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PDBsum entry 1seh
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Structural insights into the catalytic mechanism of phosphate ester hydrolysis by dutpase.
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Authors
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O.Barabás,
V.Pongrácz,
J.Kovári,
M.Wilmanns,
B.G.Vértessy.
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Ref.
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J Biol Chem, 2004,
279,
42907-42915.
[DOI no: ]
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PubMed id
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Abstract
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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.
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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.
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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.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2004,
279,
42907-42915)
copyright 2004.
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Secondary reference #1
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Title
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Atomic resolution structure of escherichia coli dutpase determined ab initio.
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Authors
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A.González,
G.Larsson,
R.Persson,
E.Cedergren-Zeppezauer.
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Ref.
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Acta Crystallogr D Biol Crystallogr, 2001,
57,
767-774.
[DOI no: ]
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PubMed id
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Figure 4.
Figure 4 Detail of the 1.05 Å structure, showing parts of the
polypeptide chain at the active site, including Tyr93 and one
molecule of glycerol (Glyc139). The 2mF[o] - DF[c]
electron-density map is contoured at 2  (in
blue) and 4 (coral).
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Figure 5.
Figure 5 View of the mercury-binding site in a 2mF[o] - DF[c]
electron-density map contoured at 3 and 10 (in
blue and orange, respectively) showing clearly the multiple
conformations of the Hg atom (red spheres) bound to the S  of
Cys36.
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The above figures are
reproduced from the cited reference
with permission from the IUCr
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