Cloned (Comment) | Organism |
---|---|
overexpression of wild-type and mutant enzymes in Escherichia coli strain MIC2067(DE3) | Geobacillus stearothermophilus |
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 | 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 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 | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | activates, required | Geobacillus stearothermophilus | |
Mn2+ | activates, required | Geobacillus stearothermophilus |
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 | UniProt | Comment | Textmining |
---|---|---|---|
Geobacillus stearothermophilus | Q6L6Q4 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzymes from Escherichia coli strain MIC2067(DE3) | Geobacillus stearothermophilus |
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 | 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 | Comment | Organism |
---|---|---|
ribonuclease H3 | - |
Geobacillus stearothermophilus |
RNase H3 | - |
Geobacillus stearothermophilus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Geobacillus stearothermophilus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8.5 | - |
assay at | Geobacillus stearothermophilus |
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 |