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Literature summary extracted from

  • Schwalm, E.; Grove, T.; Booker, S.; Boal, A.
    Crystallographic capture of a radical S-adenosylmethionine enzyme in the act of modifying tRNA (2016), Science, 352, 309-312 .
    View publication on PubMedView publication on EuropePMC

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.1.1.192 C118A enzyme mutant complexed with tRNAGlu substrate, X-ray diffraction structure determination and analysis at 2.4 A resolution, structure modelling Escherichia coli
2.1.1.192 structure of a key intermediate in the RlmN reaction, in which a Cys118Ala variant of the protein is crosslinked to a tRNAGlu substrate through the terminal methylene carbon of a formerly methylcysteinyl residue and C2 of A37. RlmN contacts the entire length of tRNAGlu, accessing A37 using an induced-fit strategy that completely unfolds the tRNA anticodon stem loop Escherichia coli

Protein Variants

EC Number Protein Variants Comment Organism
2.1.1.192 C118A mutant is unable to resolve a covalent protein/RNA intermediate during catalysis and becomes cross-linked to the nucleic acid Escherichia coli
2.1.1.192 C118A site-directed mutagenesis, analysis of the structure of a key intermediate in the RlmN reaction, in which a C118A variant of the protein is cross-linked to a tRNAGlu substrate Escherichia coli

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 Escherichia coli
-
 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 Escherichia coli
-
-
-

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
-
 Escherichia coli S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin
-
 ?

Synonyms

EC Number Synonyms Comment Organism
2.1.1.192 RlmN
-
 Escherichia coli
2.1.1.224 Cfr
-
 Escherichia coli
2.1.1.224 radical S-adenosylmethionine enzyme
-
 Escherichia coli

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
-
 Escherichia coli
2.1.1.224 reduced [2Fe-2S] ferredoxin
-
 Escherichia coli
2.1.1.224 S-adenosyl-L-methionine
-
 Escherichia coli

General Information

EC Number General Information Comment Organism
2.1.1.192 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. Structure analysis of the key intermediate in the RlmN reaction, in which a Cys118->Ala variant of the protein is cross-linked to a tRNAGlu substrate through the terminal methylene carbon of a formerly methylcysteinyl residue and C2 of A37. RlmN contacts the entire length of tRNAGlu, accessing A37 using an induced-fit strategy that completely unfolds the tRNA anticodon stem loop, which is likely critical for recognition of both tRNA and rRNA substrates. The most extensive RlmN-tRNA interactions involve the anti-codon stem loop (ACSL) of tRNAGlu near A37. The protein binds in the minor groove of the ACSL and interacts more intimately with the nucleobases. Binding structure, overview Escherichia coli
2.1.1.192 physiological function RlmN is a radical S-adenosylmethionine (SAM) enzyme that is best known for catalyzing the methylation of C2 of adenosine 2503 (A2503) (1-3) in domain V of 23S rRNA. But RlmN is a dual-specificity RNA methylase that modifies C2 of adenosine 2503 (A2503) in 23S rRNA and C2 of adenosine 37 (A37) in several Escherichia coli tRNAs. RlmN thus joins a pseudouridine synthase, RluA, as a known dual-specificity RNA modification enzymes capable of acting both on ribosomal and on transfer RNA Escherichia coli
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 Escherichia coli
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 Escherichia coli