Cloned (Comment) | Organism |
---|---|
type 2 enzyme, overexpression of wild-type in Escherichia coli BL21(DE3), and of selenomethionine mutant in Escherichia coli strain B834, a methionine-auxotroph BL21(DE3) derivative | Archaeoglobus fulgidus |
Crystallization (Comment) | Organism |
---|---|
crystallization of 40 mg/ml purified recombinant wild-type type 2 enzyme, and of recombinant selenomethionine enzyme mutant, with or without bound metal ions, hanging drop vapour diffusion method, mixing with equal volume of precipitant solution containing 10% PEG monomethylether 5000, 10% butanol, 20 mM sodium citrate, 100 mM Tris, pH 8.5, 3 days, formation of a cobalt-hexammine chloride complex, X-ray diffraction structure determination and analysis at 1.95-2.7 A resolution | Archaeoglobus fulgidus |
Protein Variants | Comment | Organism |
---|---|---|
K143A | site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme | Archaeoglobus fulgidus |
R146A | site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme | Archaeoglobus fulgidus |
R46A | site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme | Archaeoglobus fulgidus |
Y164A | site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme | Archaeoglobus fulgidus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.00006 | - |
RNA-DNA*DNA hybrid | recombinant wild-type enzyme, pH 8.0, 30°C | Archaeoglobus fulgidus | |
0.0016 | - |
RNA-DNA*DNA hybrid | recombinant mutant R146A, pH 8.0, 30°C | Archaeoglobus fulgidus | |
0.00188 | - |
RNA-DNA*DNA hybrid | recombinant mutant K143A, pH 8.0, 30°C | Archaeoglobus fulgidus | |
0.00268 | - |
RNA-DNA*DNA hybrid | recombinant mutant Y164A, pH 8.0, 30°C | Archaeoglobus fulgidus | |
0.0036 | - |
RNA-DNA*DNA hybrid | recombinant mutant R46A, pH 8.0, 30°C | Archaeoglobus fulgidus |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | dependent on | Archaeoglobus fulgidus | |
additional information | metal coordination in the active site | Archaeoglobus fulgidus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
RNA-DNA*DNA hybrid + H2O | Archaeoglobus fulgidus | RNA primer recognition and removal during DNA replication | DNA*DNA + 5'-phosphomononucleotides | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Archaeoglobus fulgidus | O29634 | type 2 enzyme | - |
Purification (Comment) | Organism |
---|---|
recombinant enzymes from Escherichia coli | Archaeoglobus fulgidus |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
Endonucleolytic cleavage to a 5'-phosphomonoester | active site structure, substrate binding groove, molecular mechanism for specific RNA*DNA hybrid substrate recognition, binding, and cleavage, a general nuclease activity is necessary for catalysis | Archaeoglobus fulgidus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | cleavage specificity | Archaeoglobus fulgidus | ? | - |
? | |
RNA-DNA single-stranded chimera + H2O | - |
Archaeoglobus fulgidus | ? | - |
? | |
RNA-DNA*DNA hybrid + H2O | RNA primer recognition and removal during DNA replication | Archaeoglobus fulgidus | DNA*DNA + 5'-phosphomononucleotides | - |
? | |
RNA-DNA*DNA hybrid + H2O | 7 M urea-denatured 18mer, no activity with untreated hybrid, specific removal of the RNA portion of the duplex | Archaeoglobus fulgidus | 8mer oligonucleotide | - |
? | |
RNA-DNA*DNA hybrid + H2O | 7 M urea-denatured 37mer, no activity with untreated hybrid, specific removal of the RNA portion of the duplex | Archaeoglobus fulgidus | oligonucleotides | a 20mer, a 10mer, a 9mer, and a 7mer | ? |
Subunits | Comment | Organism |
---|---|---|
More | two-domain architecture | Archaeoglobus fulgidus |
Synonyms | Comment | Organism |
---|---|---|
More | the enzyme belongs to the RNase H nuclease family | Archaeoglobus fulgidus |
ribonuclease H | - |
Archaeoglobus fulgidus |
RNase H | - |
Archaeoglobus fulgidus |
RNase HII | - |
Archaeoglobus fulgidus |
type 2 RNase H | - |
Archaeoglobus fulgidus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Archaeoglobus fulgidus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.133 | - |
RNA-DNA*DNA hybrid | recombinant wild-type enzyme, pH 8.0, 30°C | Archaeoglobus fulgidus | |
0.192 | - |
RNA-DNA*DNA hybrid | recombinant mutant R146A, pH 8.0, 30°C | Archaeoglobus fulgidus | |
0.2 | - |
RNA-DNA*DNA hybrid | recombinant mutant R46A and K143A, pH 8.0, 30°C | Archaeoglobus fulgidus | |
0.208 | - |
RNA-DNA*DNA hybrid | recombinant mutant Y164A, pH 8.0, 30°C | Archaeoglobus fulgidus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Archaeoglobus fulgidus |