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Information on EC 2.1.1.224 - 23S rRNA (adenine2503-C8)-methyltransferase
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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
-
peptidyl
- staphylococcus
- aureus
-
oxazolidinone
-
linezolid-resistant
-
methicillin-resistant
-
veterinary
-
5\'-deoxyadenosyl
-
enterococci
- viperin
The enzyme appears in viruses and cellular organisms
Reaction Schemes
2
+
+
2
=
+
+
+
+
2
2
+
adenine2503 in 23S rRNA
+
2
=
+
+
+
8-methyladenine2503 in 23S rRNA
+
2
Synonyms
cfr methyltransferase, radical-sam enzyme, cfr-like protein, cfr rrna methyltransferase, 
more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
Cfr
Cfr methyltransferase
ClbA
ClbB
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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
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 [2Fe-2S] ferredoxin = S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin
the enzyme catalyzes adenosine methylation by using a radical mechanism for substrate activation. The radical chemistry is enabled by the [4Fe-4S] cluster
-
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 methyl group is not transferred directly from S-adenosyl-L-methionine to the RNA. Reaction proceeds by a ping-pong mechanism involving intermediate methylation of a conserved cysteine residue
-
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
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
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)
-
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
reaction mechanism modeling overview
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PATHWAY SOURCE
PATHWAYS
MetaCyc
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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].
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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 + 2-methyladenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2,8-dimethyladenine2503 in 23S rRNA
-
S-adenosylmethionine is both the methyl donor and the source of a 5'-deoxyadenosyl radical, which activates the substrate for methylation
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
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA
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
-
-
-
-
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 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
-
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 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
-
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
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
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
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
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
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
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
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
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
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
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA
C6EKJ2
-
-
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA
-
Cfr is an plasmid-acquired methyltransferase that protects cells from the action of antibiotics
-
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-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. 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
identification of the reaction products as 8-methyladenine2503, and 2,8-dimethyladenine2503
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 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
-
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA
-
-
-
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA
-
-
-
-
?
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
V5ZEZ1
-
-
-
?
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
-
-
-
-
?
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
-
-
-
?
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
-
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)
-
-
?
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
two S-adenosyl-L-methionine molecules serve as the cofactor by providing the 5'-deoxyadenosyl radical for substrate activation and the methyl
-
-
?
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
-
-
-
?
additional information
?
-
C6EKJ2
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
-
-
-
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
-
-
-
additional information
?
-
methylation at C8 is similar to that at C2, cf. EC 2.1.1.192
-
-
-
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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
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA
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
Q9FBG4
-
-
-
-
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 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
-
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA
-
-
-
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA
-
-
-
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA
Q5WJ42
-
-
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA
C0ZJA6
-
-
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA
C0ZJA6
-
-
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA
-
Cfr is an plasmid-acquired methyltransferase that protects cells from the action of antibiotics
-
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA
-
-
-
-
?
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA
-
-
-
-
?
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
V5ZEZ1
-
-
-
?
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
-
-
-
-
?
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
A5HBL2
-
-
-
?
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
Q9FBG4
-
-
-
?
additional information
?
-
C6EKJ2
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
-
-
-
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
-
-
-
additional information
?
-
A5HBL2
methylation at C8 is similar to that at C2, cf. EC 2.1.1.192
-
-
-
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Ferredoxin
-
enzyme Cfr contains a single [4Fe-4S] cluster that binds two separate molecules of S-adenosyl-L-methionine during the reaction cycle
-
S-adenosyl-L-methionine
-
enzyme Cfr contains a single [4Fe-4S] cluster that binds two separate molecules of S-adenosyl-L-methionine during the reaction cycle
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Fe-S-clusters
-
contains a 4Fe-4S cluster. Radical-SAM enzymes contain a [4Fe-4S]+ cluster that is coordinated by the three conserved cysteine thiolate side chains in the CX3CX2C motif and one molecule of S-adenosyl-L-methionine
Fe2+
-
4Fe-4S cluster. Fe-S clusters are of structural, as well as functional, importance to Cfr. 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. Anaerobic purification from Azotobacter vinelandii Cfr
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
energy profile and thermodynamics, overview
-
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene cfr, while cfr is typically plasmid borne, in CM05 it is located on the chromosome and is coexpressed with ermB as part of the mlr operon
UniProt
brenda
Cfr is plasmid-encoded, the Escherichia coli JW2501-1 strain, that does not contain the RlmN MTase that normally mediates the m2A2503 modification, is used as the host for plasmids expressing the mutated Cfr proteins and isolation of total RNA. This is done to avoid the weak primer extension stop from the m2A2503 methylation that overlaps the Cfr-mediated m8A2503 stop and therefore interferes with assessment of the activity of the mutated Cfr proteins
-
-
brenda
gene cfr, while cfr is typically plasmid borne, in CM05 it is located on the chromosome and is coexpressed with ermB as part of the mlr operon
UniProt
brenda
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
additional information
additional information
-
in some clinical isolates, gene cfr is coexpressed with gene erm, which encodes a methyltransferase targeting another 23S rRNA residue, A2058
brenda
additional information
-
in some clinical isolates, gene cfr is coexpressed with gene erm, which encodes a methyltransferase targeting another 23S rRNA residue, A2058
-
brenda
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
physiological function
additional information
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
evolution
-
ClbA acts via the same mechanism as the Cfr methyltransferase
evolution
ClbC acts via the same mechanism as the Cfr methyltransferase
evolution
ClbB acts via the same mechanism as the Cfr methyltransferase
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
-
ClbA acts via the same mechanism as the Cfr methyltransferase
-
evolution
-
ClbB acts via the same mechanism as the Cfr methyltransferase
-
physiological function
-
the Cfr rRNA methyltransferase confers resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A antibiotics
physiological function
-
cfr confers combined resistance to chloramphenicol, florfenicol and clindamycin
physiological function
C6EKJ2
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
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
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
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
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
physiological function
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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
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
-
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)
physiological function
V5ZEZ1
the Cfr RNA methyltransferase causes multiple resistances to peptidyl transferase inhibitors by methylation of A2503 23S rRNA
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
-
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
-
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
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additional information
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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
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
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
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Show AA Sequence and Transmembrane Helices (138 entries)
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
41088
x * 41088, fully methylated protein, calculated from amino acid sequence
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
-
Cfr in solution, secondary structure analysis, circular dichroism spectroscopy and analytical gel filtration, overview
?
