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Literature summary extracted from

  • Awai, T.; Ochi, A.; Ihsanawati, A.; Sengoku, T.; Hirata, A.; Bessho, Y.; Yokoyama, S.; Hori, H.
    Substrate tRNA recognition mechanism of a multisite-specific tRNA methyltransferase, Aquifex aeolicus Trm1, based on the X-ray crystal structure (2011), J. Biol. Chem., 286, 35236-35246.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.1.1.202 gene MJ0026, expression in Escherichia coli strain BL21-CodonPlus(DE3)-RIL Methanocaldococcus jannaschii

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.1.1.202 Trm4 free and in complex with sinefungin, hanging-drop vapor diffusion, 0.001 ml of protein solution containing 10.8 mg/ml protein with 0.001 ml of crystallization solution containing 100 mM Tris-HCl, pH 7.4, 200 mM MgCl2, 25% PEG 3350, and 6% v/v isopropanol for MJ0026 alone, and 20 100 mM Tris-HCl, pH 7.4, 200 mM MgCl2, 25% PEG 3350, 3 mM sinefungin, and 10 mM cytidine for Mj0026 with sinefungin, cryoprotectant 20% glycerol and 20% PEG 400, respectively, X-ray diffraction structure determination and analysis at 1.27 A and 2.3 A resolutions, respectively Methanocaldococcus jannaschii

Protein Variants

EC Number Protein Variants Comment Organism
2.1.1.215 D130A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Aquifex aeolicus
2.1.1.215 D132A site-directed mutagenesis, inactive mutant Aquifex aeolicus
2.1.1.215 D84A site-directed mutagenesis, almost inactive mutant Aquifex aeolicus
2.1.1.215 E113A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Aquifex aeolicus
2.1.1.215 E6A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Aquifex aeolicus
2.1.1.215 F134A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Aquifex aeolicus
2.1.1.215 F140A site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Aquifex aeolicus
2.1.1.215 F27A site-directed mutagenesis, almost inactive mutant Aquifex aeolicus
2.1.1.215 H110A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Aquifex aeolicus
2.1.1.215 H219A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Aquifex aeolicus
2.1.1.215 H274A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Aquifex aeolicus
2.1.1.215 I65A site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Aquifex aeolicus
2.1.1.215 I85A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Aquifex aeolicus
2.1.1.215 K170A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Aquifex aeolicus
2.1.1.215 K283A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Aquifex aeolicus
2.1.1.215 L60A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Aquifex aeolicus
2.1.1.215 N29A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Aquifex aeolicus
2.1.1.215 R179A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Aquifex aeolicus
2.1.1.215 R192A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Aquifex aeolicus
2.1.1.215 R31A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Aquifex aeolicus
2.1.1.215 R36A site-directed mutagenesis, inactive mutant Aquifex aeolicus
2.1.1.215 R66A site-directed mutagenesis, almost inactive mutant Aquifex aeolicus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.1.1.215 0.00017
-
 S-adenosyl-L-methionine pH 7.5, 55°C, recombinant mutant I85A Aquifex aeolicus
2.1.1.215 0.00017
-
 S-adenosyl-L-methionine pH 7.5, 55°C, recombinant wild-type enzyme Aquifex aeolicus
2.1.1.215 0.0002
-
 S-adenosyl-L-methionine pH 7.5, 55°C, recombinant mutant I65A Aquifex aeolicus
2.1.1.215 0.0003
-
 S-adenosyl-L-methionine pH 7.5, 55°C, recombinant mutant E6A Aquifex aeolicus
2.1.1.215 0.0003
-
 S-adenosyl-L-methionine pH 7.5, 55°C, recombinant mutant N29A Aquifex aeolicus
2.1.1.215 0.00067
-
 S-adenosyl-L-methionine pH 7.5, 55°C, recombinant mutants L60A, F140A, and E113A Aquifex aeolicus
2.1.1.215 0.0008
-
 S-adenosyl-L-methionine pH 7.5, 55°C, recombinant mutant F134A Aquifex aeolicus
2.1.1.215 0.0013
-
 S-adenosyl-L-methionine pH 7.5, 55°C, recombinant mutant D130A Aquifex aeolicus
2.1.1.215 0.023
-
 S-adenosyl-L-methionine pH 7.5, 55°C, recombinant mutant F27A Aquifex aeolicus
2.1.1.215 0.045
-
 S-adenosyl-L-methionine pH 7.5, 55°C, recombinant mutant R66A Aquifex aeolicus
2.1.1.215 0.33
-
 S-adenosyl-L-methionine pH 7.5, 55°C, recombinant mutant D84A Aquifex aeolicus

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.1.1.215 Mg2+ required Aquifex aeolicus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.1.1.202 S-adenosyl-L-methionine + cytosine40 in tRNA precursor Methanocaldococcus jannaschii
-
 S-adenosyl-L-homocysteine + 5-methylcytosine40 in tRNA precursor
-
 ?
2.1.1.202 S-adenosyl-L-methionine + cytosine48 in tRNA Methanocaldococcus jannaschii
-
 S-adenosyl-L-homocysteine + 5-methylcytosine48 in tRNA
-
 ?
2.1.1.215 4 S-adenosyl-L-methionine + guanine26/guanine27 in tRNA Aquifex aeolicus
-
 4 S-adenosyl-L-homocysteine + N2-dimethylguanine26/N2-dimethylguanine27 in tRNA
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.1.1.202 Methanocaldococcus jannaschii
-
 gene MJ0026
-
2.1.1.215 Aquifex aeolicus
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.1.1.202 recombinant MJ0026 from Escherichia coli strain BL21-CodonPlus(DE3)-RIL by heat treatment, anion exchange and hydroxyapatite chromatography, and dialysis Methanocaldococcus jannaschii

