Cloned (Comment) | Organism |
---|---|
gene rlmN, recombinant expression of tagged enzyme in Escherichia coli | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
C118A | loss of tRNA methylation activity | Escherichia coli |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 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 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | - |
- |
- |
Escherichia coli | P36979 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant tagged enzyme from Escherichia coli by metal chelating affinity chromatography, anion exchange chromatography, and ultrafiltration | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 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 S-adenosyl-L-methionine + adenine37 in tRNAArg(ACG) + 2 reduced [2Fe-2S] ferredoxin | low activity with tRNAArg(ACG) | Escherichia coli | 2 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine37 in tRNAArg(ACG) + 2 oxidized [2Fe-2S] ferredoxin | - |
? | |
2 S-adenosyl-L-methionine + adenine37 in tRNAAsp(GUC) + 2 reduced [2Fe-2S] ferredoxin | very low activity with tRNAAsp(GUC) | Escherichia coli | 2 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine37 in tRNAAsp(GUC) + 2 oxidized [2Fe-2S] ferredoxin | - |
? | |
2 S-adenosyl-L-methionine + adenine37 in tRNAGln(CUG) + 2 reduced [2Fe-2S] ferredoxin | moderate activity with tRNAGln(CUG) | Escherichia coli | 2 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine37 in tRNAGln(CUG) + 2 oxidized [2Fe-2S] ferredoxin | - |
? | |
2 S-adenosyl-L-methionine + adenine37 in tRNAGln(UUG) + 2 reduced [2Fe-2S] ferredoxin | high activity with tRNAGln(UUG) | Escherichia coli | 2 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine37 in tRNAGln(UUG) + 2 oxidized [2Fe-2S] ferredoxin | - |
? | |
2 S-adenosyl-L-methionine + adenine37 in tRNAGlu(UUC) + 2 reduced [2Fe-2S] ferredoxin | low activity with tRNAGlu(UUC) | Escherichia coli | 2 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine37 in tRNAGlu(UUC) + 2 oxidized [2Fe-2S] ferredoxin | - |
? | |
2 S-adenosyl-L-methionine + adenine37 in tRNAHis(GUG) + 2 reduced [2Fe-2S] ferredoxin | best tRNA substrate | Escherichia coli | 2 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine37 in tRNAHis(GUG) + 2 oxidized [2Fe-2S] ferredoxin | - |
? | |
additional information | enzyme additionally methylates adenine37 in tRNA such as EScherichia coli tRNAArgACG, tRNAAspGUC, tRNAGlnCUG, tRNAGlnUUG, tRNAGluUUC, and tRNAHisGUG. tRNA methylation requirements are consistent with radical SAM reactivity | Escherichia coli | ? | - |
- |
|
additional information | only a subset of tRNAs that contain an adenosine at position 37 are substrates for RlmN, structural comparison of substrates and non-substrates, and generation of chimeric tRNA substrates, substrate specificity, detailed overview. No activity with tRNAGly(CCC), in vitro methylation of chimeric constructs | Escherichia coli | ? | - |
- |
Synonyms | Comment | Organism |
---|---|---|
RlmN | - |
Escherichia coli |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
reduced [2Fe-2S] ferredoxin | - |
Escherichia coli | |
S-adenosyl-L-methionine | - |
Escherichia coli | |
[4Fe-4S] cluster | - |
Escherichia coli |
General Information | Comment | Organism |
---|---|---|
evolution | unlike methylation of nitrogen or oxygen atoms, methylation of cytosine or uridine at the C5 position requires a different mechanism, because the target position is not nucleophilic. Covalent catalysis via a Michael addition activates the C5 carbon and accounts for methylation at these sites. In contrast, methylation at the unreactive C2 and C8 positions of adenosines requires a different enzymatic mechanism and is catalyzed by members of the radical S-adenosyl-L-methionine (SAM) superfamily | Escherichia coli |
additional information | determinants of tRNA recognition by the radical SAM enzyme RlmN, overview. Usage of in vitro transcribed tRNAs as model substrates to interrogate RNA recognition by RlmN. Structure and sequence of RNA influence methylation, identifying position 38 of tRNAs as a critical determinant of substrate recognition, tRNA methylation requirements are consistent with radical S-adenosyl-L-methionine (SAM) reactivity. Studies on RlmN and Cfr, two bacterial radical SAM methylating enzymes, have established key mechanistic features of radical SAM methylation of RNA. A unique feature of these enzymes is their ability to utilize both homolytic and heterolytic reactivity of SAM to carry out methylation of the C2 and C8 amidine carbons of adenosine. The first equivalent of SAM is used to methylate a conserved cysteine residue (C355), unassociated with the four iron-four sulfur ([4Fe-4S]) cluster, to form a protein-bound methyl thioether. A second equivalent of SAM, coordinated by the [4Fe-4S] cluster in these proteins, is then cleaved homolytically to generate a 5'-deoxyadenosyl radical, a canonical feature of radical SAM catalysis. This highly reactive radical species then abstracts a hydrogen atom from the premethylated cysteine 355 to form a thiomethylene radical. The methylene radical then adds into the substrate carbon to form a covalent RNA-protein adduct, which has been trapped by mutagenesis and characterized spectroscopically. A second conserved cysteine residue resolves the covalent RNA-protein intermediate, forming the methylated product | Escherichia coli |
physiological function | RlmN is a bacterial radical SAM methylating enzyme, that has the unusual ability to modify two distinct types of RNA: 23S rRNA and tRNA. In rRNA, RlmN installs a methyl group at the C2 position of A2503 of 23S rRNA, while in tRNA the modification occurs at nucleotide A37, immediately adjacent to the anticodon triplet | Escherichia coli |