Cloned (Comment) | Organism |
---|---|
recombinant expression of GST-tagged enzyme in Escherichia coli strain BL21(DE3) | Schizosaccharomyces pombe |
Crystallization (Comment) | Organism |
---|---|
purified recombinant enzyme Dnmt2, sitting-drop vapor diffusion technique, mixing of equal volumes of 2 mg/ml protein solution and crystallization buffer containing 50 mM MES, pH 6.5, 1% w/v PEG 4000, and 4 mM MgCl2, 4°C, 5-7 days, X-ray diffraction structure determination and analysis at 1.7 A resolution, molecular replacement using the structure of Entamoebae histolytica Dnmt2 homologue EhMeth (PBD ID 3QV2), modelling | Schizosaccharomyces pombe |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Q34tRNAAsp exhibits an only slightly increased affinity for Dnmt2 in comparison to unmodified G34tRNA. Kinetic analysis, overview. Quantitative analysis of Dnmt2 tRNA complex formation | Schizosaccharomyces pombe | |
0.00688 | - |
cytosine38 in Q34tRNAAsp | pH 7.5, temperature not specified in the publication | Schizosaccharomyces pombe | |
0.01559 | - |
cytosine38 in G34tRNAAsp | pH 7.5, temperature not specified in the publication | Schizosaccharomyces pombe |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
S-adenosyl-L-methionine + cytosine38 in G34tRNAAsp | Schizosaccharomyces pombe | - |
S-adenosyl-L-homocysteine + 5-methylcytosine38 in G34tRNAAsp | - |
? | |
S-adenosyl-L-methionine + cytosine38 in G34tRNAAsp | Schizosaccharomyces pombe ATCC 24843 | - |
S-adenosyl-L-homocysteine + 5-methylcytosine38 in G34tRNAAsp | - |
? | |
S-adenosyl-L-methionine + cytosine38 in G34tRNAAsp | Schizosaccharomyces pombe 972 | - |
S-adenosyl-L-homocysteine + 5-methylcytosine38 in G34tRNAAsp | - |
? | |
S-adenosyl-L-methionine + cytosine38 in Q34tRNAAsp | Schizosaccharomyces pombe | preferred substrate | S-adenosyl-L-homocysteine + 5-methylcytosine38 in Q34tRNAAsp | - |
? | |
S-adenosyl-L-methionine + cytosine38 in Q34tRNAAsp | Schizosaccharomyces pombe ATCC 24843 | preferred substrate | S-adenosyl-L-homocysteine + 5-methylcytosine38 in Q34tRNAAsp | - |
? | |
S-adenosyl-L-methionine + cytosine38 in Q34tRNAAsp | Schizosaccharomyces pombe 972 | preferred substrate | S-adenosyl-L-homocysteine + 5-methylcytosine38 in Q34tRNAAsp | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Schizosaccharomyces pombe | P40999 | - |
- |
Schizosaccharomyces pombe 972 | P40999 | - |
- |
Schizosaccharomyces pombe ATCC 24843 | P40999 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant GST-tagged enzyme from Escherichia coli strain BL21(DE3) by ultracentrifugation, glutathione affinity chromatography, and tag cleavage through PreScission protease, followed by heparin affinity chromatography, gel filtration, and ultrafiltration | Schizosaccharomyces pombe |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | Q34tRNAAsp exhibits an only slightly increased affinity for Dnmt2 in comparison to unmodified G34tRNA | Schizosaccharomyces pombe | ? | - |
- |
|
additional information | Q34tRNAAsp exhibits an only slightly increased affinity for Dnmt2 in comparison to unmodified G34tRNA | Schizosaccharomyces pombe ATCC 24843 | ? | - |
- |
|
additional information | Q34tRNAAsp exhibits an only slightly increased affinity for Dnmt2 in comparison to unmodified G34tRNA | Schizosaccharomyces pombe 972 | ? | - |
- |
|
S-adenosyl-L-methionine + cytosine38 in G34tRNAAsp | - |
Schizosaccharomyces pombe | S-adenosyl-L-homocysteine + 5-methylcytosine38 in G34tRNAAsp | - |
? | |
S-adenosyl-L-methionine + cytosine38 in G34tRNAAsp | - |
Schizosaccharomyces pombe ATCC 24843 | S-adenosyl-L-homocysteine + 5-methylcytosine38 in G34tRNAAsp | - |
? | |
S-adenosyl-L-methionine + cytosine38 in G34tRNAAsp | - |
Schizosaccharomyces pombe 972 | S-adenosyl-L-homocysteine + 5-methylcytosine38 in G34tRNAAsp | - |
? | |
S-adenosyl-L-methionine + cytosine38 in Q34tRNAAsp | preferred substrate | Schizosaccharomyces pombe | S-adenosyl-L-homocysteine + 5-methylcytosine38 in Q34tRNAAsp | - |
? | |
S-adenosyl-L-methionine + cytosine38 in Q34tRNAAsp | preferred substrate | Schizosaccharomyces pombe ATCC 24843 | S-adenosyl-L-homocysteine + 5-methylcytosine38 in Q34tRNAAsp | - |
? | |
S-adenosyl-L-methionine + cytosine38 in Q34tRNAAsp | preferred substrate | Schizosaccharomyces pombe 972 | S-adenosyl-L-homocysteine + 5-methylcytosine38 in Q34tRNAAsp | - |
? |
Synonyms | Comment | Organism |
---|---|---|
Dnmt2 | - |
Schizosaccharomyces pombe |
spDnmt2 | - |
Schizosaccharomyces pombe |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Schizosaccharomyces pombe |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
S-adenosyl-L-methionine | - |
Schizosaccharomyces pombe |
General Information | Comment | Organism |
---|---|---|
additional information | enzyme Dnmt2 from Schizosaccharomyces pombe contains the entire active site loop. The interaction with tRNA is analyzed by means of mass spectrometry using UV cross-linked Dnmt2-tRNA complex. Cross-link data and computational docking of Dnmt2 and tRNAAsp reveal Q34 positioned adjacent to the S-adenosylmethionine occupies the active site, suggesting that the observed increase of Dnmt2 catalytic efficiency by queuine originates from optimal positioning of the substrate molecules and residues relevant for methyl transfer. Observation of displacement of the cytosine from the codon-anticodon helix and distortion of the latter, hinting to a role of the Q-modification in translational accuracy. Analysis of spDnmt2 electrostatic surface potentials unveils predominantly positively charged surface around the SAH indicating possible interaction with the tRNA phosphate backbone. But structure exposes a strongly negatively charged cavity in close proximity to the sulfur of S-adenosyl-L-homocysteine formed by the conserved catalytic residue Glu121 | Schizosaccharomyces pombe |
physiological function | enzyme Dnmt2 methylates cytosine at position 38 of tRNAAsp. A correlation between the presence of the hypermodified nucleoside queuosine (Q) at position 34 of tRNAAsp and the Dnmt2 dependent C38 methylation has recently been found in vivo for Schizosaccharomcyces pombe. Dnmt2 shows an increase for in vitro transcribed tRNAAsp containing Q34 compared to the unmodified substrate, structural basis for the Q-dependency, overview. The C38 methylation of tRNAAsp in Schizosaccharomcyces pombe depends on the presence of queuosine (Q), a hypermodified nucleoside at position 34 of tRNAAsp, tRNAAsn, tRNATyr, and tRNAHis10 | Schizosaccharomyces pombe |