This monomeric radical SAM enzyme is present in only certain bacterial taxa (Chlamydiae/Verrucomicrobia group, Planctomycetes and Staphylococci), and in eykaryota containing plastids, like green plants, Alveolates, Choanoflagellida. It is absent in E.coli. Phylogenetic analysis of the Cfr(m8A formation)/RlmN(m2A formation) family suggests that the RlmN subfamily is likely the ancestral form, whereas the Cfr subfamily arose via duplication and horizontal gene transfer. Cfr carries out the AdoMet-dependent methylation of an amidine carbon of an adenine target (formation of m8A) at position 2503 (E. coli numbering) of the Peptidyl Transferase Center (Domain V) of 23S rRNA. The enzymatic activity of Cfr (m8A formation) is coordinated with the formation of m2A-2503 by RlmN (when present). In the absence of RlmN, Cfr has the ability to catalyze also the methylation of C-2 atom to form a dimethyl m2,8A-2503 derivative. It is mainly the enzymatic formation of m8A that confers antibiotic (florfenicol/ chloramphenicol) resistance in bacteria. As RlmN, Cfr belongs to Radical SAM superfamily and contains the characteristic cysteine-rich CX(3)CX(2)C motif. The methyl group is installed by a two-step event, first involving the methylation of a conserved Cys residue (formation of S-methylcysteinyl derivative) by AdoMet, followed by transfer of the activated methyl group to atom C8 of adenine-2503. Thus, AdoMet is both the methyl donor and the source of a 5′-deoxyadenosyl radical, which activates the substrate for methylation. Therefore, Cfr, as its homolog RlmN are methyl synthases rather than a classic methyltransferases.
Methylation site was determined for E. coli rRNA.
Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria.
Giessing AM, Jensen SS, Rasmussen A, Hansen LH, Gondela A, Long K, Vester B, Kirpekar F
A new mechanism for chloramphenicol, florfenicol and clindamycin resistance: methylation of 23S ribosomal RNA at A2503.
Biochemical and Computational Analysis of the Substrate Specificities of Cfr and RlmN Methyltransferases
Emerging themes in radical SAM chemistry.
Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria.
Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria.
RlmN and Cfr are radical SAM enzymes involved in methylation of ribosomal RNA
RNA methylation by radical SAM enzymes RlmN and Cfr proceeds via methylene transfer and hydride shift.
Structural basis for methyl transfer by a radical SAM enzyme.