2.1.1.224 2 S-adenosyl-L-methionine + 2-methyladenine2503 in 23S rRNA S-adenosylmethionine is both the methyl donor and the source of a 5'-deoxyadenosyl radical, which activates the substrate for methylation Escherichia coli S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2,8-dimethyladenine2503 in 23S rRNA incubation of Cfr with the wild-type 23S rRNA, already modified at the C2 position by the endogenous RlmN, provides 2,8-dimethyladenosine as the sole product ? 416087 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA - Mammaliicoccus sciuri 2 S-adenosyl-L-homocysteine + 2,8-dimethyladenine2503 in 23S rRNA - ? 407003 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA - Staphylococcus aureus S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA - ? 415543 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA - Bacillus amyloliquefaciens S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA - ? 415543 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA - Escherichia coli K-12 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA - ? 415543 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA - Brevibacillus brevis S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA - ? 415543 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA - Alkalihalobacillus clausii S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA - ? 415543 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA - Bacillus amyloliquefaciens S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA verification of RNA methylation at A2503 in 23S rRNA by primer extension method ? 415543 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA - Brevibacillus brevis S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA verification of RNA methylation at A2503 in 23S rRNA by primer extension method ? 415543 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA - Alkalihalobacillus clausii S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA verification of RNA methylation at A2503 in 23S rRNA by primer extension method ? 415543 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA Cfr is an plasmid-acquired methyltransferase that protects cells from the action of antibiotics Escherichia coli S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA - ? 415543 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA S-adenosylmethionine is both the methyl donor and the source of a 5'-deoxyadenosyl radical, which activates the substrate for methylation. The enzyme 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 Escherichia coli S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA identification of the reaction products as 8-methyladenine2503, and 2,8-dimethyladenine2503 ? 415543 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA the newly introduced methyl group is assembled from an S-adenosyl-L-methionine (SAM)-derived methylene fragment and a hydrogen atom that had migrated from the substrate amidine carbon. Rather than activating the adenosine nucleotide of the substrate by hydrogen atom abstraction from an amidine carbon, the 5'-deoxyadenosyl radical abstracts hydrogen from the second equivalent of SAM to form the SAM-derived radical cation. This species, or its corresponding sulfur ylide, subsequently adds into the substrate, initiating hydride shift and S-adenosylhomocysteine elimination to complete the formation of the methyl group Escherichia coli S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA - ? 415543 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA the enzyme methylates the 8 position of 23S rRNA residue A2503 to confer resistance to multiple antibiotic classes acting upon the large subunit of the bacterial ribosome Azotobacter vinelandii S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA - ? 415543 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA in the presence of sodium dithionite reconstituted Cfr is both reducible and able to cleave S-adenosyl-L-methionine to 5'-deoxyadeonsine, DOA Azotobacter vinelandii S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA - ? 415543 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA - Bacillus amyloliquefaciens FZB42 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA - ? 415543 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA - Bacillus amyloliquefaciens FZB42 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA verification of RNA methylation at A2503 in 23S rRNA by primer extension method ? 415543 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA - Brevibacillus brevis NBRC 100599 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA - ? 415543 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA - Brevibacillus brevis NBRC 100599 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA verification of RNA methylation at A2503 in 23S rRNA by primer extension method ? 415543 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA - Staphylococcus aureus RN4220 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA - ? 415543 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin - Escherichia coli S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin - ? 454145 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin - Staphylococcus sp. S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin - ? 454145 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin - Clostridioides difficile S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin - ? 454145 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin - Mammaliicoccus sciuri S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin - ? 454145 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin - Staphylococcus aureus S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin - ? 454145 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin - Bacillus amyloliquefaciens S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin - ? 454145 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin - Enterococcus faecalis S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin - ? 454145 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin - Paenibacillus lautus S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin - ? 454145 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin - Enterocloster bolteae S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin - ? 