PDBsum entry 2hr6

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protein ligands metals links
Hydrolase PDB id
Protein chain
137 a.a. *
Waters ×156
* Residue conservation analysis
PDB id:
Name: Hydrolase
Title: Crystal structure of dutpase in complex with substrate analo and manganese
Structure: Deoxyuridine 5'-triphosphate nucleotidohydrolase. Chain: a. Synonym: dutpase, dutp pyrophosphatase. Engineered: yes
Source: Escherichia coli. Organism_taxid: 562. Gene: dut. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.
1.84Å     R-factor:   0.152     R-free:   0.173
Authors: O.Barabas,J.Kovari,R.Tapai,B.G.Vertessy
Key ref:
J.Kovári et al. (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. PubMed id: 17932923 DOI: 10.1002/prot.21757
19-Jul-06     Release date:   31-Jul-07    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P06968  (DUT_ECOLI) -  Deoxyuridine 5'-triphosphate nucleotidohydrolase
151 a.a.
137 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.  - dUTP diphosphatase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: dUTP + H2O = dUMP + diphosphate
Bound ligand (Het Group name = DUD)
matches with 85.71% similarity
+ H(2)O
= dUMP
+ diphosphate
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     nucleus   2 terms 
  Biological process     nucleotide metabolic process   5 terms 
  Biochemical function     hydrolase activity     4 terms  


DOI no: 10.1002/prot.21757 Proteins 71:308-319 (2008)
PubMed id: 17932923  
Methylene substitution at the alpha-beta bridging position within the phosphate chain of dUDP profoundly perturbs ligand accommodation into the dUTPase active site.
J.Kovári, O.Barabás, B.Varga, A.Békési, F.Tölgyesi, J.Fidy, J.Nagy, B.G.Vértessy.
dUTP pyrophosphatase, a preventive DNA repair enzyme, contributes to maintain the appropriate cellular dUTP/dTTP ratio by catalyzing dUTP hydrolysis. dUTPase is essential for viability in bacteria and eukaryotes alike. Identification of species-specific antagonists of bacterial dUTPases is expected to contribute to the development of novel antimicrobial agents. As a first general step, design of dUTPase inhibitors should be based on modifications of the substrate dUTP phosphate chain, as modifications in either base or sugar moieties strongly impair ligand binding. Based on structural differences between bacterial and human dUTPases, derivatization of dUTP-analogous compounds will be required as a second step to invoke species-specific character. Studies performed with dUTP analogues also offer insights into substrate binding characteristics of this important and structurally peculiar enzyme. In this study, alpha,beta-methylene-dUDP was synthesized and its complex with dUTPase was characterized. Enzymatic phosphorylation of this substrate analogue by pyruvate kinase was not possible in contrast to the successful enzymatic phosphorylation of alpha,beta-imino-dUDP. One explanation for this finding is that the different bond angles and the presence of the methylene group may preclude formation of a catalytically competent complex with the kinase. Crystal structure of E. coli dUTPase:alpha,beta-methylene-dUDP and E. coli dUTPase:dUDP:Mn complexes were determined and analyzed in comparison with previous data. Results show that the "trans" alpha-phosphate conformation of alpha,beta-methylene-dUDP differs from the catalytically competent "gauche" alpha-phosphate conformation of the imino analogue and the oxo substrate, manifested in the shifted position of the alpha-phosphorus by more than 3 A. The three-dimensional structures determined in this work show that the binding of the methylene analogue with the alpha-phosphorus in the "gauche" conformation would result in steric clash of the methylene group with the protein atoms. In addition, the metal ion cofactor was not bound in the crystal of the complex with the methylene analogue while it was clearly visible as coordinated to dUDP, arguing that the altered phosphate chain conformation also perturbs metal ion complexation. Isothermal calorimetry titrations indicate that the binding affinity of alpha,beta-methylene-dUDP toward dUTPase is drastically decreased when compared with that of dUDP. In conclusion, the present data suggest that while alpha,beta-methylene-dUDP seems to be practically nonhydrolyzable, it is not a strong binding inhibitor of dUTPase probably due to the altered binding mode of the phosphate chain. Results indicate that in some cases methylene analogues may not faithfully reflect the competent substrate ligand properties, especially if the methylene hydrogens are in steric conflict with the protein.
  Selected figure(s)  
Figure 1.
Figure 1. (A) Reaction scheme of dUTP hydrolysis as catalyzed by dUTPases.[1] (B) The crystal structure of homotrimeric E. coli dUTPase: , -imino-dUTP:Mg complex (PDB ID: 1RN8). The three subunits of the enzyme are shown with ribbon model in green, blue, and yellow. The , -imino-dUTP ligands in the three identical active sites are in atom coloring bonds model (carbon, dark gray; oxygen, red; phosphorus, orange; nitrogen, blue; magnesium, purple).
Figure 2.
Figure 2. (A) Synthesis of 5 -O-tosyldeoxyuridine. (B) Synthesis of , -methylene-dUDP.
  The above figures are reprinted by permission from John Wiley & Sons, Inc.: Proteins (2008, 71, 308-319) copyright 2008.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
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
20197546 Q.Ye, S.W.Crawley, Y.Yang, G.P.Côté, and Z.Jia (2010).
Crystal structure of the alpha-kinase domain of Dictyostelium myosin heavy chain kinase A.
  Sci Signal, 3, ra17.
PDB codes: 3lkm 3lla 3lmh 3lmi
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.  
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
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.