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Information on EC 3.6.1.23 - dUTP diphosphatase and Organism(s) Mycobacterium tuberculosis and UniProt Accession P9WNS5
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3.6.1.23 dUTP diphosphataseIUBMB Comments
The enzyme catalyses the Mg2+-dependent hydrolysis of dUTP to dUMP, providing the substrate for EC 2.1.1.45, thymidylate synthase, leading to production of thymidine nucleotides. By reducing the effective ratio of dUTP to TTP, the enzyme also reduces the possibility of dUTP incorporation into DNA.
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Word Map
- 3.6.1.23
- uracil
- dump
- trimer
- thymidylate
-
misincorporation
- glycosylase
-
herpes
-
uracil-dna
- simplex
- drug development
-
homotrimeric
- herpesviruses
-
integrase
-
fluoropyrimidines
-
uracil-containing
- lentiviruses
- pyrophosphatases
-
sapis
-
ebv-encoded
- herv-k
- 2'-deoxyuridine
-
varicella-zoster
- fiv
- diagnostics
- medicine
- pharmacology
- synthesis
The taxonomic range for the selected organisms is: Mycobacterium tuberculosis
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
dutpase, deoxyuridine triphosphatase, deoxyuridine triphosphate nucleotidohydrolase, dut-n, deoxyuridine 5'-triphosphate nucleotidohydrolase, deoxyuridine 5'-triphosphate, dutp pyrophosphatase, dutp nucleotidohydrolase, orf 54, dcd-dut, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
dUTPase
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desoxyuridine 5'-triphosphatase
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-
-
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desoxyuridine 5'-triphosphate nucleotidohydrolase
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-
-
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desoxyuridine-triphosphatase
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-
-
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dUTP pyrophosphatase
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-
-
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dUTPase
P18
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-
-
-
PIP4
-
-
-
-
additional information
the enzyme belongs to the Nudix superfamily of enzymes
dUTPase
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-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
dUTP + H2O = dUMP + diphosphate
structural organization and substrate binding modes
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
phosphorous acid anhydride hydrolysis
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-
-
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PATHWAY SOURCE
PATHWAYS
BRENDA
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-, -, -, -, -
SYSTEMATIC NAME
IUBMB Comments
dUTP nucleotidohydrolase
The enzyme catalyses the Mg2+-dependent hydrolysis of dUTP to dUMP, providing the substrate for EC 2.1.1.45, thymidylate synthase, leading to production of thymidine nucleotides. By reducing the effective ratio of dUTP to TTP, the enzyme also reduces the possibility of dUTP incorporation into DNA.
CAS REGISTRY NUMBER
COMMENTARY
37289-34-2
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
dUTP + H2O
dUMP + diphosphate
-
-
-
?
dUTP + H2O
dUMP + diphosphate
removes dUTP from the nucleotide triphosphate pool and therefore prevents the incorporation of uracil into the DNA
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-
?
dUTP + H2O
dUMP + diphosphate
dUTPase is essential to eliminate dUTP for DNA integrity and provide dUMP for thymidylate biosynthesis
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-
?
dUTP + H2O
dUMP + diphosphate
development of a robust and continuous dUTPase enzyme activity assay method using phenolred indicator, overview
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-
?
additional information
?
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biological functions in housekeeping, overview
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-
?
additional information
?
-
substrate specificity, no hydrolysis of canonical NTPs, overview
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-
?
additional information
?
-
the bifunctional dCTP deaminase:dUTPase shows very similar affinities for dCTP and deoxyuridine triphosphate as substrates, overview
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-
?
additional information
?
-
the enzyme cannot hydrolyze alpha-beta-imido-dUTP
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-
?
additional information
?
-
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the enzyme cannot hydrolyze alpha-beta-imido-dUTP
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-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
additional information
?
-
biological functions in housekeeping, overview
-
-
?
dUTP + H2O
dUMP + diphosphate
removes dUTP from the nucleotide triphosphate pool and therefore prevents the incorporation of uracil into the DNA
-
-
?
