PDBsum entry 1rnj

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Hydrolase PDB id
Protein chain
140 a.a. *
TRS ×2
Waters ×221
* Residue conservation analysis
PDB id:
Name: Hydrolase
Title: Crystal structure of inactive mutant dutpase complexed with analogue imido-dutp
Structure: Deoxyuridine 5'-triphosphate nucleotidohydrolase. Chain: a. Synonym: dutpase, dutp pyrophosphatase. Engineered: yes. Mutation: yes
Source: Escherichia coli. Organism_taxid: 562. Gene: dut, dnas, sof, b3640. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.
Biol. unit: Trimer (from PDB file)
1.70Å     R-factor:   0.158     R-free:   0.187
Authors: O.Barabas,V.Pongracz,J.Kovari,M.Wilmanns,B.G.Vertessy
Key ref:
O.Barabás et al. (2004). Structural insights into the catalytic mechanism of phosphate ester hydrolysis by dUTPase. J Biol Chem, 279, 42907-42915. PubMed id: 15208312 DOI: 10.1074/jbc.M406135200
01-Dec-03     Release date:   07-Sep-04    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P06968  (DUT_ECOLI) -  Deoxyuridine 5'-triphosphate nucleotidohydrolase
151 a.a.
140 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 5 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.  - dUTP diphosphatase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: dUTP + H2O = dUMP + diphosphate
+ H(2)O
Bound ligand (Het Group name = DUP)
matches with 65.52% similarity
+ diphosphate
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     cytosol   1 term 
  Biological process     nucleotide metabolic process   5 terms 
  Biochemical function     hydrolase activity     4 terms  


DOI no: 10.1074/jbc.M406135200 J Biol Chem 279:42907-42915 (2004)
PubMed id: 15208312  
Structural insights into the catalytic mechanism of phosphate ester hydrolysis by dUTPase.
O.Barabás, V.Pongrácz, J.Kovári, M.Wilmanns, B.G.Vértessy.
dUTPase is essential to keep uracil out of DNA. Crystal structures of substrate (dUTP and alpha,beta-imino-dUTP) and product complexes of wild type and mutant dUTPases were determined to reveal how an enzyme responsible for DNA integrity functions. A kinetic analysis of wild type and mutant dUTPases was performed to obtain relevant mechanistic information in solution. Substrate hydrolysis is shown to be initiated via in-line nucleophile attack of a water molecule oriented by an activating conserved aspartate residue. Substrate binding in a catalytically competent conformation is achieved by (i) multiple interactions of the triphosphate moiety with catalysis-assisting Mg2+, (ii) a concerted motion of residues from three conserved enzyme motifs as compared with the apoenzyme, and (iii) an intricate hydrogen-bonding network that includes several water molecules in the active site. Results provide an understanding for the catalytic role of conserved residues in dUTPases.
  Selected figure(s)  
Figure 3.
FIG. 3. Identification of the nucleophile water. A, simulated annealed omit electron density map, restricted to exclusively show the exact position of the catalytic water molecule in the wild type dUTPase: , -imino-dUTP:Mg2+ structure. The figure also shows the hydrogen-bonding network involving the phosphate chain in this complex structure. In addition to the catalytic water, Mg2+-coordinating waters, W1, W2, W4, W15, and W21, also participate in the primary hydrogen-bonding interactions. B, superimposed structures of wild type (dark tones) and Asp90 Asn mutant (light tones) dUTPase: , -imino-dUTP:Mg2+ complexes. Note that the only remarkable difference between the superimposed structures is the disappearance of W[cat] from the mutant complex. Atomic color code: carbon, dark/light gray; oxygen, dark/light red (pink); phosphorus, dark/light orange (yellow); nitrogen, dark/light blue; magnesium, dark/light purple. C, superimposed structures of Asp90 Asn mutant dUTPase: dUTP:Mg2+ (dark tones) and Asp90 Asn mutant dUTPase: , -imino-dUTP:Mg2+ (light tones) complexes. Note the close identity in the positions of the nucleotide ligands. D, apoenzyme retains a water closely corresponding to the W[cat] position. 3-Fold superimposition of the apoenzyme (green carbons and water, otherwise standard atom coloring), enzyme-substrate (dark tones), and enzyme-product (light tones) structures. Note the position of the catalytic water from the apoenzyme to the enzyme-substrate and enzyme-product complexes. E, F, and G, simulated annealed omit electron density maps for the substrates in wild type E. coli dUTPase: , -imino-dUTP:Mg2+, the Asp90 Asn E. coli dUTPase: , -imino-dUTP: Mg2+, and the Asp90 Asn E. coli dUTPase:dUTP:Mg2+ structures, respectively. Maps are restricted to show the nucleotide ligand, the Mg2+, the three water molecules coordinating to the metal ion, as well as the catalytic water, if present.
Figure 4.