x * 41085, electrospray mass spectrometry; x * 41088, fully methylated protein, calculated from amino acid sequence
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C112A
C115A
C6EKJ2
mutation of the cysteines in the presumed radical S-adenosyl-L-methionine motif CxxxCxxC abolishes Cfr activity
C116A
C119A
C122A
C6EKJ2
mutation of the cysteines in the presumed radical S-adenosyl-L-methionine motif CxxxCxxC abolishes Cfr activity
R25A
-
mutation lowers the resistance to both florfenicol and tiamulin considerably
S189A
-
mutation lowers the resistance to both florfenicol and tiamulin considerably
C119A
-
site-directed mutagenesis, the mutant Cfr is inactive, and mutant cells show no resistance against antibiotics
C338A
-
site-directed mutagenesis, Cys338Ala Cfr binds S-adenosyl-L-methionine with equivalent affinity, oxidation of the [4Fe-4S] cluster is not observed
C119A
-
site-directed mutagenesis, the mutant Cfr is inactive, and mutant cells show no resistance against antibiotics
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C338A
the mutant binds S-adenosyl-L-methionine with wild type affinity, while oxidation of the [4Fe-4S] cluster is not observed
additional information
C119A
C6EKJ2
mutation of the cysteines in the presumed radical S-adenosyl-L-methionine motif CxxxCxxC abolishes Cfr activity
additional information
insertion of the cfr region into the CM05 chromosome occurs at a beta/six site I recognition site. Identification of a subpopulation of cfr-negative CM05, CM05DELTA, arising from recombination between duplicate ermB regions, overview
additional information
-
insertion of the cfr region into the CM05 chromosome occurs at a beta/six site I recognition site. Identification of a subpopulation of cfr-negative CM05, CM05DELTA, arising from recombination between duplicate ermB regions, overview
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PURIFICATION/commentary
ORGANISM
UNIPROT
LITERATURE
C-terminally His6-tagged wild-type enzyme and mutant enzymes C112A, C116A and C119A
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recombinant His6-tagged Cfr from Escherichia coli strain BL21Star by nickel affinity chromatography and gel filtration
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CLONED/commentary
ORGANISM
UNIPROT
LITERATURE
expressed as C-terminally His6-tagged enzyme, wild-type and mutant enzymes C125A, C129A and C132A
-
gene cfr, operon isc, co-expression with isc proteins, expression of His6-tagged Cfr under control of an arabinose-inducible promoter in Escherichia coli strain BL21Star
-
gene cfr, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain +Rosetta2(DE3)
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gene cfr, sequence comparisons and phylogenetic analysis, primer extension analysis, cloning and expression in Escherichia coli strains TOP10 and AS19
gene cfr, The Cfr-like protein in Peptoclostridium difficile is hosted by a transposon, and the same gene is found in other strains, sequence comparisons, overview. Recombinant expression in Escherichia coli strain AS19 which then shows elevated MIC values for five classes of antibiotics, and recombinant expression in rlmN-deficient Escherichia coli strain JW2505-1
V5ZEZ1
gene cfr, while cfr is typically plasmid borne, in CM05 it is located on the chromosome and is coexpressed with ermB as part of the mlr operon. DNA and amino acid sequence determination and analysis and genetic organization, overview. The cfr-containing mlr operon is located within a 15.5-kb plasmid-like insertion into 23S rRNA allele 4. In the LZDs colonies, designated CM05DELTA, a recombination event involving the two ermB genes had occurred, resulting in the deletion of cfr and the 3= flanking region, cfr-istAS-istBS-ermB
gene clbA, DNA and amino acid sequence determination and analysis, phylogenetic analysis, expression in Escherichia coli strain AS19 using plasmid pLJ10
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gene clbB, DNA and amino acid sequence determination and analysis, phylogenetic analysis, expression in Escherichia coli strain AS19 using plasmid pLJ10
gene clbC, DNA and amino acid sequence determination and analysis, phylogenetic analysis, expression in Escherichia coli strain AS19 using plasmid pLJ10
gene gfr, ermB and cfr genes are naturally coexpressed under the Perm promoter in the mlr operon, expression of mlr results in modification of A2058 and A2503 in 23S rRNA and renders cells resistant to all clinically relevant antibiotics that target the large ribosomal subunit
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Long, K.S.; Poehlsgaard, J.; Kehrenberg, C.; Schwarz, S.; Vester, B.
