Literature summary extracted from

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 (2011), Science, 332, 1089-1092 .

Natural Substrates/ Products (Substrates)

EC Number	Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
2.1.1.192	2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin	Escherichia coli	-	S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin	-	?
2.1.1.192	2 S-adenosyl-L-methionine + adenine37 in tRNA + 2 reduced [2Fe-2S] ferredoxin	Escherichia coli	-	2 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine37 in tRNA + 2 oxidized [2Fe-2S] ferredoxin	-	?
2.1.1.224	2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin	Staphylococcus aureus	-	S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin	-	?

Organism

EC Number	Organism	UniProt	Comment	Textmining
2.1.1.192	Escherichia coli	P36979	-	-
2.1.1.224	Staphylococcus aureus	A5HBL2	-	-

Substrates and Products (Substrate)

EC Number	Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
2.1.1.192	2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin	-	Escherichia coli	S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin	-	?
2.1.1.192	2 S-adenosyl-L-methionine + adenine37 in tRNA + 2 reduced [2Fe-2S] ferredoxin	-	Escherichia coli	2 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine37 in tRNA + 2 oxidized [2Fe-2S] ferredoxin	-	?
2.1.1.224	2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin	-	Staphylococcus aureus	S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin	-	?
2.1.1.224	additional information	the enzyme employs a [4Fe-4S] cluster to supply the requisite electron for reductive cleavage of SAM, usually to L-methionine and a 5'-deoxyadenosyl 5'-radical. SAM is the source of both the 5'-deoxyadenosyl 5'-radical and the appended methyl group	Staphylococcus aureus	?	-	-

Synonyms

EC Number	Synonyms	Comment	Organism
2.1.1.192	RlmN	-	Escherichia coli
2.1.1.224	Cfr	-	Staphylococcus aureus

Cofactor

EC Number	Cofactor	Comment	Organism	Structure
2.1.1.192	reduced [2Fe-2S] ferredoxin	-	Escherichia coli
2.1.1.192	S-adenosyl-L-methionine	SAM, crystal structures of RlmN and RlmN with SAM show that a single molecule of SAM coordinates the [4Fe-4S] cluster. Residue Cys 355 is S-methylated and located proximal to the SAM methyl group, suggesting that SAM involved in the initial methyl transfer binds at the same site	Escherichia coli
2.1.1.192	[4Fe-4S] cluster	a single molecule of SAM coordinates the [4Fe-4S] cluster	Escherichia coli
2.1.1.224	reduced [2Fe-2S] ferredoxin	-	Staphylococcus aureus
2.1.1.224	S-adenosyl-L-methionine	-	Staphylococcus aureus

General Information

EC Number	General Information	Comment	Organism
2.1.1.192	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	Escherichia coli
2.1.1.192	additional information	structure modelling and structure-function analysis of RlmN compared to Cfr from Staphylococcus aureus (EC 2.1.1.224), Escherichia coli RlmN and Staphylococcus aureus Cfr are mapped onto the RlmN structure, detailed overview. The RlmN reactions proceed by a ping-pong mechanism. The methyl group from one SAM molecule is initially appended to a conserved Cys355 in RlmN 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. Finally, this covalent intermediate is resolved by formation of a disulfide bond between the methyl-carrying Cys (mCys) residue and a second conserved Cys118. Nature and location of the rRNA binding site. The electrostatic surface potential for RlmN suggests the substrate may approach the active site from the bottom of the barrel. This extensive surface involves the core barrel, its extensions, and the extra domain, implicating the accessory elements in substrate interaction	Escherichia coli
2.1.1.192	physiological function	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. Enzymes RlmN and Cfr catalyze methylation of a 23S rRNA nucleotide (adenosine 2503, A2503) ultimately located within the peptidyltransferase center of the 50S subunit of the bacterial ribosome near the entrance to the nascent peptide exit tunnel. RlmN methylates the C2 position of A2503, a housekeeping modification important in translational fidelity and the nascent peptide response. 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. RlmN accomplishes its complex reaction with structural economy, harnessing the two most important reactivities of SAM within a single site	Escherichia coli
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	Staphylococcus aureus
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	Staphylococcus aureus
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	Staphylococcus aureus
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	Staphylococcus aureus
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	Staphylococcus aureus

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Release 2023.1 (January 2023)