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Literature summary for 2.7.7.49 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)

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

Protein Variants

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 [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

Metals/Ions Comment Organism Structure
Mg2+ required Thermus aquaticus

Molecular Weight [Da]

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/ Products (Substrates)

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

Organism UniProt Comment Textmining
Thermus aquaticus
-
-
-

Purification (Commentary)

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 and Products (Substrate)

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

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

Synonyms

Synonyms Comment Organism
DNA polymerase I
-
Thermus aquaticus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
assay at Thermus aquaticus

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
9
-
assay at Thermus aquaticus

kcat/KM [mM/s]

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