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3.6.1.23: dUTP diphosphatase

This is an abbreviated version!
For detailed information about dUTP diphosphatase, go to the full flat file.

Word Map on EC 3.6.1.23

Reaction

dUTP
+
H2O
=
dUMP
 +
diphosphate

Synonyms

DCD-DUT, dCTP deaminase-dUTPase, dCTP deaminase:dUTPase, deoxyuridine 5'-triphosphate, deoxyuridine 5'-triphosphate nucleotide hydrolase, deoxyuridine 5'-triphosphate nucleotidohydrolase, deoxyuridine 5'-triphosphate pyrophosphatase, deoxyuridine 50-triphosphate nucleotidohydrolase, deoxyuridine 5’-triphosphate nucleotidohydrolase, deoxyuridine nucleotidohydrolase, deoxyuridine triphosphatase, deoxyuridine triphosphate nucleotidohydrolase, deoxyuridine triphosphate pyrophosphatase, deoxyuridine-5'-triphosphate nucleotidohydrolase, deoxyuridine-triphosphate pyrophosphatase, desoxyuridine 5'-triphosphatase, desoxyuridine 5'-triphosphate nucleotidohydrolase, desoxyuridine-triphosphatase, DURP, DUT, DUT-N, DUT1, dUTP nucleotidohydrolase, dUTP pyrophosphatase, dUTPae, dUTPase, dUTPase-dUDPase, dUTPase-related protein, E165R, More, ORF 54, ORF8, P18, PIP4, Pr95, STSV2_30, Tb927.7.5160, U45, YncF, YosS

ECTree

     3 Hydrolases
         3.6 Acting on acid anhydrides
             3.6.1 In phosphorus-containing anhydrides
                3.6.1.23 dUTP diphosphatase

