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Literature summary extracted from

  • Vichier-Guerre, S.; Jestin, J.L.
    Characterisation of a DNA polymerase highly mutated along the template binding interface (2010), Mol. Biotechnol., 46, 58-62.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.7.7.7 expression of mutant DNA polymerase I in Escherichia coli strain BL21 AI cytoplasm Thermus aquaticus
2.7.7.49 expression of mutant DNA polymerase I in Escherichia coli strain BL21 AI cytoplasm Thermus aquaticus

Protein Variants

EC Number Protein Variants Comment Organism
2.7.7.7 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 2.7.7.49 Thermus aquaticus
2.7.7.49 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.7.7.7 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
2.7.7.7 0.022
-
 dATP pH 9.0, 37°C, recombinant mutant enzyme Thermus aquaticus
2.7.7.7 0.024
-
 dCTP pH 9.0, 37°C, recombinant mutant enzyme Thermus aquaticus
2.7.7.7 0.14
-
 dTTP pH 9.0, 37°C, recombinant mutant enzyme Thermus aquaticus
2.7.7.49 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
2.7.7.49 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
2.7.7.49 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
2.7.7.49 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

EC Number Metals/Ions Comment Organism Structure
2.7.7.7 Mg2+ required Thermus aquaticus
2.7.7.49 Mg2+ required Thermus aquaticus

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.7.7.7 62865
-
 x * 62879, mutant DNA polymerase I, sequence calculation, x * 62865, mutant DNA polymerase I, mass spectrometry Thermus aquaticus
2.7.7.7 62879
-
 x * 62879, mutant DNA polymerase I, sequence calculation, x * 62865, mutant DNA polymerase I, mass spectrometry Thermus aquaticus
2.7.7.49 62865
-
 x * 62879, mutant DNA polymerase I, sequence calculation, x * 62865, mutant DNA polymerase I, mass spectrometry Thermus aquaticus
2.7.7.49 62879
-
 x * 62879, mutant DNA polymerase I, sequence calculation, x * 62865, mutant DNA polymerase I, mass spectrometry Thermus aquaticus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.7.7.7 deoxynucleoside triphosphate + DNAn Thermus aquaticus
-
 diphosphate + DNAn+1
-
 ?
2.7.7.49 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

EC Number Organism UniProt Comment Textmining
2.7.7.7 Thermus aquaticus
-
-
-
2.7.7.49 Thermus aquaticus
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.7.7.7 recombinant mutant DNA polymerase I from Escherichia coli strain BL21 AI cytoplasm by metal ion and heparin affinity chromatography Thermus aquaticus
2.7.7.49 recombinant mutant DNA polymerase I from Escherichia coli strain BL21 AI cytoplasm by metal ion and heparin affinity chromatography Thermus aquaticus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.7.7.7 dATP + DNAn
-
 Thermus aquaticus diphosphate + DNAn+1
-
 ?
2.7.7.7 dCTP + DNAn
-
 Thermus aquaticus diphosphate + DNAn+1
-
 ?
2.7.7.7 deoxynucleoside triphosphate + DNAn
-
 Thermus aquaticus diphosphate + DNAn+1
-
 ?
2.7.7.7 dTTP + DNAn
-
 Thermus aquaticus diphosphate + DNAn+1
-
 ?
2.7.7.7 additional information the mutant enzyme shows single deoxynucleotide 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 ?
-
 ?
2.7.7.49 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
-
 ?
2.7.7.49 dATP + DNAn enzyme mutant N483Q/S486Q/T539N/Y545Q/D547T/P548Q/A570Q/D578Q/A597T/W604R/S612N/V730L/R736Q/S739N/M747R Thermus aquaticus diphosphate + DNAn+1
-
 ?
2.7.7.49 dCTP + DNAn enzyme mutant N483Q/S486Q/T539N/Y545Q/D547T/P548Q/A570Q/D578Q/A597T/W604R/S612N/V730L/R736Q/S739N/M747R Thermus aquaticus diphosphate + DNAn+1
-
 ?
2.7.7.49 dTTP + DNAn enzyme mutant N483Q/S486Q/T539N/Y545Q/D547T/P548Q/A570Q/D578Q/A597T/W604R/S612N/V730L/R736Q/S739N/M747R Thermus aquaticus diphosphate + DNAn+1
-
 ?
2.7.7.49 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

EC Number Subunits Comment Organism
2.7.7.7 ? x * 62879, mutant DNA polymerase I, sequence calculation, x * 62865, mutant DNA polymerase I, mass spectrometry Thermus aquaticus
2.7.7.49 ? x * 62879, mutant DNA polymerase I, sequence calculation, x * 62865, mutant DNA polymerase I, mass spectrometry Thermus aquaticus

Synonyms

EC Number Synonyms Comment Organism
2.7.7.7 DNA polymerase I
-
 Thermus aquaticus
2.7.7.49 DNA polymerase I
-
 Thermus aquaticus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.7.7.7 37
-
 assay at Thermus aquaticus
2.7.7.49 37
-
 assay at Thermus aquaticus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.7.7.7 0.89
-
 dCTP pH 9.0, 37°C, recombinant mutant enzyme Thermus aquaticus
2.7.7.7 0.96
-
 dATP pH 9.0, 37°C, recombinant mutant enzyme Thermus aquaticus
2.7.7.7 1.4
-
 dTTP pH 9.0, 37°C, recombinant mutant enzyme Thermus aquaticus
2.7.7.49 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
2.7.7.49 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
2.7.7.49 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

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.7.7.7 9
-
 assay at Thermus aquaticus
2.7.7.49 9
-
 assay at Thermus aquaticus

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.7.7.7 9.9
-
 dTTP pH 9.0, 37°C, recombinant mutant enzyme Thermus aquaticus
2.7.7.7 37
-
 dCTP pH 9.0, 37°C, recombinant mutant enzyme Thermus aquaticus
2.7.7.7 44
-
 dATP pH 9.0, 37°C, recombinant mutant enzyme Thermus aquaticus
2.7.7.49 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
2.7.7.49 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
2.7.7.49 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