Cloned (Comment) | Organism |
---|---|
recombinant expression of N-terminally His-tagged enzyme Trm10 in Escherichia coli strain BL21/Gold(DE3) | Saccharomyces cerevisiae |
recombinant expression of N-terminally His-tagged enzyme Trm10 in Escherichia coli strain BL21/Gold(DE3), selenomethionine-labeled spTrm10_74 (residues 74-281) is expressed in Escherichia coli strain B834, recombinant wild-type and mutant Q226A TrmT10C (residues 40-403) is coexpressed with His-tagged protein SDR5C1 | Schizosaccharomyces pombe |
Crystallization (Comment) | Organism |
---|---|
purified enzyme free or in complex with S-adenosyl-L-homocysteine, X-ray diffraction structure determination and analysis at 2.0-2.5 A resolution, molecular replacement | Schizosaccharomyces pombe |
purified enzyme in complex with S-adenosyl-L-homocysteine, X-ray diffraction structure determination and analysis at 1.8 A resolution, molecular replacement | Saccharomyces cerevisiae |
Protein Variants | Comment | Organism |
---|---|---|
D207N | site-directed mutagenesis, inactive mutant | Schizosaccharomyces pombe |
D210N | site-directed mutagenesis, inactive mutant | Saccharomyces cerevisiae |
K110E/R121E/R127E | site-directed mutagenesis, inactive mutant | Schizosaccharomyces pombe |
K153E/R147E | site-directed mutagenesis, inactive mutant | Schizosaccharomyces pombe |
K208A | site-directed mutagenesis, mutation of a nucleoside binding pocket residue, the mutant shows 28% reduced activity compared to the wild-type enzyme | Schizosaccharomyces pombe |
N209A | site-directed mutagenesis, the mutant shows increased Km for tRNA compared to the wild-type | Schizosaccharomyces pombe |
N212A | site-directed mutagenesis, the mutant shows increased Km for tRNA compared to the wild-type | Saccharomyces cerevisiae |
Q118A | site-directed mutagenesis, mutation of a nucleoside binding pocket residue, inactive mutant | Schizosaccharomyces pombe |
T244A | site-directed mutagenesis, mutation of a nucleoside binding pocket residue, the mutant shows 65% reduced activity compared to the wild-type enzyme | Schizosaccharomyces pombe |
V206A | site-directed mutagenesis, mutation of a nucleoside binding pocket residue, the mutant shows 81% reduced activity compared to the wild-type enzyme | Schizosaccharomyces pombe |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | kinetic mechanism of Trm10, overview | Saccharomyces cerevisiae | |
additional information | - |
additional information | kinetic mechanism of Trm10, overview | Schizosaccharomyces pombe | |
0.0032 | - |
guanine9 in tRNAGly | tRNA from Schizosaccharomyces pombe, recombinant enzyme, pH and temperature not specified in the publication | Schizosaccharomyces pombe | |
0.0053 | - |
guanine9 in tRNAGly | tRNA from Schizosaccharomyces pombe, recombinant enzyme, pH and temperature not specified in the publication | Saccharomyces cerevisiae |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
S-adenosyl-L-methionine + guanine9 in tRNAGly | Saccharomyces cerevisiae | Trm10 is specific for G9 of tRNAGly | S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNAGly | - |
? | |
S-adenosyl-L-methionine + guanine9 in tRNAGly | Schizosaccharomyces pombe | Trm10 is specific for G9 of tRNAGly | S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNAGly | - |
? | |
S-adenosyl-L-methionine + guanine9 in tRNAGly | Schizosaccharomyces pombe 972 | Trm10 is specific for G9 of tRNAGly | S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNAGly | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Saccharomyces cerevisiae | Q12400 | - |
- |
Schizosaccharomyces pombe | O14214 | - |
- |
Schizosaccharomyces pombe 972 | O14214 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant N-terminally His-tagged enzyme Trm10 from Escherichia coli strain BL21/Gold(DE3) by nickel affinity chromatography, gel filtration, and anion exchange chromatography, followed by dialysis | Saccharomyces cerevisiae |
recombinant N-terminally His-tagged enzyme Trm10 from Escherichia coli strain BL21/Gold(DE3) by nickel affinity chromatography, gel filtration, and anion exchange chromatography, followed by dialysis. Purification of the recombinant TrmT10C-SDR5C1 wild-type and mutant complexes by nickel affinity chromatography | Schizosaccharomyces pombe |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
S-adenosyl-L-methionine + guanine9 in tRNA = S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNA | catalytic mechanism and recognition of tRNA substrate, overview. The N-terminal extension of Trm10 is necessary for substrate tRNA recognition | Saccharomyces cerevisiae | |
