical S-Adenosylmethionine Methylases. Methods Enzymol 595:303-329
Lanz, Nicholas D; Lee, Kyung-Hoon; Horstmann, Abigail K et al. (2016) Characterization of Lipoyl Synthase from Mycobacterium tuberculosis. Biochemistry 55:1372-83
Landgraf, Bradley J; McCarthy, Erin L; Booker, Squire J (2016) Radical S-Adenosylmethionine Enzymes in Human Health and Disease. Annu Rev Biochem 85:485-514
Blaszczyk, Anthony J; Silakov, Alexey; Zhang, Bo et al. (2016) Spectroscopic and Electrochemical Characterization of the Iron-Sulfur and Cobalamin Cofactors of TsrM, an Unusual Radical S-Adenosylmethionine Methylase. J Am Chem Soc 138:3416-26
Schwalm, Erica L; Grove, Tyler L; Booker, Squire J et al. (2016) Crystallographic capture of a radical S-adenosylmethionine enzyme in the act of modifying tRNA. Science 352:309-12
Pandelia, Maria-Eirini; Lanz, Nicholas D; Booker, Squire J et al. (2015) Mössbauer spectroscopy of Fe/S proteins. Biochim Biophys Acta 1853:1395-405
Bauerle, Matthew R; Schwalm, Erica L; Booker, Squire J (2015) Mechanistic diversity of radical S-adenosylmethionine (SAM)-dependent methylation. J Biol Chem 290:3995-4002
Lanz, Nicholas D; Rectenwald, Justin M; Wang, Bo et al. (2015) Characterization of a Radical Intermediate in Lipoyl Cofactor Biosynthesis. J Am Chem Soc 137:13216-9

Showing the most recent 10 out of 16 publications