dUTP + H2O
dUMP + diphosphate
dUTPase is essential to eliminate dUTP for DNA integrity and provide dUMP for thymidylate biosynthesis
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
Mg2+
required for stabilization of the active form of the enzyme
Mg2+
required for full activity, In the absence of divalent cations, dUTPase retains about 50% of the catalytic rate observed in the presence of Mg2+
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2'-deoxyuridine 5'-(alpha,beta-imido)triphosphate
best inhibitor
thymidine triphosphate
binding of thymidine triphosphate leads to disordered C-terminal arranged as a lid covering the active site, and the enzyme adapts an inactive conformation as a result of structural changes in the active site, mechanism, overview
additional information
fast screening for binding of potential inhibitors to the active site
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additional information
-
fast screening for binding of potential inhibitors to the active site
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
steady-state kinetic analysis of the dual activities of the bifunctional enzyme, overview
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0.0009
dUTP
wild type enzyme, in 1 mM HEPES pH 7.5, 100 mM KCl, and 5 mM MgCl2, at 20°C
0.0011
dUTP
DELTA-loop mutant enzyme, in 1 mM HEPES pH 7.5, 100 mM KCl, and 5 mM MgCl2, at 20°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.024
dUTP
mutant enzyme D28N/H145W, in 1 mM HEPES pH 7.5 containing 100 mM KCl and 5 mM MgCl2, at 20°C
0.88
dUTP
DELTA-loop mutant enzyme, in 1 mM HEPES pH 7.5, 100 mM KCl, and 5 mM MgCl2, at 20°C
1.2
dUTP
mutant enzyme H145W, in 1 mM HEPES pH 7.5 containing 100 mM KCl and 5 mM MgCl2, at 20°C
1.22
dUTP
wild type enzyme, in 1 mM HEPES pH 7.5, 100 mM KCl, and 5 mM MgCl2, at 20°C
3.7 - 5.8
dUTP
pH 7.5, 25°C, recombinant wild-type and mutant enzymes
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
dUTPase in complex with the isosteric substrate analogue, alpha,beta-imido-dUTP, and Mg2+, about 0.223 mM dUTPase, 1.25 mM alpha,beta-imido-dUTP and 10 mM MgCl2 in 10 mM Tris-HCl, pH7.0, 50 mM NaCl, and 0.1 mM TCEP buffer is mixed with different reservoir solutions, X-ray diffraction structure determination and analysis at 1.5 A resolution, molecular replacement
mutant enzymes H145W and H145A in complex with alpha,beta-imido-dUTP, hanging drop vapor diffusion method, using 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1.20-1.75 M ammonium sulfate and 10% (v/v) glycerol in a 1:1 ratio
mutant enzymes R140K/H145W and G143STOP, hanging drop vapor diffusion method, using 0.1 M Tris/HCl pH 7.5, 1.5 M ammonium sulfate, and 12.5% (v/v) glycerol
purified recombinant bifunctional dCTP deaminase:dUTPase in complex with inhibitor thymidine triphosphate, hanging drop vapor diffusion method, 15°C, 0.004 ml of enzyme solution containing 1.8 mg/ml enzyme, 20 mM MgCl2, 5 mM dTTP, 50 mM HEPES, pH 6.8, is mixed with 0.002 ml of reservoir solution, cotaining 45% PEG 400, 200 mM MgCl2 and 100 mM HEPES, pH 7.5, and equilibrated over 1 ml of reservoir solution, 1 day, for the free enzyme crystallization is used: 1.9 mg/ml enzyme and 50 mM HEPES, pH 6.8, mixed with 0.002 ml of reservoir solution, 20% PEG 8000, 50 mM MgCl2 and 100 mM HEPES, pH 7.5, and equilibrated over 1 ml of reservoir solution, for 1 day to 6 weeks, X-ray diffraction structure determination and analysis at 2.0-2.5 A resolution
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D28N/H145W
the Asp/Asn mutation within Motif I results in approximately 50fold decrease of steady-state rate and significantly weakens the interaction of the protein with alpha,beta-imido-dUTP (15fold) whereas dUMP binding is only slightly affected
G143STOP
the mutant shows severely reduced activity compared to the wild type enzyme
H145A
the mutant shows decreased catalytic activity compared to the wild type enzyme
H145W
R40K/H145W
the mutant shows severely reduced activity compared to the wild type enzyme
T138STOP
the mutant shows severely reduced activity compared to the wild type enzyme
additional information
H145W
site-directed mutagenesis, the replacement introduces a 244 sensitive fluorescent label into the binding site. The steady-state kinetics show no difference in the case of the mutant and wild-type enzyme
H145W
the mutant shows strongly reduced activity compared to the wild type enzyme
H145W
the mutant shows decreased catalytic activity compared to the wild type enzyme
H145W
the mutation has no significant effect on the catalytic properties of dUTPase
additional information
the DELTA-loop deletion mutant is created by deletion of the five (Ala133-Ser137) loop-specific amino acids and shows reduced kcat and increased Km values for dUTP
additional information
-
the DELTA-loop deletion mutant is created by deletion of the five (Ala133-Ser137) loop-specific amino acids and shows reduced kcat and increased Km values for dUTP
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme from Escherichia coli strain BL21(DE3) by streptomycin precipitation, anion exchange chromatography, dialysis, and ammonium sulfate fractionation to homogeneity
recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21-Gold (DE3) as His-tag fusion protein
expression of His6-tagged enzyme in Escherichia coli strain BL21(DE3)
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
drug development
dUTPase is a promising antituberculotic drug target
medicine
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Chan, S.; Segelke, B.; Lekin, T.; Krupka, H.; Cho, U.S.; Kim, M.Y.; So, M.; Kim, C.Y.; Naranjo, C.M.; Rogers, Y.C.; Park, M.S.; Waldo, G.S.; Pashkov, I.; Cascio, D.; Perry, J.L.; Sawaya, M.R.
