title: Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria.
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 m(2)A2503 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M. giessing@bmb.sdu.dk
authors.forename: Anders Michael Bernth
authors.initials: AM
authors.lastname: Giessing
authors.forename: Søren Skov
authors.initials: SS
authors.lastname: Jensen
authors.forename: Anette
authors.initials: A
authors.lastname: Rasmussen
authors.forename: Lykke Haastrup
authors.initials: LH
authors.lastname: Hansen
authors.forename: Andrzej
authors.initials: A
authors.lastname: Gondela
authors.forename: Katherine
authors.initials: K
authors.lastname: Long
authors.forename: Birte
authors.initials: B
authors.lastname: Vester
authors.forename: Finn
authors.initials: F
authors.lastname: Kirpekar
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008745:Methylation; D008780:Methyltransferases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D012338:RNA, Ribosomal, 23S; C070330:8-methyladenosine; C534716:Cfr protein, E coli; D008780:Methyltransferases; D000241:Adenosine