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Literature summary extracted from

  • Tchigvintsev, A.; Singer, A.U.; Flick, R.; Petit, P.; Brown, G.; Evdokimova, E.; Savchenko, A.; Yakunin, A.F.
    Structure and activity of the Saccharomyces cerevisiae dUTP pyrophosphatase DUT1, an essential housekeeping enzyme (2011), Biochem. J., 437, 243-253.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.6.1.23 expressed in Escherichia coli BL21-Gold(DE3) cells Saccharomyces cerevisiae

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.6.1.23 hanging drop vapor diffusion method, apo-enzyme using 0.1 M ammonium sulfate, 0.1 M Bis-Tris (pH 5.5), and 25% (w/v) PEG3350. In complex with dUMP using 0.2 M sodium acetate, 0.1 M Tris-HCl (pH 8.5), and 30% (w/v) PEG4000. In complex with alpha,beta-imido dUTP using 30% Jeffamine ED-2001 (pH 7.0), 0.1 M potassium-HEPES (pH 7.0) and 3 mM MgCl2 Saccharomyces cerevisiae

Protein Variants

EC Number Protein Variants Comment Organism
3.6.1.23 D32A the mutant exhibits negligible activity Saccharomyces cerevisiae
3.6.1.23 D85A the mutant exhibits negligible activity Saccharomyces cerevisiae
3.6.1.23 D87A the mutant exhibits negligible activity Saccharomyces cerevisiae
3.6.1.23 D87A/R137A/D85A inactive Saccharomyces cerevisiae
3.6.1.23 F142A the mutant exhibits very low activity Saccharomyces cerevisiae
3.6.1.23 Q114A the mutant shows essentially the wild type affinity for dUTP and greatly reduced activity Saccharomyces cerevisiae
3.6.1.23 R111A the mutant has reduced activity and lower substrate affinity (increased Km) compared to the wild type enzyme Saccharomyces cerevisiae
3.6.1.23 R137A the mutant exhibits negligible activity Saccharomyces cerevisiae
3.6.1.23 R68A the mutant exhibits very low activity Saccharomyces cerevisiae
3.6.1.23 S69A the mutant exhibits negligible activity Saccharomyces cerevisiae
3.6.1.23 Y88A the mutant protein is equally active against both dUTP and UTP and has reduced activity and lower substrate affinity (increased Km) compared to the wild type enzyme Saccharomyces cerevisiae

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.6.1.23 EDTA less than 10% residual activity at 1 mM Saccharomyces cerevisiae

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.6.1.23 0.0132
-
 dUTP wild type enzyme, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C Saccharomyces cerevisiae
3.6.1.23 0.0142
-
 dUTP mutant enzyme Q114A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C Saccharomyces cerevisiae
3.6.1.23 0.0315
-
 dUTP mutant enzyme R111A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C Saccharomyces cerevisiae
3.6.1.23 0.035
-
 UTP mutant enzyme Y88A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C Saccharomyces cerevisiae
3.6.1.23 0.041
-
 dUTP mutant enzyme Y88A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C Saccharomyces cerevisiae
3.6.1.23 0.0447
-
 dITP wild type enzyme, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C Saccharomyces cerevisiae

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.6.1.23 Co2+ strictly dependent on a bivalent metal cation like Co2+ Saccharomyces cerevisiae
3.6.1.23 Mg2+ strictly dependent on a bivalent metal cation like Mg2+ which supports the highest rate of dUTP hydrolysis Saccharomyces cerevisiae
3.6.1.23 Mn2+ strictly dependent on a bivalent metal cation like Mn2+ Saccharomyces cerevisiae
3.6.1.23 Ni2+ strictly dependent on a bivalent metal cation like Ni2+ Saccharomyces cerevisiae
3.6.1.23 Zn2+ strictly dependent on a bivalent metal cation like Zn2+ Saccharomyces cerevisiae

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
3.6.1.23 18100
-
 3 * 18100, His tagged enzyme, calculated from amino acid sequence Saccharomyces cerevisiae
3.6.1.23 56600
-
 gel filtration Saccharomyces cerevisiae

Organism

EC Number Organism UniProt Comment Textmining
3.6.1.23 Saccharomyces cerevisiae P33317
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.6.1.23 nickel affinity resin column chromatography and Superdex-200 gel filtration Saccharomyces cerevisiae

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.6.1.23 dITP + H2O shows also significant activity against dITP Saccharomyces cerevisiae dIMP + diphosphate
-
 ?
3.6.1.23 dUTP + H2O
-
 Saccharomyces cerevisiae dUMP + diphosphate
-
 ?
3.6.1.23 additional information alpha,beta-imido dUTP is a non-hydrolysable substrate Saccharomyces cerevisiae ?
-
 ?
3.6.1.23 UTP + H2O substrate for mutant enzyme Y88A Saccharomyces cerevisiae UMP + diphosphate
-
 ?

Subunits

EC Number Subunits Comment Organism
3.6.1.23 homotrimer 3 * 18100, His tagged enzyme, calculated from amino acid sequence Saccharomyces cerevisiae

Synonyms

EC Number Synonyms Comment Organism
3.6.1.23 DUT1
-
 Saccharomyces cerevisiae
3.6.1.23 dUTP pyrophosphatase
-
 Saccharomyces cerevisiae
3.6.1.23 dUTPase
-
 Saccharomyces cerevisiae

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.6.1.23 1.3
-
 dUTP mutant enzyme Q114A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C Saccharomyces cerevisiae
3.6.1.23 1.3
-
 dITP wild type enzyme, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C Saccharomyces cerevisiae
3.6.1.23 3.3
-
 dUTP mutant enzyme R111A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C Saccharomyces cerevisiae
3.6.1.23 5.3
-
 UTP mutant enzyme Y88A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C Saccharomyces cerevisiae
3.6.1.23 7.4
-
 dUTP mutant enzyme Y88A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C Saccharomyces cerevisiae
3.6.1.23 9.6
-
 dUTP wild type enzyme, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C Saccharomyces cerevisiae

pH Range

EC Number pH Minimum pH Maximum Comment Organism
3.6.1.23 7 9
-
 Saccharomyces cerevisiae

General Information

EC Number General Information Comment Organism
3.6.1.23 physiological function the enzyme plays a key role in preventing uracil incorporation into DNA Saccharomyces cerevisiae

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.6.1.23 29
-
 dITP wild type enzyme, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C Saccharomyces cerevisiae
3.6.1.23 90
-
 dUTP mutant enzyme Q114A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C Saccharomyces cerevisiae
3.6.1.23 100
-
 dUTP mutant enzyme R111A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C Saccharomyces cerevisiae
3.6.1.23 150
-
 UTP mutant enzyme Y88A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C Saccharomyces cerevisiae
3.6.1.23 180
-
 dUTP mutant enzyme Y88A, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C Saccharomyces cerevisiae
3.6.1.23 740
-
 dUTP wild type enzyme, in 50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM MnCl2, 0.5 mM NiCl2, at 30°C Saccharomyces cerevisiae