Adenosine C2 methyltransferase (RlmN)

 

23S rRNA (adenine(2503)-C(2))-methyltransferase (RlmN) specifically methylates position 2 of adenine 2503 in 23S rRNA and position 2 of adenine 37 in tRNAs. m2A2503 modification seems to play a crucial role in the proofreading step occurring at the peptidyl transferase centre and thus would serve to optimise ribosomal fidelity. Unmodified tRNA is not a suitable substrate for RlmN, which suggests that RlmN works in a late step during tRNA maturation. RlmN is a member of the Radical SAM superfamily.

 

Reference Protein and Structure

Sequence
P36979 UniProt (2.1.1.192) IPR027492 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
3rfa - X-ray structure of RlmN from Escherichia coli in complex with S-adenosylmethionine (2.05 Å) PDBe PDBsum 3rfa
Catalytic CATH Domains
3.20.20.70 CATHdb (see all for 3rfa)
Cofactors
Tetra-mu3-sulfido-tetrairon (1)
Click To Show Structure

Enzyme Reaction (EC:2.1.1.192)

S-adenosyl-L-methionine zwitterion
CHEBI:59789ChEBI
+
di-mu-sulfido-diiron(1+)
CHEBI:33738ChEBI
+
adenosine 5'-monophosphate(1-) residue
CHEBI:74411ChEBI
+
di-mu-sulfido-diiron(2+)
CHEBI:33737ChEBI
→
5'-deoxyadenosine
CHEBI:17319ChEBI
+
S-adenosyl-L-homocysteine zwitterion
CHEBI:57856ChEBI
+
2-methyladenosine 5'-monophosphate(1-) residue
CHEBI:74497ChEBI
+
L-methionine zwitterion
CHEBI:57844ChEBI
Alternative enzyme names: RlmN, YfgB, Cfr,

Enzyme Mechanism

Introduction

The first SAM molecule donates its methyl group to Cys355. SAH dissociates and a second molecule of SAM binds. This second molecule binds at the apical iron of the [4Fe-4S]-cluster. SAM binds at this free iron and undergoes homolytic cleavage to form the 5'-deoxyadenosyl radical essential for the reaction. This radical then abstracts a hydrogen atom from the methyl group attached to Cys355. RNA then binds, the methyl radical attacks the adenine base to form a new radical species. Glu105 abstracts a proton from the adenine base and a single electron is lost to ferredoxin. Cys118 then forms a disulfide bond with Cys355, eliminating the methylated RNA. The anionic species formed then abstracts a proton from Glu105 to form the final species. The enzyme's active site is regenerated through the reduction of the disulfide bond with the gain of two protons and loss of two electrons; the exact mechanism of this regeneration is currently unclear.

Catalytic Residues Roles

UniProt PDB* (3rfa)
Cys118 Cys118B Nucleophile, initiates reductive cleavage of S-S bond covalent catalysis
Glu105 Glu105B Acts as a general acid/base. proton shuttle (general acid/base)
Cys129, Cys125, Cys132 Cys129B, Cys125B, Cys132B Forms the canonical CxxCxxxC motif that defines membership of the radical SAM superfamily. Binds [4Fe-4S]-AdoMet cluster. metal ligand
Cys355 Smc355B Nucleophile, forms methylthioether with methyl group from SAM covalent catalysis
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

References

  1. Zhang Q et al. (2012), Acc Chem Res, 45, 555-564. Radical-mediated enzymatic methylation: a tale of two SAMS. DOI:10.1021/ar200202c. PMID:22097883.
  2. Schwalm EL et al. (2016), Science, 352, 309-312. Crystallographic capture of a radical S-adenosylmethionine enzyme in the act of modifying tRNA. DOI:10.1126/science.aad5367. PMID:27081063.
  3. Silakov A et al. (2014), J Am Chem Soc, 136, 8221-8228. Characterization of a cross-linked protein-nucleic acid substrate radical in the reaction catalyzed by RlmN. DOI:10.1021/ja410560p. PMID:24806349.
  4. Fujimori DG (2013), Curr Opin Chem Biol, 17, 597-604. Radical SAM-mediated methylation reactions. DOI:10.1016/j.cbpa.2013.05.032. PMID:23835516.
  5. McCusker KP et al. (2012), J Am Chem Soc, 134, 18074-18081. Covalent intermediate in the catalytic mechanism of the radical S-adenosyl-L-methionine methyl synthase RlmN trapped by mutagenesis. DOI:10.1021/ja307855d. PMID:23088750.
  6. Benítez-Páez A et al. (2012), RNA, 18, 1783-1795. The Escherichia coli RlmN methyltransferase is a dual-specificity enzyme that modifies both rRNA and tRNA and controls translational accuracy. DOI:10.1261/rna.033266.112. PMID:22891362.
  7. Grove TL et al. (2011), J Am Chem Soc, 133, 19586-19589. Cfr and RlmN contain a single [4Fe-4S] cluster, which directs two distinct reactivities for S-adenosylmethionine: methyl transfer by SN2 displacement and radical generation. DOI:10.1021/ja207327v. PMID:21916495.
  8. Boal AK et al. (2011), Science, 332, 1089-1092. Structural basis for methyl transfer by a radical SAM enzyme. DOI:10.1126/science.1205358. PMID:21527678.
  9. Grove TL et al. (2011), Science, 332, 604-607. A radically different mechanism for S-adenosylmethionine-dependent methyltransferases. DOI:10.1126/science.1200877. PMID:21415317.
  10. Yan F et al. (2011), Proc Natl Acad Sci U S A, 108, 3930-3934. RNA methylation by radical SAM enzymes RlmN and Cfr proceeds via methylene transfer and hydride shift. DOI:10.1073/pnas.1017781108. PMID:21368151.
  11. Yan F et al. (2010), J Am Chem Soc, 132, 3953-3964. RlmN and Cfr are radical SAM enzymes involved in methylation of ribosomal RNA. DOI:10.1021/ja910850y. PMID:20184321.
  12. Toh SM et al. (2008), RNA, 14, 98-106. The methyltransferase YfgB/RlmN is responsible for modification of adenosine 2503 in 23S rRNA. DOI:10.1261/rna.814408. PMID:18025251.

Step 1.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys118B covalent catalysis
Smc355B covalent catalysis
Cys125B activator, metal ligand
Cys129B metal ligand
Cys132B metal ligand
Glu105B proton shuttle (general acid/base)

Chemical Components

Step 1.

Contributors

Gemma L. Holliday