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Literature summary extracted from

  • Johannsson, S.; Neumann, P.; Wulf, A.; Welp, L.M.; Gerber, H.D.; Krull, M.; Diederichsen, U.; Urlaub, H.; Ficner, R.
    Structural insights into the stimulation of S. pombe Dnmt2 catalytic efficiency by the tRNA nucleoside queuosine (2018), Sci. Rep., 8, 8880 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.1.1.204 recombinant expression of GST-tagged enzyme in Escherichia coli strain BL21(DE3) Schizosaccharomyces pombe

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.1.1.204 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.1.1.204 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
2.1.1.204 0.00688
-
 cytosine38 in Q34tRNAAsp pH 7.5, temperature not specified in the publication Schizosaccharomyces pombe
2.1.1.204 0.01559
-
 cytosine38 in G34tRNAAsp pH 7.5, temperature not specified in the publication Schizosaccharomyces pombe

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.1.1.204 S-adenosyl-L-methionine + cytosine38 in G34tRNAAsp Schizosaccharomyces pombe
-
 S-adenosyl-L-homocysteine + 5-methylcytosine38 in G34tRNAAsp
-
 ?
2.1.1.204 S-adenosyl-L-methionine + cytosine38 in G34tRNAAsp Schizosaccharomyces pombe 972
-
 S-adenosyl-L-homocysteine + 5-methylcytosine38 in G34tRNAAsp
-
 ?
2.1.1.204 S-adenosyl-L-methionine + cytosine38 in G34tRNAAsp Schizosaccharomyces pombe ATCC 24843
-
 S-adenosyl-L-homocysteine + 5-methylcytosine38 in G34tRNAAsp
-
 ?
2.1.1.204 S-adenosyl-L-methionine + cytosine38 in Q34tRNAAsp Schizosaccharomyces pombe preferred substrate S-adenosyl-L-homocysteine + 5-methylcytosine38 in Q34tRNAAsp
-
 ?
2.1.1.204 S-adenosyl-L-methionine + cytosine38 in Q34tRNAAsp Schizosaccharomyces pombe 972 preferred substrate S-adenosyl-L-homocysteine + 5-methylcytosine38 in Q34tRNAAsp
-
 ?
2.1.1.204 S-adenosyl-L-methionine + cytosine38 in Q34tRNAAsp Schizosaccharomyces pombe ATCC 24843 preferred substrate S-adenosyl-L-homocysteine + 5-methylcytosine38 in Q34tRNAAsp
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.1.1.204 Schizosaccharomyces pombe P40999
-
-
2.1.1.204 Schizosaccharomyces pombe 972 P40999
-
-
2.1.1.204 Schizosaccharomyces pombe ATCC 24843 P40999
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.1.1.204 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 and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.1.1.204 additional information Q34tRNAAsp exhibits an only slightly increased affinity for Dnmt2 in comparison to unmodified G34tRNA Schizosaccharomyces pombe ?
-
 ?
2.1.1.204 additional information Q34tRNAAsp exhibits an only slightly increased affinity for Dnmt2 in comparison to unmodified G34tRNA Schizosaccharomyces pombe 972 ?
-
 ?
2.1.1.204 additional information Q34tRNAAsp exhibits an only slightly increased affinity for Dnmt2 in comparison to unmodified G34tRNA Schizosaccharomyces pombe ATCC 24843 ?
-
 ?
2.1.1.204 S-adenosyl-L-methionine + cytosine38 in G34tRNAAsp
-
 Schizosaccharomyces pombe S-adenosyl-L-homocysteine + 5-methylcytosine38 in G34tRNAAsp
-
 ?
2.1.1.204 S-adenosyl-L-methionine + cytosine38 in G34tRNAAsp
-
 Schizosaccharomyces pombe 972 S-adenosyl-L-homocysteine + 5-methylcytosine38 in G34tRNAAsp
-
 ?
2.1.1.204 S-adenosyl-L-methionine + cytosine38 in G34tRNAAsp
-
 Schizosaccharomyces pombe ATCC 24843 S-adenosyl-L-homocysteine + 5-methylcytosine38 in G34tRNAAsp
-
 ?
2.1.1.204 S-adenosyl-L-methionine + cytosine38 in Q34tRNAAsp preferred substrate Schizosaccharomyces pombe S-adenosyl-L-homocysteine + 5-methylcytosine38 in Q34tRNAAsp
-
 ?
2.1.1.204 S-adenosyl-L-methionine + cytosine38 in Q34tRNAAsp preferred substrate Schizosaccharomyces pombe 972 S-adenosyl-L-homocysteine + 5-methylcytosine38 in Q34tRNAAsp
-
 ?
2.1.1.204 S-adenosyl-L-methionine + cytosine38 in Q34tRNAAsp preferred substrate Schizosaccharomyces pombe ATCC 24843 S-adenosyl-L-homocysteine + 5-methylcytosine38 in Q34tRNAAsp
-
 ?

Synonyms

EC Number Synonyms Comment Organism
2.1.1.204 Dnmt2
-
 Schizosaccharomyces pombe
2.1.1.204 spDnmt2
-
 Schizosaccharomyces pombe

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.1.1.204 7.5
-
 assay at Schizosaccharomyces pombe

Cofactor

EC Number Cofactor Comment Organism Structure
2.1.1.204 S-adenosyl-L-methionine
-
 Schizosaccharomyces pombe

General Information

EC Number General Information Comment Organism
2.1.1.204 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
2.1.1.204 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