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Information on EC 2.1.1.224 - 23S rRNA (adenine2503-C8)-methyltransferase and Organism(s) Mammaliicoccus sciuri and UniProt Accession Q9FBG4
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2.1.1.224 23S rRNA (adenine2503-C8)-methyltransferaseIUBMB Comments
This enzyme is a member of the 'AdoMet radical' (radical SAM) family. S-Adenosyl-L-methionine acts as both a radical generator and as the source of the appended methyl group. It contains an [4Fe-4S] cluster [3,6,7]. Cfr is an plasmid-acquired methyltransferase that protects cells from the action of antibiotics . The enzyme methylates adenosine at position 2503 of 23S rRNA by a radical mechanism, transferring a CH2 group from S-adenosyl-L-methionine while retaining the hydrogen at the C-8 position of the adenine. Cfr first transfers an CH2 group to a conserved cysteine (Cys338 in Staphylococcus aureus) , the generated radical from a second S-adenosyl-L-methionine then attacks the methyl group, exctracting a hydrogen. The formed radical forms a covalent intermediate with the adenine group of the tRNA . The enzyme will also methylate 2-methyladenine produced by the action of EC 2.1.1.192 [23S rRNA (adenine2503-C2)-methyltransferase].
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Word Map
- 2.1.1.224
- linezolid
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oxazolidinone
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5\'-deoxyadenosyl
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thiopeptide
-
optra
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linezolid-resistant
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phenicols
-
cobalamin-dependent
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phlopsa
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pleuromutilins
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nosiheptide
- 5'-deoxyadenosine
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poxta
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ribosome-targeting
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cobiialamin
The taxonomic range for the selected organisms is: Mammaliicoccus sciuri
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Reaction Schemes
2
+
+
2
=
+
+
+
+
2
2
+
adenine2503 in 23S rRNA
+
2
=
+
+
+
8-methyladenine2503 in 23S rRNA
+
2
Synonyms
cfr(c), cfr-like, antibiotic resistance protein, cfr methyltransferase, radical s-adenosylmethionine enzyme, radical-sam enzyme, cfr(e), radical s-adenosylmethionine methylase, cfr-like protein, rs methylase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin = S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin
the enzyme uses a mechanism involving radical S-adenosyl methionine to methylate RNA via an intermediate with a methylated cysteine in the enzyme and a transient cross-linking to the RNA
PATHWAY SOURCE
PATHWAYS
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-
SYSTEMATIC NAME
IUBMB Comments
S-adenosyl-L-methionine:23S rRNA (adenine2503-C8)-methyltransferase
This enzyme is a member of the 'AdoMet radical' (radical SAM) family. S-Adenosyl-L-methionine acts as both a radical generator and as the source of the appended methyl group. It contains an [4Fe-4S] cluster [3,6,7]. Cfr is an plasmid-acquired methyltransferase that protects cells from the action of antibiotics [1]. The enzyme methylates adenosine at position 2503 of 23S rRNA by a radical mechanism, transferring a CH2 group from S-adenosyl-L-methionine while retaining the hydrogen at the C-8 position of the adenine. Cfr first transfers an CH2 group to a conserved cysteine (Cys338 in Staphylococcus aureus) [7], the generated radical from a second S-adenosyl-L-methionine then attacks the methyl group, exctracting a hydrogen. The formed radical forms a covalent intermediate with the adenine group of the tRNA [8]. The enzyme will also methylate 2-methyladenine produced by the action of EC 2.1.1.192 [23S rRNA (adenine2503-C2)-methyltransferase].
CAS REGISTRY NUMBER
COMMENTARY
39369-30-7
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
2 S-adenosyl-L-homocysteine + 2,8-dimethyladenine2503 in 23S rRNA
-
-
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin
-
-
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [4Fe-4S] ferredoxin
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [4Fe-4S] ferredoxin
-
-
-
?
S-adenosyl-L-methionine + adenine2503 in 23S rRNA + reduced [2Fe-2S] ferredoxin
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + oxidized [2Fe-2S] ferredoxin
-
-
-
?
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
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-
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin
-
-
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [4Fe-4S] ferredoxin
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [4Fe-4S] ferredoxin
-
-
-
?
S-adenosyl-L-methionine + adenine2503 in 23S rRNA + reduced [2Fe-2S] ferredoxin
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + oxidized [2Fe-2S] ferredoxin
-
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
physiological function
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
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
evolution
evolutionary relationship between the Cfr and RlmN (EC 2.1.1.192) enzymes, phylogenetic analysis, overview
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
physiological function
antibiotic resistance effects of wild-type and mutant enzymes, overview
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
41088
x * 41088, fully methylated protein, calculated from amino acid sequence
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
?
x * 41088, fully methylated protein, calculated from amino acid sequence
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
homology modeling of structure and comparison with RlmN from Echerichia coli, EC 2.1.1.192
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C338A
the mutant binds S-adenosyl-L-methionine with wild type affinity, while oxidation of the [4Fe-4S] cluster is not observed
additional information
additional information
construction of chimera between Cfr and RlmN from Echerichia coli to analyze C2/C8 and C2 methylation specificity, respectively. The catalytic site is expected to be responsible for the C2/C8 specificity. Almost all replacements show no function in the primer extension assay, apart from a few that have a weak effect
additional information
construction of chimeric mutants of gene cfr, cfr1234567rrlmN and rlmN1234567rcfr, and construction of mixed genes containing gene fragment of cfr and rlmN genes, overview. Analysis of effects of the mutations on catalytic activity and antibiotics resistance
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene cfr, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain JW2501-1 (rlmN knockout) and AS19
gene cfr, sequence comparisons and phylogenetic analysis, primer extension analysis, cloning and expression in Escherichia coli strains TOP10 and AS19
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Atkinson, G.C.; Hansen, L.H.; Tenson, T.; Rasmussen, A.; Kirpekar, F.; Vester, B.
Distinction between the Cfr methyltransferase conferring antibiotic resistance and the housekeeping RlmN methyltransferase
Antimicrob. Agents Chemother.
57
4019-4026
2013
Mammaliicoccus sciuri (Q9FBG4)
Challand, M.R.; Salvadori, E.; Driesener, R.C.; Kay, C.W.; Roach, P.L.; Spencer, J.
Cysteine methylation controls radical generation in the Cfr radical AdoMet rRNA methyltransferase
PLoS ONE
8
e67979
2013
Mammaliicoccus sciuri (Q9FBG4), Staphylococcus aureus
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Release 2023.1 (January 2023)
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