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Literature summary for 2.1.1.218 extracted from

  • Van Laer, B.; Roovers, M.; Wauters, L.; Kasprzak, J.; Dyzma, M.; Deyaert, E.; Singh, R.; Feller, A.; Bujnicki, J.; Droogmans, L.; Versees, W.
    Structural and functional insights into tRNA binding and adenosine N1-methylation by an archaeal Trm10 homologue (2016), Nucleic Acids Res., 44, 940-953 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

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 (Commentary)

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

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

Metals/Ions Comment Organism Structure
Mg2+ required for activity, best at 5 mM Sulfolobus acidocaldarius

Molecular Weight [Da]

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/ Products (Substrates)

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

Organism UniProt Comment Textmining
Sulfolobus acidocaldarius Q4J894
-
-
Sulfolobus acidocaldarius ATCC 33909 Q4J894
-
-

Purification (Commentary)

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 and Products (Substrate)

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

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

Synonyms Comment Organism
adenosine-specific Trm10
-
Sulfolobus acidocaldarius
SaTrm10
-
Sulfolobus acidocaldarius
Trm10
-
Sulfolobus acidocaldarius

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Sulfolobus acidocaldarius

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Sulfolobus acidocaldarius

Cofactor

Cofactor Comment Organism Structure
S-adenosyl-L-methionine
-
Sulfolobus acidocaldarius

General Information

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