Cloned (Comment) | Organism |
---|---|
expression of mutant DNA polymerase I in Escherichia coli strain BL21 AI cytoplasm | Thermus aquaticus |
Protein Variants | Comment | Organism |
---|---|---|
N483Q/S486Q/T539N/Y545Q/D547T/P548Q/A570Q/D578Q/A597T/W604R/S612N/V730L/R736Q/S739N/M747R | selection of a polymerase with 15 mutations, mostly located at the template binding interface, by directed evolution of Thermus aquaticus DNA polymerase I, the mutant enzyme is a single variant of the Stoffel fragment of Taq DNA polymerase I, the enzyme shows broad template specificity and is a thermostable DNA-dependent and RNA-dependent DNA-polymerase, see also EC 2.7.7.7 | Thermus aquaticus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | kinetic constants for DNA-dependent and RNA-dependent DNA polymerization Michaelis-Menten mechanism and kinetic model of the mutant enzyme, overview | Thermus aquaticus | |
0.099 | - |
dATP | pH 9.0, 37°C, recombinant mutant enzyme N483Q/S486Q/T539N/Y545Q/D547T/P548Q/A570Q/D578Q/A597T/W604R/S612N/V730L/R736Q/S739N/M747R | Thermus aquaticus | |
0.167 | - |
dCTP | pH 9.0, 37°C, recombinant mutant enzyme N483Q/S486Q/T539N/Y545Q/D547T/P548Q/A570Q/D578Q/A597T/W604R/S612N/V730L/R736Q/S739N/M747R | Thermus aquaticus | |
0.379 | - |
dTTP | pH 9.0, 37°C, recombinant mutant enzyme N483Q/S486Q/T539N/Y545Q/D547T/P548Q/A570Q/D578Q/A597T/W604R/S612N/V730L/R736Q/S739N/M747R | Thermus aquaticus |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Thermus aquaticus |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
62865 | - |
x * 62879, mutant DNA polymerase I, sequence calculation, x * 62865, mutant DNA polymerase I, mass spectrometry | Thermus aquaticus |
62879 | - |
x * 62879, mutant DNA polymerase I, sequence calculation, x * 62865, mutant DNA polymerase I, mass spectrometry | Thermus aquaticus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
2'-deoxyribonucleoside triphosphate + DNAn | Thermus aquaticus | enzyme mutant N483Q/S486Q/T539N/Y545Q/D547T/P548Q/A570Q/D578Q/A597T/W604R/S612N/V730L/R736Q/S739N/M747R | diphosphate + DNAn+1 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Thermus aquaticus | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant mutant DNA polymerase I from Escherichia coli strain BL21 AI cytoplasm by metal ion and heparin affinity chromatography | Thermus aquaticus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2'-deoxyribonucleoside triphosphate + DNAn | enzyme mutant N483Q/S486Q/T539N/Y545Q/D547T/P548Q/A570Q/D578Q/A597T/W604R/S612N/V730L/R736Q/S739N/M747R | Thermus aquaticus | diphosphate + DNAn+1 | - |
? | |
dATP + DNAn | enzyme mutant N483Q/S486Q/T539N/Y545Q/D547T/P548Q/A570Q/D578Q/A597T/W604R/S612N/V730L/R736Q/S739N/M747R | Thermus aquaticus | diphosphate + DNAn+1 | - |
? | |
dCTP + DNAn | enzyme mutant N483Q/S486Q/T539N/Y545Q/D547T/P548Q/A570Q/D578Q/A597T/W604R/S612N/V730L/R736Q/S739N/M747R | Thermus aquaticus | diphosphate + DNAn+1 | - |
? | |
dTTP + DNAn | enzyme mutant N483Q/S486Q/T539N/Y545Q/D547T/P548Q/A570Q/D578Q/A597T/W604R/S612N/V730L/R736Q/S739N/M747R | Thermus aquaticus | diphosphate + DNAn+1 | - |
? | |
additional information | the mutant enzyme shows single nucleotide additions with dCTP, dATP and dTTP, but not with dGTP as it results in addition of two successive base incorporations on the chosen template 2 hybridised to the DNA primer 1, thereby invalidating the single-turnover kinetic model, Michaelis-Menten mechanism, overview | Thermus aquaticus | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
? | x * 62879, mutant DNA polymerase I, sequence calculation, x * 62865, mutant DNA polymerase I, mass spectrometry | Thermus aquaticus |
Synonyms | Comment | Organism |
---|---|---|
DNA polymerase I | - |
Thermus aquaticus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Thermus aquaticus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.25 | - |
dATP | pH 9.0, 37°C, recombinant mutant enzyme, N483Q/S486Q/T539N/Y545Q/D547T/P548Q/A570Q/D578Q/A597T/W604R/S612N/V730L/R736Q/S739N/M747R | Thermus aquaticus | |
0.67 | - |
dTTP | pH 9.0, 37°C, recombinant mutant enzyme, N483Q/S486Q/T539N/Y545Q/D547T/P548Q/A570Q/D578Q/A597T/W604R/S612N/V730L/R736Q/S739N/M747R | Thermus aquaticus | |
0.83 | - |
dCTP | pH 9.0, 37°C, recombinant mutant enzyme, N483Q/S486Q/T539N/Y545Q/D547T/P548Q/A570Q/D578Q/A597T/W604R/S612N/V730L/R736Q/S739N/M747R | Thermus aquaticus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
9 | - |
assay at | Thermus aquaticus |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1.8 | - |
dTTP | pH 9.0, 37°C, recombinant mutant enzyme N483Q/S486Q/T539N/Y545Q/D547T/P548Q/A570Q/D578Q/A597T/W604R/S612N/V730L/R736Q/S739N/M747R | Thermus aquaticus | |
2.5 | - |
dATP | pH 9.0, 37°C, recombinant mutant enzyme N483Q/S486Q/T539N/Y545Q/D547T/P548Q/A570Q/D578Q/A597T/W604R/S612N/V730L/R736Q/S739N/M747R | Thermus aquaticus | |
4.9 | - |
dCTP | pH 9.0, 37°C, recombinant mutant enzyme N483Q/S486Q/T539N/Y545Q/D547T/P548Q/A570Q/D578Q/A597T/W604R/S612N/V730L/R736Q/S739N/M747R | Thermus aquaticus |