RlmN and Cfr are radical SAM enzymes involved in methylation of ribosomal RNA

J Am Chem Soc. 2010 Mar 24;132(11):3953-64. doi: 10.1021/ja910850y.


Posttranscriptional modifications of ribosomal RNA (rRNA) nucleotides are a common mechanism of modulating the ribosome's function and conferring bacterial resistance to ribosome-targeting antibiotics. One such modification is methylation of an adenosine nucleotide within the peptidyl transferase center of the ribosome mediated by the endogenous methyltransferase RlmN and its evolutionarily related resistance enzyme Cfr. These methyltransferases catalyze methyl transfer to aromatic carbon atoms of the adenosine within a complex 23S rRNA substrate to form the 2,8-dimethylated product. RlmN and Cfr are members of the Radical SAM superfamily and contain the characteristic cysteine-rich CX(3)CX(2)C motif. We demonstrate that both enzymes are capable of accommodating the requisite [4Fe-4S] cluster. S-Adenosylmethionine (SAM) is both the methyl donor and the source of a 5'-deoxyadenosyl radical, which activates the substrate for methylation. Detailed analyses of the rRNA requirements show that the enzymes can utilize protein-free 23S rRNA as a substrate, but not the fully assembled large ribosomal subunit, suggesting that the methylations take place during the assembly of the ribosome. The key recognition elements in the 23S rRNA are helices 90-92 and the adjacent single stranded RNA that encompasses A2503. To our knowledge, this study represents the first in vitro description of a methyl transfer catalyzed by a member of the Radical SAM superfamily, and it expands the catalytic repertoire of this diverse enzyme class. Furthermore, by providing information on both the timing of methylation and its substrate requirements, our findings have important implications for the functional consequences of Cfr-mediated modification of rRNA in the acquisition of antibiotic resistance.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenosine / metabolism
  • Amino Acid Motifs
  • Biocatalysis
  • Carbon / chemistry
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / isolation & purification
  • Escherichia coli Proteins / metabolism*
  • Hydrogen / chemistry
  • Methyltransferases / chemistry
  • Methyltransferases / genetics
  • Methyltransferases / isolation & purification
  • Methyltransferases / metabolism*
  • Models, Molecular
  • Protein Conformation
  • RNA, Ribosomal / chemistry
  • RNA, Ribosomal / metabolism*
  • S-Adenosylmethionine / metabolism*


  • Escherichia coli Proteins
  • RNA, Ribosomal
  • Carbon
  • S-Adenosylmethionine
  • Hydrogen
  • Cfr protein, E coli
  • Methyltransferases
  • Adenosine