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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM101957-02
Application #
8461575
Study Section
Special Emphasis Panel (ZRG1-BCMB-P (02))
Program Officer
Anderson, Vernon
Project Start
2012-05-01
Project End
2016-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
2
Fiscal Year
2013
Total Cost
$261,691
Indirect Cost
$78,341
Name
Pennsylvania State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
003403953
City
University Park
State
PA
Country
United States
Zip Code
16802
Silakov, Alexey; Grove, Tyler L; Radle, Matthew I et al. (2014) Characterization of a cross-linked protein-nucleic acid substrate radical in the reaction catalyzed by RlmN. J Am Chem Soc 136:8221-8
Christensen, Quin H; Grove, Tyler L; Booker, Squire J et al. (2013) A high-throughput screen for quorum-sensing inhibitors that target acyl-homoserine lactone synthases. Proc Natl Acad Sci U S A 110:13815-20
Grove, Tyler L; Livada, Jovan; Schwalm, Erica L et al. (2013) A substrate radical intermediate in catalysis by the antibiotic resistance protein Cfr. Nat Chem Biol 9:422-7