FIG. 4. Interaction mapping in enzyme-substrate (A), and enzyme-product complexes (B). Interactions are shown only for the phosphate chain moiety of the ligand. Due to the close similarity of the nucleotide interactions in the three enzyme-substrate complexes determined in the present study (cf. Fig. 3 and Table I), the map was selected to show the actual distances as found in the wild type dUTPase: , -imino-dUTP: Mg2+ (X = N) complex where W[cat] is also present. In the Asp90 Asn mutant dUTPase: , -imino-dUTP:Mg2+ (X = N) and Asp90 Asn mutant dUTPase:dUTP: Mg2+ (X = O) complex, the only significant differences are that (i) W[cat] is absent and Asp90O 2 becomes AsnN 2 and (ii) in the Asp90 Asn mutant dUTPase:dUTP: Mg2+ (X = O) complex, the X-Ser72O interaction is absent. Changes in all other distances are within ±0.2 Å.
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 42907-42915) copyright 2004.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20601405 I.Pecsi, I.Leveles, V.Harmat, B.G.Vertessy, and J.Toth (2010).
Aromatic stacking between nucleobase and enzyme promotes phosphate ester hydrolysis in dUTPase.
  Nucleic Acids Res, 38, 7179-7186.
PDB codes: 3hza 3loj
20823546 J.García-Nafría, L.Burchell, M.Takezawa, N.J.Rzechorzek, M.J.Fogg, and K.S.Wilson (2010).
The structure of the genomic Bacillus subtilis dUTPase: novel features in the Phe-lid.
  Acta Crystallogr D Biol Crystallogr, 66, 953-961.
PDB codes: 2xcd 2xce
18837522 B.G.Vértessy, and J.Tóth (2009).
Keeping uracil out of DNA: physiological role, structure and catalytic mechanism of dUTPases.
  Acc Chem Res, 42, 97.  
  19342774 G.L.Li, J.Wang, L.F.Li, and X.D.Su (2009).
Crystallization and preliminary X-ray analysis of three dUTPases from Gram-positive bacteria.
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 65, 339-342.  
19586911 L.Freeman, M.Buisson, N.Tarbouriech, A.Van der Heyden, P.Labbé, and W.P.Burmeister (2009).
The flexible motif V of Epstein-Barr virus deoxyuridine 5'-triphosphate pyrophosphatase is essential for catalysis.
  J Biol Chem, 284, 25280-25289.
PDB codes: 2we0 2we1 2we2 2we3
17932923 J.Kovári, O.Barabás, B.Varga, A.Békési, F.Tölgyesi, J.Fidy, J.Nagy, and B.G.Vértessy (2008).
Methylene substitution at the alpha-beta bridging position within the phosphate chain of dUDP profoundly perturbs ligand accommodation into the dUTPase active site.
  Proteins, 71, 308-319.
PDB codes: 2hr6 2hrm
17452782 A.Samal, N.Schormann, W.J.Cook, L.J.DeLucas, and D.Chattopadhyay (2007).
Structures of vaccinia virus dUTPase and its nucleotide complexes.
  Acta Crystallogr D Biol Crystallogr, 63, 571-580.
PDB codes: 2okb 2okd 2oke 2ol0 2ol1
17450564 I.Berente, E.Czinki, and G.Náray-Szabó (2007).
A combined electronegativity equalization and electrostatic potential fit method for the determination of atomic point charges.
  J Comput Chem, 28, 1936-1942.  
17169987 V.Németh-Pongrácz, O.Barabás, M.Fuxreiter, I.Simon, I.Pichová, M.Rumlová, H.Zábranská, D.Svergun, M.Petoukhov, V.Harmat, E.Klement, E.Hunyadi-Gulyás, K.F.Medzihradszky, E.Kónya, and B.G.Vértessy (2007).
Flexible segments modulate co-folding of dUTPase and nucleocapsid proteins.
  Nucleic Acids Res, 35, 495-505.
PDB codes: 2d4l 2d4m 2d4n
16441668 A.Guranowski, E.StarzyƄska, M.Pietrowska-Borek, J.Jemielity, J.Kowalska, E.Darzynkiewicz, M.J.Thompson, and G.M.Blackburn (2006).
Methylene analogues of adenosine 5'-tetraphosphate. Their chemical synthesis and recognition by human and plant mononucleoside tetraphosphatases and dinucleoside tetraphosphatases.
  FEBS J, 273, 829-838.  
16617146 M.Guillet, P.A.Van Der Kemp, and S.Boiteux (2006).
dUTPase activity is critical to maintain genetic stability in Saccharomyces cerevisiae.
  Nucleic Acids Res, 34, 2056-2066.  
16908222 S.U.Lari, C.Y.Chen, B.G.Vertéssy, J.Morré, and S.E.Bennett (2006).
Quantitative determination of uracil residues in Escherichia coli DNA: Contribution of ung, dug, and dut genes to uracil avoidance.
  DNA Repair (Amst), 5, 1407-1420.  
16154087 N.Tarbouriech, M.Buisson, J.M.Seigneurin, S.Cusack, and W.P.Burmeister (2005).
The monomeric dUTPase from Epstein-Barr virus mimics trimeric dUTPases.
  Structure, 13, 1299-1310.
PDB codes: 2bsy 2bt1
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.