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PDBsum entry 2we3
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References listed in PDB file
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Key reference
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Title
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The flexible motif V of epstein-Barr virus deoxyuridine 5'-Triphosphate pyrophosphatase is essential for catalysis.
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Authors
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L.Freeman,
M.Buisson,
N.Tarbouriech,
A.Van der heyden,
P.Labbé,
W.P.Burmeister.
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Ref.
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J Biol Chem, 2009,
284,
25280-25289.
[DOI no: ]
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PubMed id
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Abstract
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Deoxyuridine 5'-triphosphate pyrophosphatases (dUTPases) are ubiquitous enzymes
essential for hydrolysis of dUTP, thus preventing its incorporation into DNA.
Although Epstein-Barr virus (EBV) dUTPase is monomeric, it has a high degree of
similarity with the more frequent trimeric form of the enzyme. In both cases,
the active site is composed of five conserved sequence motifs. Structural and
functional studies of mutants based on the structure of EBV dUTPase gave new
insight into the mechanism of the enzyme. A first mutant allowed us to exclude a
role in enzymatic activity for the disulfide bridge involving the beginning of
the disordered C terminus. Sequence alignments revealed two groups of dUTPases,
based on the position in sequence of a conserved aspartic acid residue close to
the active site. Single mutants of this residue in EBV dUTPase showed a highly
impaired catalytic activity, which could be partially restored by a second
mutation, making EBV dUTPase more similar to the second group of enzymes.
Deletion of the flexible C-terminal tail carrying motif V resulted in a protein
completely devoid of enzymatic activity, crystallizing with unhydrolyzed
Mg(2+)-dUTP complex in the active site. Point mutations inside motif V
highlighted the essential role of lid residue Phe(273). Magnesium appears to
play a role mainly in substrate binding, since in absence of Mg(2+), the K(m) of
the enzyme is reduced, whereas the k(cat) is less affected.
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Figure 1.
EBV dUTPase sequence and structure.A, sequence alignment of
different dUTPases with known structures and the one from C.
glutamaticum. Residue numbers are given above the sequence for
the human enzyme and below the sequences for the mycobacterial
and EBV enzymes. The linker region is highlighted with cyan
letters. The five conserved motifs are highlighted. Key residues
of motif V are printed in red. A pink background highlights
residues of motif V where side chain hydroxyl and main chain
amide contact γ-phosphate in the crystal structure (13, 14).
Green background, main chain amide interaction with β- and
γ-phosphate; residues that have been deleted in the ΔV mutant
are underlined. The conserved residues interacting with the
motif V arginine of human-like dUTPases are printed in blue;
those interacting with the motif V arginine of
mycobacterium-like dUTPases are shown in magenta. B, structure
of EBV dUTPase in complex with α,β-imido-dUTP and Mg^2+
(Protein Data Bank entry 2bt1). The catalytic residue Asp^76 is
shown together with Asp^131 next to it. The four visible
sequence motifs of the active site are colored according to Fig.
1A, and the loop connecting the two domains (residues 114–125)
is colored in cyan. The bound α,β-imido-dUTP molecule is shown
as sticks and colored according to atom types. C, the ΔV
structure (pink) in complex with dUTP (sticks, atom colors) and
Mg^2+. The disordered part of the connecting loop is located
between the two arrows. Superposed is the dUMP-bound WT dUTPase
structure (Protein Data Bank entry 2bsy; gray, connecting loop
in cyan, motif I in green, disordered part dotted).
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Figure 4.
Structure of the active site with bound ligands.A, stereoview
of dUTP, Mg^2+, and its coordinating (green lines) water
molecules bound to the active site of EBV dUTPase ΔV mutant
(pink). The arginine residue of a neighbor symmetry mate
(Arg^37) is shown in orange. B, M. tuberculosis dUTPase
structure with bound α,β-imido-dUTP and the arginine and the
histidine lid residues of motif V (Protein Data Bank entry 2py4)
(14). Important hydrogen bonds and salt bridges are shown in the
color of their partner molecule. C, F[o] − F[c] electron
density contoured at 2.5 σ level. dUTP, Mg^2+, and water
molecules have been left out of the structure factor
calculation. D, structure of the EBV dUTPase ΔV mutant
Mg^2+-dUTP complex. The accessible surface is shown colored
according to the electrostatic potential of the protein (from
negative (red) to positive (blue)) calculated with apbs (34).
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2009,
284,
25280-25289)
copyright 2009.
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Secondary reference #1
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Title
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The monomeric dutpase from epstein-Barr virus mimics trimeric dutpases.
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Authors
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N.Tarbouriech,
M.Buisson,
J.M.Seigneurin,
S.Cusack,
W.P.Burmeister.
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Ref.
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Structure, 2005,
13,
1299-1310.
[DOI no: ]
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PubMed id
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Figure 3.
Figure 3. Superposition of the Two dUTPase-like Domains of
EBV dUTPase
Stereoview in which domain I is colored in
green and domain II is in red. The superposition used the CCP4MG
software. Secondary structure elements are labeled according to
Figure 1C, with N or C specified if the element is specific for
domains I or II.
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The above figure is
reproduced from the cited reference
with permission from Cell Press
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