2.1.1.224	evolution	analysis of RNA methylation by phylogenetically diverse Cfr radical SAM enzymes reveals an ironbinding accessory domain in a clostridial enzyme. Sequence comparisons and phylogenetic analysis and tree, overview. Cfr homologues from Bacillus amyloliquefaciens, Enterococcus faecalis, Paenibacillus lautus, and Clostridioides difficile act as C8 adenine RNA methylases in biochemical assays	-, 758141	
2.1.1.224	evolution	analysis of RNA methylation by phylogenetically diverse Cfr radical SAM enzymes reveals an ironbinding accessory domain in a clostridial enzyme. Sequence comparisons and phylogenetic analysis and tree, overview. Cfr homologues from Bacillus amyloliquefaciens, Enterococcus faecalis, Paenibacillus lautus, and Clostridioides difficile act as C8 adenine RNA methylases in biochemical assays. Clostridioides difficile Cfr contains an additional Cys-rich C-terminal domain that binds a mononuclear Fe2+ ion in a rubredoxin-type Cys4 motif, which has an important purpose for the observed C-terminal iron in the native fusion protein. Bioinformatic analysis of the Clostridioides difficile Cfr Cys-rich domain shows that it is widespread (about 1400 homologues) as a stand-alone gene in pathogenic or commensal Bacilli and Clostridia, with >10% encoded adjacent to a predicted radical SAM RNA methylase	-, 758141	
2.1.1.224	evolution	bioinformatics analysis of the Cfr/RlmN family establishes their significant evolutionary link with radical-S-adenosyl-L-methionine enzymes. The RlmN subfamily is likely the ancestral form, whereas the Cfr subfamily arose via duplication and horizontal gene transfer	705983	
2.1.1.224	evolution	cfr(C) is part of a putative 24 kb-transposon, which generated a detectable circular intermediate. An element differing by a single nucleotide is found in Clostridium difficile DA00203 from GenBank data, consistent with a recent horizontal transfer	-, 756922	
2.1.1.224	evolution	ClbA acts via the same mechanism as the Cfr methyltransferase	-, 718601	
2.1.1.224	evolution	ClbB acts via the same mechanism as the Cfr methyltransferase	-, 718601	
2.1.1.224	evolution	ClbC acts via the same mechanism as the Cfr methyltransferase	718601	
2.1.1.224	evolution	enzyme Cfr belongs to the radical SAM (RS) superfamily of enzymes, catalysts that use S-adenosyl-L-methionine (SAM) as an oxidant to perform difficult and often complex transformations by radical mechanisms. RS superfamily enzymes employ a [4Fe-4S] cluster to supply the requisite electron for reductive cleavage of SAM, usually to L-methionine and a 5'-deoxyadenosyl 5'-radical. Similar enzyme RlmN (EC 2.1.1.192) is proposed to be an evolutionary precursor to Cfr. Residues conserved among both enzymes in a pairwise alignment of Escherichia coli RlmN and Staphylococcus aureus Cfr are mapped onto the RlmN structure. The catalytic residues in the active site are strictly conserved as are most of the surrounding residues within the core of the barrel, supporting the proposal that the enzymes use a common mechanism for C-methylation. The high degree of sequence conservation near the active site suggests that methylation site specificity during the reaction may be controlled in part by more distant structural elements. In Cfr, two large conformationally flexible regions in the RlmN structure are absent	758461	
2.1.1.224	evolution	evolutionary relationship between the Cfr and RlmN (EC 2.1.1.192) enzymes, phylogenetic analysis, overview	758128	
2.1.1.224	evolution	RlmN and Cfr belong to the radical SAM (RS) superfamily of enzymes. RlmN is proposed to be an evolutionary precursor to Cfr. The catalytic residues in theactive site are strictly conserved as are most of the surrounding residues within the core of the barrel	758461	
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	additional information	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	additional information	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	additional information	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	additional information	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	additional information	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	
2.1.1.224	additional information	the presence of a methyl group on Cfr Cys338 is a key determinant of the activity of the enzyme towards S-adenosyl-L-methionine, thus enabling a single active site to support two distinct modes of S-adenosyl-L-methionine cleavage	735095	
2.1.1.224	physiological function	antibiotic resistance effects of wild-type and mutant enzymes, overview	758128	
2.1.1.224	physiological function	Cfr confers a phenotype with resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A antibiotics. The RlmN knock out strain JW2501-1 is less sensitive to the antibiotics than standard laboratory strain HB101	706726	
