Literature summary for 2.1.1.224 extracted from

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 (2010), Protein Expr. Purif., 74, 204-210.

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Activating Compound

Activating Compound	Comment	Organism	Structure
Sodium dithionite	-	Azotobacter vinelandii

Cloned(Commentary)

Cloned (Comment)	Organism
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	Azotobacter vinelandii

Metals/Ions

Metals/Ions	Comment	Organism	Structure
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	Azotobacter vinelandii

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA	Azotobacter vinelandii	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	S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA	-	?

Organism

Organism	UniProt	Comment	Textmining
Azotobacter vinelandii	-	isc operon, gene cfr	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His6-tagged Cfr from Escherichia coli strain BL21Star by nickel affinity chromatography and gel filtration	Azotobacter vinelandii

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
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	-	?
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	-	?

Subunits

Subunits	Comment	Organism
monomer	Cfr in solution, secondary structure analysis, circular dichroism spectroscopy and analytical gel filtration, overview	Azotobacter vinelandii

Synonyms

Synonyms	Comment	Organism
Cfr	-	Azotobacter vinelandii
radical-S-adenosyl-L-methionine enzyme	-	Azotobacter vinelandii
radical-SAM enzyme	-	Azotobacter vinelandii
radical-SAM rRNA methyltransferase	-	Azotobacter vinelandii

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Azotobacter vinelandii

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
9.5	-	assay at	Azotobacter vinelandii

Cofactor

Cofactor	Comment	Organism	Structure
S-adenosyl-L-methionine	-	Azotobacter vinelandii

General Information

General Information	Comment	Organism
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	Azotobacter vinelandii

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Release 2023.1 (January 2023)