Crystallization

Crystallization on EC 3.6.1.23 - dUTP diphosphatase

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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
apo state and in complex with dUMP, hanging drop vapor diffusion method, using 0.1 M ammonium citrate tribasic (pH 7.0), 12% (w/v) polyethylene glycol 3350 at 18°C (apoenzyme) or 0.1 M citric acid (pH 3.5), 14% (w/v) polyethylene glycol 1000 (enzyme in complex with dUMP)
in complex with dUMP and Mg2+, sitting drop vapor diffusion method, using 0.16 M sodium chloride, 0.08 M HEPES pH 7.5, 0.02 M sodium citrate tribasic dihydrate, 17% (w/v) polyethylene glycol 3350
hanging drop vapor diffusion method, using 50 mM Tris-HCl at pH 7.4 and 5 mM of the non-hydrolyzable ligand analog 2'-deoxyuridine 5'-[(alpha,beta)-imido]-triphosphate
purified recombinant enzyme, hanging drop vapour diffusion method, 25°C, 0.001 ml of protein solution containing 10 mg/ml protein in 50 mM Tris-HCl, pH 7.4, is mixed with 0.001 ml of reservoir solution containing 2 M ammonium sulfate and 0.1 M Tris-HCl, pH 8.5, X-ray diffraction structure determination and analysis at 2.2-2.3 A resolution, molecular replacement
in complex with dUMP. dU-PPi-Mg2+ triggers the ordering of both the C-terminal arm and a loop (residues 18-26) which is uniquely disordered in the Bacillus subtilis dUTPases. The dUMP complex suggests two stages in substrate release
isoform YncF complexed with dU-PPi-Mg2+. dU-PPi-Mg2+ triggers the ordering of both the C-terminal arm and a loop (residues 18-26) which is uniquely disordered in the Bacillus subtilis dUTPases. The dUMP complex suggests two stages in substrate release
purified recombinant selenomethionine-labelled YosS and YncF, hanging drop vapour diffusion method, for YosS: 30 mg/ml protein in 20 mM Tris-HCl, and 150 mM NaCl, pH 7.5, with 2.0 M ammonium sulfate, 0.2 M potassium/sodium tartrate, 0.1 M trisodium citrate, pH 5.6, in 3 days, for YncF: 20 mg/ml protein in 20 mM Tris-HCl, and 150 mM NaCl, pH 7.5, with 0.2 M trimethylamine N-oxide dehydrate, 15% w/v PEG MME 2000, 0.1 M Tris-HCl, pH 9.0, in two weeks, X-ray diffraction structure determination and analysis at 2.3 A and 1.7 A resolution, respectively
-
vapour diffusion method, cocrystallized with dUDP and Sr2+
-
hanging drop vapor diffusion method, using 12% (w/v) polyethylene glycol 4000, 10% (w/v) 2-propanol and 0.3 M sodium citrate pH 5.65 for the His-tagged enzyme, using 11% (w/v) polyethylene glycol 4000, 10% (w/v) 2-propanol and 0.3 M sodium citrate pH 5.65 for the His-tagged enzyme in complex with dUTP, or using 10% (w/v) polyethylene glycol 4000, 10% (w/v) 2-propanol and 0.3 M sodium citrate pH 5.6 for the mutant enzyme E81S/T84R in complex with dUTP
-
hanging and sitting drop vapor diffusion method, stable monomers observed in crystal phase
-
purified recombinant core enzyme lacking the 28-residue-long C-terminal fly-specific segment, sitting drop vapor diffusion method, 0.002 ml of 3 mg/ml protein in 50 mM Tris-HCl buffer, pH 7.0, also containing 50 mM NaCl and 1 mM DTT, 1.2 mM dUDP and 10 mM MgCl2 are mixed with an equal volume of reservoir solution containing 50 mM sodium succinate buffer, pH 4.6-4.8, and 200 mM NaCl and 30-35% v/v 2-methyl-2,4-pentanediol, 4°C, X-ray diffraction structure determination and analysis at 1.88 A resolution
structure of dUTPase trimer, to 2.1 A resolution. The phage-specific insert folds into a small beta-pleated mini-domain reaching out from the dUTPase core surface. The insert mini-domains jointly coordinate a single Mg2+ ion per trimer at the entrance to the threefold inner channel. Residue Asp95, is the metal-ion-coordinating moiety potentially involved in correct positioning of the insert. The insert has no major role in dUTP binding or cleavage
Dubowvirus dv11
enzyme in complex with the substrate analogue dUDP
-
hanging-drop vapor diffusion method
purified recombinant enzyme in complex with inhibitors, i.e. dUTPase-alpha,beta-methylene-dUDP and dUTPase-dUDP-Mn complexes, 20°C, hanging-drop vapor diffusion, 3 mg/ml of enzyme in 10 mM Tris/HCl buffer, pH 7.0, and 50 mM NaCl, as well as 1.25 mM alpha,beta-methylene-dUDP or dUDP and 10 mM MgCl2 or MnCl2, respectively, mixing with an equal volume of reservoir solution 0.1 M Tris/HCl buffer, pH 7.8, containing 18-20% PEG 3350, and 400 mM Na-acetate, X-ray diffraction structure determination and analysis at 1.7-1.84 resolution, analysis of the different alpha-phosphate sites, overview