Reaction

EC Number Reaction Comment Organism Reaction ID
2.1.1.215 4 S-adenosyl-L-methionine + guanine26/guanine27 in tRNA = 4 S-adenosyl-L-homocysteine + N2-dimethylguanine26/N2-dimethylguanine27 in tRNA reaction mechanism, overview Aquifex aeolicus
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.1.1.202 additional information highly conserved residues form a large, positively charged surface, which seems to be suitable for tRNA binding. Substrate specificity and methylation sites on tRNA, overview Methanocaldococcus jannaschii ?
-
 ?
2.1.1.202 S-adenosyl-L-methionine + cytosine40 in tRNA precursor
-
 Methanocaldococcus jannaschii S-adenosyl-L-homocysteine + 5-methylcytosine40 in tRNA precursor
-
 ?
2.1.1.202 S-adenosyl-L-methionine + cytosine48 in tRNA
-
 Methanocaldococcus jannaschii S-adenosyl-L-homocysteine + 5-methylcytosine48 in tRNA
-
 ?
2.1.1.215 4 S-adenosyl-L-methionine + guanine26/guanine27 in tRNA
-
 Aquifex aeolicus 4 S-adenosyl-L-homocysteine + N2-dimethylguanine26/N2-dimethylguanine27 in tRNA
-
 ?
2.1.1.215 4 S-adenosyl-L-methionine + guanine26/guanine27 in tRNA the T-arm in tRNA is the binding site of Trm1, multisite specificity, tRNA-docking modeling, overview Aquifex aeolicus 4 S-adenosyl-L-homocysteine + N2-dimethylguanine26/N2-dimethylguanine27 in tRNA
-
 ?
2.1.1.215 4 S-adenosyl-L-methionine + guanine26/guanine27 in tRNAPhe tRNAPhe from yeast Aquifex aeolicus 4 S-adenosyl-L-homocysteine + N2-dimethylguanine26/N2-dimethylguanine27 in tRNAPhe
-
 ?
2.1.1.215 additional information Trm1 catalyzes methyl transfers to class II tRNAs as well as all class I tRNAs, the size and sequence of the variable region are not recognized by Aquifex aeolicus Trm1, all tRNA transcripts are methylated. tRNA recognition mechanism of multisite specific Trm1, Asp132 is a catalytic center, structure-function analysis, overview Aquifex aeolicus ?
-
 ?

Subunits

EC Number Subunits Comment Organism
2.1.1.215 More Trm1 from Aquifex aeolicus contains a zinc-cysteine cluster in the C-terminal domain. The N-terminal domain is a typical catalytic domain of S-adenosyl-L-methionine-dependent methyltransferases. Overall structure, structure comparisons, and structure-function analysis, overview Aquifex aeolicus

Synonyms

EC Number Synonyms Comment Organism
2.1.1.202 MJ0026
-
 Methanocaldococcus jannaschii
2.1.1.202 RNA:m5C methyltransferase
-
 Methanocaldococcus jannaschii
2.1.1.202 TRM4
-
 Methanocaldococcus jannaschii
2.1.1.215 multisite-specific tRNA methyltransferase
-
 Aquifex aeolicus
2.1.1.215 Trm1
-
 Aquifex aeolicus
2.1.1.215 tRNA (N2,N2-guanine)-dimethyltransferase
-
 Aquifex aeolicus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.1.1.202 70
-
 assay at Methanocaldococcus jannaschii
2.1.1.215 55
-
 assay at Aquifex aeolicus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.1.1.202 7.7
-
 assay at Methanocaldococcus jannaschii
2.1.1.215 7.5
-
 assay at Aquifex aeolicus

Cofactor

EC Number Cofactor Comment Organism Structure
2.1.1.202 additional information the S-adenosyl-L-methionine analigue sinefungin is bound in a negatively charged pocket near helix alpha8, binding structure, detailed overview Methanocaldococcus jannaschii
2.1.1.202 S-adenosyl-L-methionine helix alpha8 can adopt two different conformations, thereby controlling the entry of S-adenosyl-L-methionine into the active site Methanocaldococcus jannaschii
2.1.1.215 S-adenosyl-L-methionine binding structure, overview Aquifex aeolicus

General Information

EC Number General Information Comment Organism
2.1.1.215 evolution archaeal and eukaryotic tRNA (N2,N2-guanine)-dimethyltransferase, Trm1, produces N2,N2-dimethylguanine at position 26 in tRNA. In contrast, Trm1 from Aquifex aeolicus, a hyper-thermophilic eubacterium, modifies G27 as well as G26. The overall structure of Aquifex aeolicus Trm1 is similar to that of archaeal Trm1, although there is a zinc-cysteine cluster in the C-terminal domain of Aquifex aeolicus Trm1 Aquifex aeolicus