S-adenosyl-L-methionine + guanine9 in tRNA = S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNA | catalytic mechanism and recognition of tRNA substrate, overview. The N-terminal extension of Trm10 is necessary for substrate tRNA recognition | Schizosaccharomyces pombe |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | no activity of the enzyme with three sptRNAGly mutants at Position 9 (G9A, G9C, and G9U) | Saccharomyces cerevisiae | ? | - |
? | |
additional information | no activity of the enzyme with three sptRNAGly mutants at Position 9 (G9A, G9C, and G9U) | Schizosaccharomyces pombe | ? | - |
? | |
additional information | no activity of the enzyme with three sptRNAGly mutants at Position 9 (G9A, G9C, and G9U) | Schizosaccharomyces pombe 972 | ? | - |
? | |
S-adenosyl-L-methionine + guanine9 in tRNAGly | Trm10 is specific for G9 of tRNAGly | Saccharomyces cerevisiae | S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNAGly | - |
? | |
S-adenosyl-L-methionine + guanine9 in tRNAGly | Trm10 is specific for G9 of tRNAGly | Schizosaccharomyces pombe | S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNAGly | - |
? | |
S-adenosyl-L-methionine + guanine9 in tRNAGly | Trm10 is specific for G9 of tRNAGly | Schizosaccharomyces pombe 972 | S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNAGly | - |
? |
Subunits | Comment | Organism |
---|---|---|
monomer | the enzyme adopts a globular alpha/beta structure consisting of six-stranded beta-sheets sandwiched by alpha-helices at both sides, small angle X-ray scattering analysis | Saccharomyces cerevisiae |
monomer | the enzyme adopts a globular alpha/beta structure consisting of six-stranded beta-sheets sandwiched by alpha-helices at both sides, small angle X-ray scattering analysis | Schizosaccharomyces pombe |
Synonyms | Comment | Organism |
---|---|---|
Trm10 | - |
Saccharomyces cerevisiae |
Trm10 | - |
Schizosaccharomyces pombe |
tRNA m1G9 methyltransferase | - |
Saccharomyces cerevisiae |
tRNA m1G9 methyltransferase | - |
Schizosaccharomyces pombe |
tRNA m1G9 MTase | - |
Saccharomyces cerevisiae |
tRNA m1G9 MTase | - |
Schizosaccharomyces pombe |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0027 | - |
guanine9 in tRNAGly | tRNA from Schizosaccharomyces pombe, recombinant enzyme, pH and temperature not specified in the publication | Schizosaccharomyces pombe | |
0.0028 | - |
guanine9 in tRNAGly | tRNA from Schizosaccharomyces pombe, recombinant enzyme, pH and temperature not specified in the publication | Saccharomyces cerevisiae |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
S-adenosyl-L-methionine | binding structure, overview | Saccharomyces cerevisiae | |
S-adenosyl-L-methionine | binding structure, overview | Schizosaccharomyces pombe | |
SDR5C1 protein | enzyme TrmT10C requires another protein, SDR5C1 (MRPP2), to execute its m1R9 (G9 and A9) MTase and additional RNase P activity | Schizosaccharomyces pombe |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme Trm10 belongs to the SPOUT superfamily. Trm10 behaves as a monomer in solution, whereas other members of the SPOUT superfamily all function as homodimers. The MTase domain (the catalytic domain) of the Trm10 family displays a typical SpoU-TrmD (SPOUT) fold. Trm10 from Schizosaccharomyces pombe demonstrates identical tRNA MTase activity as Trm10 from Saccharomyces cerevisiae | Saccharomyces cerevisiae |
evolution | the enzyme Trm10 belongs to the SPOUT superfamily. Trm10 behaves as a monomer in solution, whereas other members of the SPOUT superfamily all function as homodimers. The MTase domain (the catalytic domain) of the Trm10 family displays a typical SpoU-TrmD (SPOUT) fold. Trm10 from Schizosaccharomyces pombe demonstrates identical tRNA MTase activity as Trm10 from Saccharomyces cerevisiae | Schizosaccharomyces pombe |
additional information | enzyme overall structure analysis, active site structure, overview | Saccharomyces cerevisiae |
additional information | enzyme overall structure analysis, active site structure, overview | Schizosaccharomyces pombe |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.538 | - |
guanine9 in tRNAGly | tRNA from Schizosaccharomyces pombe, recombinant enzyme, pH and temperature not specified in the publication | Saccharomyces cerevisiae | |
1.66 | - |
guanine9 in tRNAGly | tRNA from Schizosaccharomyces pombe, recombinant enzyme, pH and temperature not specified in the publication | Schizosaccharomyces pombe |