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PDBsum entry 1ofq
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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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Substrate and metal complexes of 3-Deoxy-D-Arabino-Heptulosonate-7-Phosphate synthase from saccharomyces cerevisiae provide new insights into the catalytic mechanism.
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Authors
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V.König,
A.Pfeil,
G.H.Braus,
T.R.Schneider.
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Ref.
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J Mol Biol, 2004,
337,
675-690.
[DOI no: ]
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PubMed id
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Abstract
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3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthases are metal-dependent
enzymes that catalyse the first committed step in the biosynthesis of aromatic
amino acids in microorganisms and plants, the condensation of
2-phophoenolpyruvate (PEP) and d-erythrose 4-phosphate (E4P) to DAHP. The DAHP
synthases are possible targets for fungicides and represent a model system for
feedback regulation in metabolic pathways. To gain further insight into the role
of the metal ion and the catalytic mechanism in general, the crystal structures
of several complexes between the tyrosine-regulated form of DAHP synthase from
Saccharomyces cerevisiae and different metal ions and ligands have been
determined. The crystal structures provide evidence that the simultaneous
presence of a metal ion and PEP result in an ordering of the protein into a
conformation that is prepared for binding the second substrate E4P. The site and
binding mode of E4P was derived from the 1.5A resolution crystal structure of
DAHP synthase in complex with PEP, Co2+, and the E4P analogue glyceraldehyde
3-phosphate. Our data suggest that the oxygen atom of the reactive carbonyl
group of E4P replaces a water molecule coordinated to the metal ion, strongly
favouring a reaction mechanism where the initial step is a nucleophilic attack
of the double bond of PEP on the metal-activated carbonyl group of E4P.
Mutagenesis experiments substituting specific amino acids coordinating PEP, the
divalent metal ion or the second substrate E4P, result in stable but inactive
Aro4p-derivatives and show the importance of these residues for the catalytic
mechanism.
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Figure 3.
Figure 3. (a) Topology plot of DAHPS. The b-strands and
a-helices belonging to the canonical ba-barrel are shown in
light blue and dark blue, respectively; non-canonical modules
are shown in cyan. Regions of the structure that were found to
be flexible or disordered in any of the crystal forms are marked
by a grey background. The location of residues interacting with
PEP, G3P, and the metal is indicated using different flags (red
for PEP, yellow for G3P, grey for metal). (b) Schematic view of
the crystal structure of DAHPS in complex with PEP (red), G3P
(yellow), and Co2+ (grey) with phosphorus atoms shown in
magenta. (c) The same as (b) but the view is towards the
C-terminal face of the barrel.
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Figure 8.
Figure 8. Stereoview of the active site in the
DAHPS·Co2+·PEP·G3P-complex showing the
water molecules on the re-side of PEP in green. In this view,
the si-side of PEP is on top of PEP, the re-side on the bottom.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2004,
337,
675-690)
copyright 2004.
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Secondary reference #1
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Title
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Evolution of feedback-Inhibited beta /alpha barrel isoenzymes by gene duplication and a single mutation.
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Authors
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M.Hartmann,
T.R.Schneider,
A.Pfeil,
G.Heinrich,
W.N.Lipscomb,
G.H.Braus.
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Ref.
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Proc Natl Acad Sci U S A, 2003,
100,
862-867.
[DOI no: ]
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PubMed id
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Figure 2.
Fig. 2. Aro4p structure of S. cerevisiae. (A) Topology
plot of DAHP synthase from S. cerevisiae. The  -strands and
 -helices of
the central / barrel are
shown in orange and yellow, respectively. Loops on the N- and
C-terminal sides of the barrel are in cyan and green,
respectively. The additional structural elements at the N
terminus and between helix 5 and -strand 6 are also
shown in cyan. Residues involved in binding of PEP are marked in
green. Residues involved in regulation are marked in blue with
italic lettering for those identified by random screening and
normal lettering for those found by site-directed mutagenesis.
Red arrows mark the cutting points in Aro4p for the chimera
constructs (Fig. 1A). The dotted 0 strand is
part of the second monomer of a dimer and interacts with the
6a and 6b. (B) The
crystal structure of DAHP synthase from S. cerevisiae in ribbon
presentation. The crystal structures contain a molecule of PEP
bound to the active site shown as space-filling models (red and
magenta). Secondary structure elements are color-coded as
described for A. Shown are views from the C-terminal face of the
central barrel
(Left), side of the barrel (Center), and N-terminal face of the
barrel (Right). The programs MOLSCRIPT 2.1 (21) and RASTER3D
(22) were used for the presentation of DAHP synthase.
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Figure 5.
Fig. 5. Comparison of the putative effector-binding
cavity of the tyrosine- and phenylalanine-regulated DAHP
synthases. (A) Amino acid sequence alignment of a part of the
putative regulation cavity of the tyrosine- and
phenylalanine-regulated DAHP synthases of several organisms. C.
albicans, Candida albicans; A. nidulans, Aspergillus nidulans;
H. influenzae, Haemophilus influenzae; S. typhimurium,
Salmonella typhimurium. (B-D) Accessible surface plots of the
tyrosine-regulated DAHP synthase Aro4p from S. cerevisiae (S.c.,
B and C) and the phenylalanine-inhibited DAHP synthase AroG from
E. coli (E.c., D). Surface elements closer than 3 Å to
atoms belonging to the N-terminal extension or the inserted sheet are
shown in cyan. Residues identified as playing a role in
regulation (blue) and for the specificity-related residue (red,
G226 in Aro4p and S211 in AroG) are indicated. (B) The
orientation is the same as described for Fig. 2B. (C and D) The
view is rotated by 40° around the vertical and 30°
around the horizontal axis with respect to Fig. 2B. The figure
was created with DINO (ref. 23 and www.dino3d.org).
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