Cloned (Comment) | Organism |
---|---|
recombinant expression of His-tagged full-length wild-type enzyme, of truncated enzyme SaTrm10 1-249, and of enzyme mutants in Escherichia coli strain Rosetta (DE3)pLysS, recombinant expression of nontagged wild-type enzyme | Sulfolobus acidocaldarius |
Crystallization (Comment) | Organism |
---|---|
purified recombinant truncated construct of SaTrm10, lacking the C-terminal domain, native and enriched in selenomethionine (SaTrm10 1-249 and SaTrm10 1-249 SAD, respectively), hanging drop vapour diffusion, for SaTrm10 1-249: mixing of 15 mg/ml protein solution with an equal volume of crystallisation buffer containing 8% ethylene glycol, 0.1 M HEPES, pH 7.5, and 10% PEG 8000, for SaTrm10 1-249 SAD: mixing of 15 mg/ml protein solution with an equal volume of crystallisation buffer containing 2% dioxane, 0.05 M bicine, pH 9.0, and 15% PEG 20000, for full-length enzyme: mixing of 8 mg/ml protein in the presence of 1 mM SAH with an equal volume of crystallisation buffer containing 0.4 M (NH4)2SO4, 0.1 M sodium acetate, pH 4.5, and 15-20% PEG2000MME, several months, 20°C, X-ray diffraction structure determination and analysis at 2.1-2.4 A resolution, molecular replacement and modelling | Sulfolobus acidocaldarius |
Protein Variants | Comment | Organism |
---|---|---|
D184N | site-directed mutagenesis, kinetics and structure comparison | Sulfolobus acidocaldarius |
D220N | site-directed mutagenesis, kinetics and structure comparison | Sulfolobus acidocaldarius |
K121E | site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type | Sulfolobus acidocaldarius |
K185E | site-directed mutagenesis, the mutant shows very highly reduced activity compared to wild-type | Sulfolobus acidocaldarius |
K249E | site-directed mutagenesis, the mutant is almost inactive | Sulfolobus acidocaldarius |
K38E | site-directed mutagenesis, the mutant shows very highly reduced activity compared to wild-type | Sulfolobus acidocaldarius |
K5E | site-directed mutagenesis, the mutant shows moderately reduced activity compared to wild-type | Sulfolobus acidocaldarius |
K64E | site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type | Sulfolobus acidocaldarius |
K75E | site-directed mutagenesis, the mutant shows very highly reduced activity compared to wild-type | Sulfolobus acidocaldarius |
K78E | site-directed mutagenesis, the mutant shows moderately reduced activity compared to wild-type | Sulfolobus acidocaldarius |
R276E | site-directed mutagenesis, the mutant shows moderately reduced activity compared to wild-type | Sulfolobus acidocaldarius |
R288E | site-directed mutagenesis, the mutant is almost inactive | Sulfolobus acidocaldarius |
R47E | site-directed mutagenesis, the mutant shows very highly reduced activity compared to wild-type | Sulfolobus acidocaldarius |
R74E | site-directed mutagenesis, the mutant shows very highly reduced activity compared to wild-type | Sulfolobus acidocaldarius |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required for activity, best at 5 mM | Sulfolobus acidocaldarius |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
35000 | - |
native enzyme, gel filtration | Sulfolobus acidocaldarius |
38000 | - |
recombinant His-tagged enzyme, gel filtration | Sulfolobus acidocaldarius |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
S-adenosyl-L-methionine + adenine9 in tRNA | Sulfolobus acidocaldarius | - |
S-adenosyl-L-homocysteine + N1-methyladenine9 in tRNA | - |
? | |
S-adenosyl-L-methionine + adenine9 in tRNA | Sulfolobus acidocaldarius ATCC 33909 | - |
S-adenosyl-L-homocysteine + N1-methyladenine9 in tRNA | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Sulfolobus acidocaldarius | Q4J894 | - |
- |
Sulfolobus acidocaldarius ATCC 33909 | Q4J894 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged full-length wild-type enzyme, truncated enzyme SaTrm10 1-249, and enzyme mutants from Escherichia coli strain Rosetta (DE3)pLysS by nickel affinity chromatography and gel filtration, recombinant nontagged wild-type enzyme by anion exchange and cation exchange chromatography, followed by gel filtration | Sulfolobus acidocaldarius |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | model for substrate tRNA binding by SaTrm10, in silico tRNA-SaTrm10 docking, role of Asp184 and Asp220 in the catalytic mechanism of SaTrm10, tRNA binding mechanism, overview | Sulfolobus acidocaldarius | ? | - |
- |
|
additional information | model for substrate tRNA binding by SaTrm10, in silico tRNA-SaTrm10 docking, role of Asp184 and Asp220 in the catalytic mechanism of SaTrm10, tRNA binding mechanism, overview | Sulfolobus acidocaldarius ATCC 33909 | ? | - |
- |
|
S-adenosyl-L-methionine + adenine9 in tRNA | - |
Sulfolobus acidocaldarius | S-adenosyl-L-homocysteine + N1-methyladenine9 in tRNA | - |
? | |
S-adenosyl-L-methionine + adenine9 in tRNA | - |
Sulfolobus acidocaldarius ATCC 33909 | S-adenosyl-L-homocysteine + N1-methyladenine9 in tRNA | - |
? | |
S-adenosyl-L-methionine + adenine9 in tRNAiMet | docking model of tRNAiMet of Sulfolobus acidocaldarius onto SaTrm10-SAH | Sulfolobus acidocaldarius | S-adenosyl-L-homocysteine + N1-methyladenine9 in tRNAiMet | - |
? | |
S-adenosyl-L-methionine + adenine9 in tRNAiMet | docking model of tRNAiMet of Sulfolobus acidocaldarius onto SaTrm10-SAH | Sulfolobus acidocaldarius ATCC 33909 | S-adenosyl-L-homocysteine + N1-methyladenine9 in tRNAiMet | - |
? |
Subunits | Comment | Organism |
---|---|---|
monomer | 1 * 33200, native enzyme, SDS-PAGE, 1 * 35300, recombinant His-tagged enzyme, SDS-PAGE | Sulfolobus acidocaldarius |
More | monomeric, full length SaTrm10 SAH displays an overall (tRNA resembling) L-shape and can be divided in three regions: a central domain (residues 89-246) which adopts a typical SPOUT fold, an N-terminal domain (residues 1-79) and a C-terminal helical domain (residues 247-292). The N-terminal domain is formed by a curved beta-sheet and two alpha-helices which together resemble a horse shoe | Sulfolobus acidocaldarius |
Synonyms | Comment | Organism |
---|---|---|
adenosine-specific Trm10 | - |
Sulfolobus acidocaldarius |
SaTrm10 | - |
Sulfolobus acidocaldarius |
Trm10 | - |
Sulfolobus acidocaldarius |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Sulfolobus acidocaldarius |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Sulfolobus acidocaldarius |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
S-adenosyl-L-methionine | - |
Sulfolobus acidocaldarius |
General Information | Comment | Organism |
---|---|---|
additional information | tRNA binding and adenosine N1-methylation by an archaeal Trm10 homologue, structure-function analysis, overview. N1-adenosine methylation by SaTrm10 requires two catalytic aspartate residues | Sulfolobus acidocaldarius |