BRENDA - Enzyme Database

Substrate tRNA recognition mechanism of a multisite-specific tRNA methyltransferase, Aquifex aeolicus Trm1, based on the X-ray crystal structure

Awai, T.; Ochi, A.; Ihsanawati, A.; Sengoku, T.; Hirata, A.; Bessho, Y.; Yokoyama, S.; Hori, H.; J. Biol. Chem. 286, 35236-35246 (2011)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
2.1.1.202
gene MJ0026, expression in Escherichia coli strain BL21-CodonPlus(DE3)-RIL
Methanocaldococcus jannaschii
Crystallization (Commentary)
EC Number
Crystallization
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
Engineering
EC Number
Amino acid exchange
Commentary
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
Commentary
Organism
Structure
2.1.1.215
-
-
S-adenosyl-L-methionine
pH 7.5, 55C, recombinant mutant F27A
Aquifex aeolicus
2.1.1.215
0.00017
-
S-adenosyl-L-methionine
pH 7.5, 55C, recombinant mutant I85A; pH 7.5, 55C, recombinant wild-type enzyme
Aquifex aeolicus
2.1.1.215
0.0002
-
S-adenosyl-L-methionine
pH 7.5, 55C, recombinant mutant I65A
Aquifex aeolicus
2.1.1.215
0.0003
-
S-adenosyl-L-methionine
pH 7.5, 55C, recombinant mutant E6A; pH 7.5, 55C, recombinant mutant N29A
Aquifex aeolicus
2.1.1.215
0.00067
-
S-adenosyl-L-methionine
pH 7.5, 55C, recombinant mutants L60A, F140A, and E113A
Aquifex aeolicus
2.1.1.215
0.0008
-
S-adenosyl-L-methionine
pH 7.5, 55C, recombinant mutant F134A
Aquifex aeolicus
2.1.1.215
0.0013
-
S-adenosyl-L-methionine
pH 7.5, 55C, recombinant mutant D130A
Aquifex aeolicus
2.1.1.215
0.023
-
S-adenosyl-L-methionine
pH 7.5, 55C, recombinant mutant F27A
Aquifex aeolicus
2.1.1.215
0.045
-
S-adenosyl-L-methionine
pH 7.5, 55C, recombinant mutant R66A
Aquifex aeolicus
2.1.1.215
0.33
-
S-adenosyl-L-methionine
pH 7.5, 55C, recombinant mutant D84A
Aquifex aeolicus
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
2.1.1.215
Mg2+
required
Aquifex aeolicus
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
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
Primary Accession No. (UniProt)
Commentary
Textmining
2.1.1.202
Methanocaldococcus jannaschii
-
gene MJ0026
-
2.1.1.215
Aquifex aeolicus
-
-
-
Purification (Commentary)
EC Number
Commentary
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
Commentary
Organism
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
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
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
719968
Methanocaldococcus jannaschii
?
-
-
-
-
2.1.1.202
S-adenosyl-L-methionine + cytosine40 in tRNA precursor
-
719968
Methanocaldococcus jannaschii
S-adenosyl-L-homocysteine + 5-methylcytosine40 in tRNA precursor
-
-
-
?
2.1.1.202
S-adenosyl-L-methionine + cytosine48 in tRNA
-
719968
Methanocaldococcus jannaschii
S-adenosyl-L-homocysteine + 5-methylcytosine48 in tRNA
-
-
-
?
2.1.1.215
4 S-adenosyl-L-methionine + guanine26/guanine27 in tRNA
-
719968
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
719968
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
719968
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
719968
Aquifex aeolicus
?
-
-
-
-
Subunits
EC Number
Subunits
Commentary
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
Temperature Optimum [C]
EC Number
Temperature Optimum [C]
Temperature Optimum Maximum [C]
Commentary
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
Commentary
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
Commentary
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
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
2.1.1.202
gene MJ0026, expression in Escherichia coli strain BL21-CodonPlus(DE3)-RIL
Methanocaldococcus jannaschii
Cofactor (protein specific)
EC Number
Cofactor
Commentary
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
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
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
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
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] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
2.1.1.215
-
-
S-adenosyl-L-methionine
pH 7.5, 55C, recombinant mutant F27A
Aquifex aeolicus
2.1.1.215
0.00017
-
S-adenosyl-L-methionine
pH 7.5, 55C, recombinant mutant I85A; pH 7.5, 55C, recombinant wild-type enzyme
Aquifex aeolicus
2.1.1.215
0.0002
-
S-adenosyl-L-methionine
pH 7.5, 55C, recombinant mutant I65A
Aquifex aeolicus
2.1.1.215
0.0003
-
S-adenosyl-L-methionine
pH 7.5, 55C, recombinant mutant E6A; pH 7.5, 55C, recombinant mutant N29A
Aquifex aeolicus
2.1.1.215
0.00067
-
S-adenosyl-L-methionine
pH 7.5, 55C, recombinant mutants L60A, F140A, and E113A
Aquifex aeolicus
2.1.1.215
0.0008
-
S-adenosyl-L-methionine
pH 7.5, 55C, recombinant mutant F134A
Aquifex aeolicus
2.1.1.215
0.0013
-
S-adenosyl-L-methionine
pH 7.5, 55C, recombinant mutant D130A
Aquifex aeolicus
2.1.1.215
0.023
-
S-adenosyl-L-methionine
pH 7.5, 55C, recombinant mutant F27A
Aquifex aeolicus
2.1.1.215
0.045
-
S-adenosyl-L-methionine
pH 7.5, 55C, recombinant mutant R66A
Aquifex aeolicus
2.1.1.215
0.33
-
S-adenosyl-L-methionine
pH 7.5, 55C, recombinant mutant D84A
Aquifex aeolicus
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
2.1.1.215
Mg2+
required
Aquifex aeolicus
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
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
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
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
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
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
719968
Methanocaldococcus jannaschii
?
-
-
-
-
2.1.1.202
S-adenosyl-L-methionine + cytosine40 in tRNA precursor
-
719968
Methanocaldococcus jannaschii
S-adenosyl-L-homocysteine + 5-methylcytosine40 in tRNA precursor
-
-
-
?
2.1.1.202
S-adenosyl-L-methionine + cytosine48 in tRNA
-
719968
Methanocaldococcus jannaschii
S-adenosyl-L-homocysteine + 5-methylcytosine48 in tRNA
-
-
-
?
2.1.1.215
4 S-adenosyl-L-methionine + guanine26/guanine27 in tRNA
-
719968
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
719968
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
719968
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
719968
Aquifex aeolicus
?
-
-
-
-
Subunits (protein specific)
EC Number
Subunits
Commentary
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
Temperature Optimum [C] (protein specific)
EC Number
Temperature Optimum [C]
Temperature Optimum Maximum [C]
Commentary
Organism
2.1.1.202
70
-
assay at
Methanocaldococcus jannaschii
2.1.1.215
55
-
assay at
Aquifex aeolicus
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
2.1.1.202
7.7
-
assay at
Methanocaldococcus jannaschii
2.1.1.215
7.5
-
assay at
Aquifex aeolicus
General Information
EC Number
General Information
Commentary
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
General Information (protein specific)
EC Number
General Information
Commentary
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