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PDBsum entry 2o1c
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References listed in PDB file
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Key reference
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Title
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Structure and function of the e. Coli dihydroneopterin triphosphate pyrophosphatase: a nudix enzyme involved in folate biosynthesis.
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Authors
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S.B.Gabelli,
M.A.Bianchet,
W.Xu,
C.A.Dunn,
Z.D.Niu,
L.M.Amzel,
M.J.Bessman.
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Ref.
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Structure, 2007,
15,
1014-1022.
[DOI no: ]
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PubMed id
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Abstract
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Nudix hydrolases are a superfamily of pyrophosphatases, most of which are
involved in clearing the cell of potentially deleterious metabolites and in
preventing the accumulation of metabolic intermediates. We determined that the
product of the orf17 gene of Escherichia coli, a Nudix NTP hydrolase, catalyzes
the hydrolytic release of pyrophosphate from dihydroneopterin triphosphate, the
committed step of folate synthesis in bacteria. That this dihydroneopterin
hydrolase (DHNTPase) is indeed a key enzyme in the folate pathway was confirmed
in vivo: knockout of this gene in E. coli leads to a marked reduction in folate
synthesis that is completely restored by a plasmid carrying the gene. We also
determined the crystal structure of this enzyme using data to 1.8 A resolution
and studied the kinetics of the reaction. These results provide insight into the
structural bases for catalysis and substrate specificity in this enzyme and
allow the definition of the dihydroneopterin triphosphate pyrophosphatase family
of Nudix enzymes.
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Figure 2.
Figure 2. Structure of the E. coli Dihydroneopterin
Pyrophosphatase
(A) Ribbon diagram of the structure of
DHNTPase in complex with its product, pyrophosphate. Residues of
the Nudix signature sequence, Arg-29 and Glu-117, are shown as
green sticks. The secondary structural elements are labeled L
for loops, β for strands, and α for helices.
(B) Ribbon
diagram of the overlap of monomers A, B, and C (turquoise,
cornflower blue, and blue, respectively) with their
pyrophosphate and sulfate molecules.
(C) Molecular surface
of the complex structure (monomer A, closed).
(D) Ribbon
diagram of the overlap of monomer A (complex structure;
turquoise) and monomer D (ligand-free structure, open;
orange-red).
(E) Molecular surface of the ligand-free
structure (open).
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Figure 3.
Figure 3. Interactions of the Signature Sequence Nudix
Residues of DHNTPase
(Left) Hydrogen-bonding pattern of the
conserved residues (orange dashes) and recognition of the
pyrophosphate inhibitor as observed in the native complex
structure (monomers A, B, and C). (Right) Recognition of the
pyrophosphate as observed in the complex in the presence of
SmCl[3]. A 2F[o] − F[c] electron density map of the metal and
pyrophosphate is shown contoured at 1.2σ.
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The above figures are
reprinted
by permission from Cell Press:
Structure
(2007,
15,
1014-1022)
copyright 2007.
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Secondary reference #1
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Title
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Escherichia coli orf17 codes for a nucleoside triphosphate pyrophosphohydrolase member of the mutt family of proteins. Cloning, Purification, And characterization of the enzyme.
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Authors
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S.F.O'Handley,
D.N.Frick,
L.C.Bullions,
A.S.Mildvan,
M.J.Bessman.
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Ref.
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J Biol Chem, 1996,
271,
24649-24654.
[DOI no: ]
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PubMed id
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Figure 1.
Fig. 1. Expression and purification of the Orf17 protein. A
polyacrylamide gel (15%) containing 1% SDS, stained with
Coomassie Blue, contained the following: lane 1, 4 µg each
of bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic
anhydrase (31 kDa), and MutT (15 kDa); lanes 2 and 3, 1 µl
of crude extract from cells transformed with pET11b (no insert)
or pETorf17, respectively; lanes 3-5, approximately 4 µg
of Fractions I, II, or III, respectively (see ``Methods '').
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Figure 2.
Fig. 2. Products of the reaction. A standard colorimetric
assay (see ``Methods'') was scaled up 20-fold and was monitored
by HPLC to quantify dATP (  ) and dAMP
(  ). No dADP
was detected during the course of the reaction. PP[i] (  circle )
was converted to P[i] by the inorganic pyrophosphatase included
in the reaction mixture and was calculated as one-half of the
P[i] measured in the Fiske-Subbarow assay (31). No P[i] was
formed in a parallel reaction in the absence of inorganic
pyrophosphatase.
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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Secondary reference #2
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Title
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The biosynthesis of folic acid. Xii. Purification and properties of dihydroneopterin triphosphate pyrophosphohydrolase.
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Authors
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Y.Suzuki,
G.M.Brown.
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Ref.
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J Biol Chem, 1974,
249,
2405-2410.
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PubMed id
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