The Cfr rRNA methyltransferase confers resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A antibiotics
Antimicrob. Agents Chemother.
50
2500-2505
2006
Escherichia coli
brenda
Yan, F.; LaMarre, J.M.; Röhrich, R.; Wiesner, J.; Jomaa, H.; Mankin, A.S.; Fujimori, D.G.
RlmN and Cfr are radical SAM enzymes involved in methylation of ribosomal RNA
J. Am. Chem. Soc.
132
3953-3964
2010
Escherichia coli
brenda
Kehrenberg, C.; Schwarz, S.; Jacobsen, L.; Hansen, L.H.; Vester, B.
A new mechanism for chloramphenicol, florfenicol and clindamycin resistance: methylation of 23S ribosomal RNA at A2503
Mol. Microbiol.
57
1064-1073
2005
Escherichia coli
brenda
Kaminska, K.H.; Purta, E.; Hansen, L.H.; Bujnicki, J.M.; Vester, B.; Long, K.S.
Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria
Nucleic Acids Res.
38
1652-1663
2009
Escherichia coli
brenda
Giessing, A.M.; Jensen, S.S.; Rasmussen, A.; Hansen, L.H.; Gondela, A.; Long, K.; Vester, B.; Kirpekar, F.
Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria
RNA
15
327-336
2009
Escherichia coli (C6EKJ2), Escherichia coli
brenda
Yan, F.; Fujimori, D.G.
RNA methylation by radical SAM enzymes RlmN and Cfr proceeds via methylene transfer and hydride shift
Proc. Natl. Acad. Sci. USA
108
3930-3934
2011
Escherichia coli
brenda
Boal, A.K.; Grove, T.L.; McLaughlin, M.I.; Yennawar, N.H.; Booker, S.J.; Rosenzweig, A.C.
Structural basis for methyl transfer by a radical SAM enzyme
Science
332
1089-1092
2011
Escherichia coli
brenda
Grove, T.L.; Benner, J.S.; Radle, M.I.; Ahlum, J.H.; Landgraf, B.J.; Krebs, C.; Booker, S.J.
A radically different mechanism for S-adenosylmethionine-dependent methyltransferases
Science
332
604-607
2011
Escherichia coli
brenda
LaMarre, J.M.; Locke, J.B.; Shaw, K.J.; Mankin, A.S.
Low fitness cost of the multidrug resistance gene cfr
Antimicrob. Agents Chemother.
55
3714-3719
2011
Staphylococcus aureus, Staphylococcus aureus RN4220
brenda
Locke, J.B.; Rahawi, S.; Lamarre, J.; Mankin, A.S.; Shaw, K.J.
Genetic environment and stability of cfr in methicillin-resistant Staphylococcus aureus CM05
Antimicrob. Agents Chemother.
56
332-340
2012
Staphylococcus aureus (E1VCY5), Staphylococcus aureus, Staphylococcus aureus CM05 (E1VCY5), Staphylococcus aureus CM05
brenda
Hansen, L.H.; Planellas, M.H.; Long, K.S.; Vester, B.
The order Bacillales hosts functional homologs of the worrisome cfr antibiotic resistance gene
Antimicrob. Agents Chemother.
56
3563-3567
2012
Bacillus amyloliquefaciens, Bacillus amyloliquefaciens FZB42, Bacillus clausii (Q5WJ42), Brevibacillus brevis (C0ZJA6), Brevibacillus brevis NBRC 100599 (C0ZJA6)
brenda
Booth, M.P.; Challand, M.R.; Emery, D.C.; Roach, P.L.; Spencer, J.
High-level expression and reconstitution of active Cfr, a radical-SAM rRNA methyltransferase that confers resistance to ribosome-acting antibiotics
Protein Expr. Purif.
74
204-210
2010
Azotobacter vinelandii
brenda
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
Staphylococcus sciuri (Q9FBG4)
brenda
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
Staphylococcus aureus, Staphylococcus sciuri (Q9FBG4)
brenda
Hansen, L.H.; Vester, B.
A cfr-like gene from Clostridium difficile confers multiple antibiotic resistance by the same mechanism as the cfr gene
Antimicrob. Agents Chemother.
59
5841-5843
2015
Clostridioides difficile, Clostridioides difficile (V5ZEZ1)
brenda
Wang, C.; Yang, X.; Wang, E.; Li, B.
Quantum chemistry studies of adenosine 2503 methylation by S-adenosylmethionine-dependent enzymes
Int. J. Quantum Chem.
113
1409-1415
2013
Staphylococcus aureus (A5HBL2)
-
brenda
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