EC Number   |
General Information |
Reference |
|---|
   2.1.1.224 | evolution |
the cfr gene can be horizontally transferred to its hosts, as it is always found either on plasmids or together with insertion sequences. The cfr gene with only minor sequence differences are found worldwide in various bacteria isolated from humans and animals. Comparative sequence analysis identifies differentially conserved residues that indicate functional sequence divergence between the two classes of Cfr and RlmN-like sequences. The enzymes are homologous and use the same mechanism involving radical S-adenosyl methionine to methylate RNA via an intermediate involving a methylated cysteine in the enzyme and a transient cross-linking to the RNA, but they differ in which carbon atom in the adenine they methylate. The differentiation between the two classes is supported by experimental evidence from antibiotic resistance, primer extensions, and mass spectrometry. The Cfr- and RlmN-specific conserved sites provide a very good indication of whether a gene is Cfr-like or RlmN-like. Most bacteria have an rlmN-like gene and that all those that have a cfr-like gene also have an rlmN-like gene, evolutionary aspects of the bacterial distribution of Cfr and RlmN-like enzymes, overview |
733111 |
| 2.1.1.224 | evolution |
three cfr-like genes implicated in antibiotic resistance have been described, two of which, cfr(B) and cfr(C), have been sporadically detected in Clostridium difficile. The methylase activity of Cfr(C) has not been confirmed. cfr(B), cfr(C), and a cfr-like genes show only 51 to 58% protein sequence identity to Cfr and Cfr-like enzymes in clinical Clostridium difficile isolates recovered across nearly a decade in Mexico, Honduras, Costa Rica, and Chile. This resistance gene is termed cfr(E). The predicted protein sequence of Cfr(E) shows homology to C8 RNA-methylating enzymes. Enzymes Cfr(C) or Cfr(E) are determined to methylate A2503 at the C8 position. The cfr-like gene of isolate DF11 (Cfr(E))is found integrated into an undescribed MGE that shows partial hits to genomic sequences of various intestinal Firmicutes, but in all cases, shared regions do not include cfr(E) or its adjacent genes, overview |
-, 755810 |
| 2.1.1.224 | malfunction |
Clostridioides difficile Cfr contains an additional Cys-rich C-terminal domain that binds a mononuclear Fe2+ ion in a rubredoxin-type Cys4 motif. The C-terminal domain can be truncated with minimal impact on Cfr activity, but the rate of turnover is decreased upon disruption of the Fe2+-binding site by Zn2+ substitution or ligand mutation |
-, 758141 |
| 2.1.1.224 | metabolism |
methyl transfer is essential in the synthesis of cellular metabolites and clinically relevant natural products, and in the modification of RNA, DNA, lipids, and proteins |
758461 |
| 2.1.1.224 | metabolism |
several groups of antibiotics inhibit bacterial growth by binding to bacterial ribosomes. Mutations in ribosomal protein L3 have been associated with resistance to linezolid and tiamulin, which both bind at the peptidyl transferase center in the ribosome. Resistance to these and other antibiotics also occurs through methylation of 23S rRNA at position A2503 by the methyltransferase Cfr. The resistance from Cfr is, in all cases, stronger than the effects of the L3 mutations, but various effects are obtained with the combinations of Cfr and L3 mutations ranging from a synergistic to an antagonistic effect. Linezolid and tiamulin susceptibility vary greatly among the L3 mutations, while no significant effects on florfenicol and Q-D susceptibility are seen. Relative positions of L3 mutations, methylation of the 23S rRNA at position A2503, and antibiotics, three-dimensional structure model, overview. Analysis of antibiotic susceptibilities of the L3 mutant strains with and without Cfr expression |
755805 |
| 2.1.1.224 | more |
acquisition of the cfr gene does not produce any appreciable reduction in the cell growth rate, analysis of fitness cost of cfr expression, overview. Genes ermB and cfr are coexpressed under the Perm promoter in the mlr operon. Dimethylation of A2058 by the Erm methyltransferase increases the fitness cost associated with Cfr-mediated modification of A2503 |
-, 718587 |
| 2.1.1.224 | more |
although SAM is the source of the appended methyl carbon in the reactions catalyzed by RlmN and Cfr, these enzymes operate by a mechanism that is distinctly different from that of typical SAM-dependent methyltransferases. As radical SAM (RS) enzymes, RlmN and Cfr employ very similar radical-based mechanisms of catalysis, initiated by the abstraction of a hydrogen atom from a Cys-appended methyl group via a 5'-deoxyadenosyl 5'-radical. Subsequent attack of the resulting methylene radical upon the carbon atom undergoing methylation affords a protein/RNA cross-linked intermediate whose resolution requires prior proton abstraction from C2 (RlmN) or C8 (Cfr) of the substrate by an unidentified base. Conversion of the intermediate to the methylated product has also been demonstrated in the Cfr reaction. The proximity (5.0 A) of the Cys 355 side chain (the proposed site of thiyl radical formation) to the sulfur atom of Met176, a strictly conserved residue in RlmN and Cfr, might allow formation of a transient thiosulfuranyl radical |
758463 |
| 2.1.1.224 | more |
gene cfr(C) is mainly confined in Clostridium difficile within polymorphic environments suggesting that this microorganism is a reservoir for PhLOPSA resistance. In silico analysis shows cfr(C) in 19 out of 274 Clostridium difficile genomes. This gene is also detected by PCR analysis in 9 out of 80 Clostridium difficile from a laboratory strain collection according to resistance to linezolid and florfenicol |
-, 756922 |
| 2.1.1.224 | more |
mechanisms of catalytic action of Cfr and related RlmN (EC 2.1.1.192), the methylation mechanism involves a transitory methylation of Cys338 for Cfr and Cys355 for RlmN, investigation of target binding to the active sites of the two enzymes, overview. Cfr and RlmN are methylated before transfer of the methyl group to the substrate. Homology structure modelling, molecular dynamics simulations, and calculation of the binding free energy, using structure of Escherichia coli RlmN (PDB ID 3RFA), the homology model is made with the [4Fe-4S] cluster and a SAM molecule positioned in the same way as seen in the RlmN X-ray structure. Defining regions of the active site to be interchanged to investigate C8/C2 specificity |
758128 |
| 2.1.1.224 | more |
structure-function analysis of RlmN from Escherichia coli (EC 2.1.1.192) compared to Cfr, Escherichia coli RlmN and Staphylococcus aureus Cfr are mapped onto the RlmN structure, detailed overview. The Cfr reaction proceeds by a ping-pong mechanism. The methyl group from one SAM molecule is initially appended to a conserved Cys residue by a typical SN2 displacement. This SAM-derived one carbon unit is then attached to the RNA by radical addition initiated by a 5'-deoxyadenosyl 5'-radical formed from a second molecule of SAM. The expected role of the radical is to abstract a hydrogen atom from the substrate, in this case the C8 (Cfr) hydrogen atom from A2503, activating the substrate for subsequent methylation. Finally, this covalent intermediate is resolved by formation of a disulfide bond between the methyl-carrying Cys (mCys) residue and a second conserved Cys residue. Cys 355 is a key catalytic residue that is methylated in the first step of the proposed mechanism |
758461 |