Crystal structure of the Mycobacterium tuberculosis dUTPase: insights into the catalytic mechanism
J. Mol. Biol.
341
503-517
2004
Mycobacterium tuberculosis (P9WNS5), Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv (P9WNS5)
Galperin, M.Y.; Moroz, O.V.; Wilson, K.S.; Murzin, A.G.
House cleaning, a part of good housekeeping
Mol. Microbiol.
59
5-19
2006
Saccharomyces cerevisiae, Campylobacter jejuni, Escherichia coli, Leishmania major, Staphylococcus aureus, Trypanosoma cruzi, Mycobacterium tuberculosis (P9WNS5), Mycobacterium tuberculosis H37Rv (P9WNS5)
Varga, B.; Barabas, O.; Takacs, E.; Nagy, N.; Nagy, P.; Vertessy, B.G.
Active site of mycobacterial dUTPase: structural characteristics and a built-in sensor
Biochem. Biophys. Res. Commun.
373
8-13
2008
Mycobacterium tuberculosis (P9WNS5), Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv (P9WNS5)
Helt, S.S.; Thymark, M.; Harris, P.; Aagaard, C.; Dietrich, J.; Larsen, S.; Willemoes, M.
Mechanism of dTTP inhibition of the bifunctional dCTP deaminase:dUTPase encoded by Mycobacterium tuberculosis
J. Mol. Biol.
376
554-569
2008
Mycobacterium tuberculosis (P9WNS5), Mycobacterium tuberculosis H37Rv (P9WNS5)
Takacs, E.; Nagy, G.; Leveles, I.; Harmat, V.; Lopata, A.; Toth, J.; Vertessy, B.G.
Direct contacts between conserved motifs of different subunits provide major contribution to active site organization in human and mycobacterial dUTPases
FEBS Lett.
584
3047-3054
2010
Homo sapiens, Mycobacterium tuberculosis (P9WNS5), Mycobacterium tuberculosis H37Rv (P9WNS5)
Pecsi, I.; Leveles, I.; Harmat, V.; Vertessy, B.G.; Toth, J.
Aromatic stacking between nucleobase and enzyme promotes phosphate ester hydrolysis in dUTPase
Nucleic Acids Res.
38
7179-7186
2010
Homo sapiens, Mycobacterium tuberculosis (P9WNS5), Mycobacterium tuberculosis H37Rv (P9WNS5)
Ramalho, T.C.; Caetano, M.S.; Josa, D.; Luz, G.P.; Freitas, E.A.; da Cunha, E.F.
Molecular modeling of Mycobacterium tuberculosis dUTpase: docking and catalytic mechanism studies
J. Biomol. Struct. Dyn.
28
907-917
2011
Mycobacterium tuberculosis (P9WNS5), Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv (P9WNS5)
Pecsi, I.; Hirmondo, R.; Brown, A.C.; Lopata, A.; Parish, T.; Vertessy, B.G.; Toth, J.
The dUTPase enzyme is essential in Mycobacterium smegmatis
PLoS ONE
7
e37461
2012
Mycolicibacterium smegmatis (A0QW08), Mycolicibacterium smegmatis, Mycobacterium tuberculosis (P9WNS5), Mycobacterium tuberculosis, Mycolicibacterium smegmatis mc(2)155 / ATCC 700084 (A0QW08), Mycobacterium tuberculosis H37Rv (P9WNS5)
Nagy, G.N.; Suardiaz, R.; Lopata, A.; Ozohanics, O.; Vekey, K.; Brooks, B.R.; Leveles, I.; Toth, J.; Vertessy, B.G.; Rosta, E.
Structural characterization of arginine fingers identification of an arginine finger for the pyrophosphatase dUTPases
J. Am. Chem. Soc.
138
15035-15045
2016
Mycobacterium tuberculosis (P9WNS5), Mycobacterium tuberculosis H37Rv (P9WNS5)
Hizi, A.; Herzig, E.
dUTPase the frequently overlooked enzyme encoded by many retroviruses
Retrovirology
12
70
2015
bovine immunodeficiency virus, Caenorhabditis elegans, Caprine arthritis encephalitis virus, equine infectious anemia virus, Escherichia coli (P06968), feline immunodeficiency virus, Homo sapiens (P33316), Homo sapiens, Human endogenous retrovirus K, Human gammaherpesvirus 4, Human gammaherpesvirus 8, Jembrana disease virus, Mason-Pfizer monkey virus, mouse mammary tumor virus, Murid gammaherpesvirus 4, Mycobacterium tuberculosis, Plasmodium falciparum, Saccharomyces cerevisiae, Visna-maedi virus
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