1. Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria
- Author
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Søren Skov Jensen, Birte Vester, Andrzej Gondela, Anders M.B. Giessing, Anette Rasmussen, Finn Kirpekar, Katherine S. Long, and Lykke Haastrup Hansen
- Subjects
Spectrometry, Mass, Electrospray Ionization ,Adenosine ,Peptidyl transferase ,Methyltransferase ,Biology ,medicine.disease_cause ,Methylation ,Ribosome ,Catalysis ,Article ,23S ribosomal RNA ,Drug Resistance, Bacterial ,Escherichia coli ,medicine ,Molecular Biology ,Chromatography, High Pressure Liquid ,Escherichia coli Proteins ,RNA ,Methyltransferases ,Anti-Bacterial Agents ,RNA, Ribosomal, 23S ,Biochemistry ,biology.protein ,Nucleic Acid Conformation ,Radical SAM ,Chromatography, Liquid - Abstract
The Cfr methyltransferase confers combined resistance to five different classes of antibiotics that bind to the peptidyl transferase center of bacterial ribosomes. The Cfr-mediated modification has previously been shown to occur on nucleotide A2503 of 23S rRNA and has a mass corresponding to an additional methyl group, but its specific identity and position remained to be elucidated. A novel tandem mass spectrometry approach has been developed to further characterize the Cfr-catalyzed modification. Comparison of nucleoside fragmentation patterns of A2503 from Escherichia coli cfr+ and cfr− strains with those of a chemically synthesized nucleoside standard shows that Cfr catalyzes formation of 8-methyladenosine. In addition, analysis of RNA derived from E. coli strains lacking the m2A2503 methyltransferase reveals that Cfr also has the ability to catalyze methylation at position 2 to form 2,8-dimethyladenosine. The mutation of single conserved cysteine residues in the radical SAM motif CxxxCxxC of Cfr abolishes its activity, lending support to the notion that the Cfr modification reaction occurs via a radical-based mechanism. Antibiotic susceptibility data confirm that the antibiotic resistance conferred by Cfr is provided by methylation at the 8 position and is independent of methylation at the 2 position of A2503. This investigation is, to our knowledge, the first instance where the 8-methyladenosine modification has been described in natural RNA molecules.
- Published
- 2009