Q93H mutant in complex with a non-hydrolysable substrate analogue, alpha,beta-imido-dUTP, hanging drop vapor diffusion method, using 0.1 M Tris, 18-33.75 % polyethylene glycol 3350, 400 mM NaAc, pH 7.5
sitting drop vapor diffusion method, using 30% (w/v) PEG 1500
-
enzyme in complex with inhibitor alpha,beta-imino-dUTP and Mg2+, 0.282 mM dUTPase, 1.25 mM alpha,beta-imino-dUTP, and 10 mM MgCl2 in 10 mM Tris-HCl, 50 mM NaCl, and 0.1 mM TCEP, pH 7.0, are mixed with reservoir solution containing 0.1 M Na-HEPES buffer, pH 7.5, and 18-20% PEG 3350, generation of rhombohedral crystals, X-ray diffraction structure determination and analysis
purified recombinant enzyme
purified enzyme in complex with either product dUMPand Mg2+, or with substrate analogue alpha,beta-imino-dUTP, hanging drop vapour diffusion method, the reservoir solution contains 0.1 M Tris-HCl, pH 8.5, 20% PEG 3350, and 0.2 M LiSO4, a few weeks, soaking in europium nitrate solution, X-ray diffraction structure determination and analysis at 1.5A and 2.7 A resolution, respectively, single-wavelength anomalous diffraction
-
vapor diffusion method, using 0.3 M ammonium sulfate, 25% (w/v) polyethylene glycol 3350, 0.1 M HEPES, pH 7.0 (for mutant enzymes D131S/G78D and D131N), or using 150 mM malic acid, pH 7.5, and 25% (w/v) PEG 3350 (for mutant enzyme C4S), or using 0.1 M Tris, pH 8.5, 20% (w/v) polyethylene glycol 3350, 200 mM Li2SO4 (for mutant enzyme DELTAV, a 257-residue construct with the last 22 C-terminal residues removed)
in complex with nucleotides. Reaction starts with dUTPase capturing and properly positioning two substrates: dUTP and the catalytic water molecule. the catalytic water molecule is placed near the alpha-phosphate group opposite from the leaving group. Hydrolysis is then initiated by nucleophilic attack of Wthe water on the alpha-phosphate along the line of the scissile bond (in-line attack). A symmetric phosphorane-like transition state structure is formed with significant bond order in both forming and breaking bonds. Bond breaking is concomitant with inversion of the alpha-phosphate configuration and diphosphate-Mg2+ escape. The dUMP product then relaxes by rotation of the alpha-phosphate group and establishes a configuration similar to the one observed for this moiety in the E-S complex
O92810
purified recombinant C-terminal domain nucleocapsid fusion enzyme, hanging drop vapour diffusion method, 5 mg/ml protein in 50 mM Tris-HCl, pH 8.0, 0.2 M NH4Cl, 10 mM MgCl2, 5 mM DTT, and 0.5 mM PMSF, with or without 1.3 mM alpha,beta-imino-dUTP, room temperature, mixed 1:1 with reservoir solution containing 0.1 M Tris-HCl, pH 8.5, 8% w/v PEG 8000, X-ray diffraction structure determination and analysis at 1.75-2.3 A resolution
-
hanging drop vapor diffusion method, using 0.2 M potassium hydrogen phosphate and 20% (w/v) PEG3350 for mutant enzyme E145A complexed with alpha,beta-imido-dUTP, or 0.2 M potassium citrate and 20% (w/v) PEG3350 for mutant enzyme E145Q complexed with diphosphate
-
hanging drop vapor-diffusion method
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
hanging and sitting drop vapor diffusion method
mutant enzymes D28N and T138STOP
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
purified recombinant enzyme in complex with inhibitor 2',3'-dideoxy-3'-fluoro-5'-O-trityluridine, X-ray diffraction structure determination and analysis at 2.4 A resolution
-
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
purified recombinant free enzyme or recombinant enzyme complexed with substrate dUTP, hanging drop vapour diffusion method, protein and reservoir solution in a 1:1 ratio, 17°C, the reservoir solution contains 0.1 M HEPES, pH 7.5, 10% w/v PEG 8000, and 8% v/v ethylene glycol, 2 days, X-ray diffraction structure determination and analysis at 2.0 A resolution
Spbetavirus SPbeta
-
purified recombinant enzyme, sitting drop vapour diffusion method, 15 mg/ml protein in 20 mM Tris-HCl, and 150 mM NaCl, pH 7.5, with 0.2 M MgCl2, 25% w/v PEG 3350, 0.1 M Tris-HCl, pH 8.5, X-ray diffraction structure determination and analysis at 1.3 A resolution
-
transition state analogue complexes in presence of dUMP, AlF3 and MgF3-. In the dimeric enzyme, the nucleophilic attack occurs on the beta-phosphate group
enzyme in complex with inhibitor dUDP, crystal structure determination and analysis
-
vapor diffusion method
-
sitting drop vapor diffusion method, using 30% (w/v) PEG 1500