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Literature summary for 3.1.26.4 extracted from

  • Miyashita, S.; Tadokoro, T.; Angkawidjaja, C.; You, D.J.; Koga, Y.; Takano, K.; Kanaya, S.
    Identification of the substrate binding site in the N-terminal TBP-like domain of RNase H3 (2011), FEBS Lett., 585, 2313-2317.
    View publication on PubMed
Search for more literature for 3.1.26.4

Cloned(Commentary)

Cloned (Comment) Organism
overexpression of wild-type and mutant enzymes in Escherichia coli strain MIC2067(DE3) Geobacillus stearothermophilus

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant mutant Q54A-RNase H3 enzyme, X-ray diffraction structure determination and analysis at 2.6 A resolution Geobacillus stearothermophilus

Protein Variants

Protein Variants Comment Organism
K50A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Geobacillus stearothermophilus
L52A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Geobacillus stearothermophilus
additional information construction of mutant proteins of the intact protein and isolated N-domain, in which six of the seventeen residues corresponding to those involved in DNA binding of TBP are individually mutated to Ala. All of the mutants exhibit decreased enzymatic activities and/or substrate-binding affinities when compared to those of the parent proteins Geobacillus stearothermophilus
Q54A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Geobacillus stearothermophilus
S48A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Geobacillus stearothermophilus
V42A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Geobacillus stearothermophilus
Y46A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Geobacillus stearothermophilus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information kinetics of recombinant wild-type and mutant enzymes, overview Geobacillus stearothermophilus
0.00082
-
 M13 DNA/RNA hybrid pH 8.5, 30°C, Mg2+, recombinant wild-type enzyme Geobacillus stearothermophilus
0.0013
-
 M13 DNA/RNA hybrid pH 8.5, 30°C, Mg2+, recombinant mutant K50A Geobacillus stearothermophilus
0.0014
-
 M13 DNA/RNA hybrid pH 8.5, 30°C, Mg2+, recombinant mutant V42A Geobacillus stearothermophilus

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ activates, required Geobacillus stearothermophilus
Mn2+ activates, required Geobacillus stearothermophilus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
additional information Geobacillus stearothermophilus RNase H specifically hydrolyzes the RNA strand of RNA/DNA hybrids in the presence of divalent metal ions, such as Mg2+ and Mn2+ ?
-
 ?

Organism

Organism UniProt Comment Textmining
Geobacillus stearothermophilus Q6L6Q4
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain MIC2067(DE3) Geobacillus stearothermophilus

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.04
-
 purified mutant Y46A, in presence of Mn2+, pH 8.5, 30°C Geobacillus stearothermophilus
0.046
-
 purified mutant Y46A, in presence of Mg2+, pH 8.5, 30°C Geobacillus stearothermophilus
0.06
-
 purified mutant Q54A, in presence of Mn2+, pH 8.5, 30°C Geobacillus stearothermophilus
0.068
-
 purified mutant Q54A, in presence of Mg2+, pH 8.5, 30°C Geobacillus stearothermophilus
0.55
-
 purified mutant S48A, in presence of Mn2+, pH 8.5, 30°C Geobacillus stearothermophilus
0.59
-
 purified mutant L52A, in presence of Mg2+, pH 8.5, 30°C Geobacillus stearothermophilus
0.61
-
 purified mutant S48A, in presence of Mg2+, pH 8.5, 30°C Geobacillus stearothermophilus
1.1
-
 purified wild-type enzyme, in presence of Mn2+, pH 8.5, 30°C Geobacillus stearothermophilus
1.2
-
 purified mutant K50A, in presence of Mn2+, pH 8.5, 30°C Geobacillus stearothermophilus
1.5
-
 purified mutant V42A, in presence of Mg2+, pH 8.5, 30°C Geobacillus stearothermophilus
1.6
-
 purified mutant K50A, in presence of Mg2+, pH 8.5, 30°C Geobacillus stearothermophilus
1.9
-
 purified wild-type enzyme, in presence of Mg2+, pH 8.5, 30°C Geobacillus stearothermophilus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
M13 DNA/RNA hybrid + H2O substrate is 3H-labeled M13 DNA/RNA hybrid Geobacillus stearothermophilus ?
-
 ?
additional information RNase H specifically hydrolyzes the RNA strand of RNA/DNA hybrids in the presence of divalent metal ions, such as Mg2+ and Mn2+ Geobacillus stearothermophilus ?
-
 ?
additional information model for the complex between Bst-RNase H3 and RNA/DNA hybrid and substrate binding mechanism, overview Geobacillus stearothermophilus ?
-
 ?

Synonyms

Synonyms Comment Organism
ribonuclease H3
-
 Geobacillus stearothermophilus
RNase H3
-
 Geobacillus stearothermophilus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
 assay at Geobacillus stearothermophilus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8.5
-
 assay at Geobacillus stearothermophilus

General Information

General Information Comment Organism
additional information the substrate binding site is located in the N-terminal TBP-like domain of RNase H3. The N-terminal domain of RNase H3 uses the flat surface of the b-sheet for substrate binding as TBP to bind DNA. This domain may greatly change conformation upon substrate binding Geobacillus stearothermophilus