454145 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin - Bacillus amyloliquefaciens DSM-7 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin - ? 454145 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin - Clostridioides difficile T10 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin - ? 454145 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin - Paenibacillus lautus Y412MC10 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin - ? 454145 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin - Enterocloster bolteae 90B3 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin - ? 454145 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin - Enterococcus faecalis TX0645 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin - ? 454145 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin - Clostridioides difficile QCD63q42 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin - ? 454145 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin - Clostridioides difficile DF11 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin - ? 454145 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [4Fe-4S] ferredoxin - Staphylococcus aureus S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [4Fe-4S] ferredoxin - ? 434510 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [4Fe-4S] ferredoxin - Mammaliicoccus sciuri S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [4Fe-4S] ferredoxin - ? 434510 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [4Fe-4S] ferredoxin - Clostridioides difficile S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [4Fe-4S] ferredoxin - ? 434510 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [4Fe-4S] ferredoxin enzyme Cfr consumes two AdoMet equivalents per reaction cycle to support both methyl transfer to Cfr Cys338 (AdoMet1, step 1) and subsequently generation of the 59dA. radical (AdoMet2, step2) Staphylococcus aureus S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [4Fe-4S] ferredoxin - ? 434510 2.1.1.224 2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [4Fe-4S] ferredoxin two S-adenosyl-L-methionine molecules serve as the cofactor by providing the 5'-deoxyadenosyl radical for substrate activation and the methyl Staphylococcus aureus S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [4Fe-4S] ferredoxin - ? 434510 2.1.1.224 additional information Cfr confers a phenotype with resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A antibiotics. Methylation of position 8 is the dominant antibiotic resistance determinant, but indigenous modification at position 2 also contributes to low-level resistance Escherichia coli K-12 ? - ? 89 2.1.1.224 additional information enzyme Cfr methylates adenosine 2503 of the 23S rRNA in the peptidyltransferase centre (P-site) of the bacterial ribosome. In wild-type Cfr, where Cys338 is methylated, S-adenosyl-L-methionine binding leads to rapid oxidation of the [4Fe-4S] cluster and production of 5'-deoxyadenosine Staphylococcus aureus ? - ? 89 2.1.1.224 additional information methylation at C8 is similar to that at C2, cf. EC 2.1.1.192 Staphylococcus aureus ? - ? 89 2.1.1.224 additional information Cfr methylates A2503 of 23S rRNA at C8 and C2, while RlmN only performs a C2 methylation using the same mechanism of function Mammaliicoccus sciuri ? - - 89 2.1.1.224 additional information in vitro assays with Escherichia coli RNA Clostridioides difficile ? - - 89 2.1.1.224 additional information kinetic analysis of methylation of a 155mer rRNA substrate by Cfr enzymes under limiting and under excess substrate conditions initiated with a protein-based reductant, substrate sequence, overview Bacillus amyloliquefaciens ? - - 89 2.1.1.224 additional information kinetic analysis of methylation of a 155mer rRNA substrate by Cfr enzymes under limiting and under excess substrate conditions initiated with a protein-based reductant, substrate sequence, overview Enterococcus faecalis ? - - 89 2.1.1.224 additional information kinetic analysis of methylation of a 155mer rRNA substrate by Cfr enzymes under limiting and under excess substrate conditions initiated with a protein-based reductant, substrate sequence, overview Paenibacillus lautus ? - - 89 2.1.1.224 additional information kinetic analysis of methylation of a 155mer rRNA substrate by Cfr enzymes under limiting and under excess substrate conditions initiated with a protein-based reductant, substrate sequence, overview Clostridioides difficile ? - - 89 2.1.1.224 additional information the enzyme employs a [4Fe-4S] cluster to supply the requisite electron for reductive cleavage of SAM, usually to L-methionine and a 5'-deoxyadenosyl 5'-radical. SAM is the source of both the 5'-deoxyadenosyl 5'-radical and the appended methyl group Staphylococcus aureus ? - - 89 2.1.1.224 additional information kinetic analysis of methylation of a 155mer rRNA substrate by Cfr enzymes under limiting and under excess substrate conditions initiated with a protein-based reductant, substrate sequence, overview Bacillus amyloliquefaciens DSM-7 ? - - 89 2.1.1.224 additional information kinetic analysis of methylation of a 155mer rRNA substrate by Cfr enzymes under limiting and under excess substrate conditions initiated with a protein-based reductant, substrate sequence, overview Paenibacillus lautus Y412MC10 ? - - 89 2.1.1.224 additional information kinetic analysis of methylation of a 155mer rRNA substrate by Cfr enzymes under limiting and under excess substrate conditions initiated with a protein-based reductant, substrate sequence, overview Enterococcus faecalis TX0645 ? - - 89 2.1.1.224 additional information kinetic analysis of methylation of a 155mer rRNA substrate by Cfr enzymes under limiting and under excess substrate conditions initiated with a protein-based reductant, substrate sequence, overview Clostridioides difficile QCD63q42 ? - - 89 2.1.1.224 additional information in vitro assays with Escherichia coli RNA Clostridioides difficile DF11 ? - - 89 2.1.1.224 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + reduced [2Fe-2S] ferredoxin - Mammaliicoccus sciuri S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + oxidized [2Fe-2S] ferredoxin - ? 432931