2.1.1.224	physiological function	cfr confers combined resistance to chloramphenicol, florfenicol and clindamycin	705766	
2.1.1.224	physiological function	Cfr is a radical S-adenosyl-L-methionine (SAM) enzyme that confers cross-resistance to antibiotics targeting the 23S rRNA through hypermethylation of nucleotide A2503	-, 755810	
2.1.1.224	physiological function	Cfr is a radical S-adenosylmethionine (SAM) RNA methylase linked to multidrug antibiotic resistance in bacterial pathogens. It catalyzes a chemically challenging C-C bond-forming reaction to methylate C8 of A2503 (Escherichia coli numbering) of 23S rRNA during ribosome assembly	-, 758141	
2.1.1.224	physiological function	Cfr modifies C8 of A2503 in 23S rRNA conferring resistance to multiple classes of antibiotics. A2503 is also methylated at C8 by RlmN, which is both evolutionarily and mechanistically related to Cfr. Methylation of C8 of A2503 is the only known in vivo activity of Cfr, while RlmN also installs a C2 methyl group at adenosine 37	758463	
2.1.1.224	physiological function	ClbA confers resistance to antibiotics, florfenicol, clindamycin, linezolid, tiamulin, and streptogramin A/streptogramin B, to the cell, also when expressed in Escherichia coli strain AS19, overview	-, 718601	
2.1.1.224	physiological function	ClbB confers resistance to antibiotics, florfenicol, clindamycin, linezolid, tiamulin, and streptogramin A/streptogramin B, to the cell, also when expressed in Escherichia coli strain AS19, overview	-, 718601	
2.1.1.224	physiological function	ClbC confers resistance to antibiotics, florfenicol, clindamycin, linezolid, tiamulin, and streptogramin A/streptogramin B, to the cell, also when expressed in Escherichia coli strain AS19, overview	718601	
2.1.1.224	physiological function	Clostridium bolteae strain 90B3 is multiresistant to various antibiotics including ampicillin, as well as to linezolid, florfenicol, streptogramin A, and tiamulin due to methylation of 23S rRNA at A2503	-, 756922	
2.1.1.224	physiological function	enzyme Cfr methylates adenosine 2503 of the 23S rRNA in the peptidyltransferase centre (P-site) of the bacterial ribosome. This modification protects host bacteria, notably methicillin-resistant Staphylococcus aureus (MRSA), from numerous antibiotics, including agents (e.g. linezolid, retapamulin)	735095	
2.1.1.224	physiological function	gene cfr(C) confers linezolid resistance is common in Clostridium difficile. Clostridium difficile strain T10 is resistant to erythromycin, chloramphenicol, clindamycin, florfenicol, linezolid, streptogramin A, and tiamulin due to methylation of 23S rRNA at A2503	-, 756922	
2.1.1.224	physiological function	rRNA methyltransferase Cfr that methylates the conserved 23S rRNA residue A2503, located in a functionally critical region of the ribosome, confers resistance to an array of ribosomal antibiotics, including linezolid	-, 718587	
2.1.1.224	physiological function	the cfr gene encodes an rRNA methyltransferase that adds a methyl group at the C-8 position of 23S rRNA nucleotide A2503 at the peptidyl transferase center (PTC) in the ribosome. This m8A2503 modification confers resistance to more than six classes of antibiotics that bind at overlapping nonidentical sites at the PTC	755805	
2.1.1.224	physiological function	the Cfr methyltransferase primarily methylates C-8 in A2503 of 23S rRNA in the peptidyl transferase region of bacterial ribosomes. Enzyme Cfr confers resistance to antibiotics binding to the peptidyl transferase center on the ribosome, defining a PhLOPSa phenotype that reflects resistance to phenicol, lincosamide, oxazolidinone, pleuromutilin, and streptogramin A antibiotic classes. Cfr also provides resistance to some large macrolide antibiotics. The cfr gene is thus a health threat when spreading in pathogenic bacteria because many clinically important antibiotics become useless for treatment	733111	
2.1.1.224	physiological function	the Cfr RNA methyltransferase causes multiple resistances to peptidyl transferase inhibitors by methylation of A2503 23S rRNA	735450	
2.1.1.224	physiological function	the Cfr rRNA methyltransferase confers resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A antibiotics	701724	
2.1.1.224	physiological function	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. Radical-SAM enzymes use an Fe-S cluster to generate the 5'-deoxyadenosyl radical from SAM, enabling them to modify intrinsically unreactive centres such as adenosine C8	720968	
2.1.1.224	physiological function	the radical SAM (RS) enzymes RlmN and Cfr methylate 23S ribosomal RNA, modifying the C2 or C8 position of adenosine 2503. The methyl groups are installed by a two-step sequence involving initial methylation of a conserved Cys residue (RlmN Cys 355) by SAM. Methyl transfer to the substrate requires reductive cleavage of a second equivalent of SAM. Cfr confers antibiotic resistance by methylating C8 of A2503	758461