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PDBsum entry 2nod
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Oxidoreductase
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PDB id
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2nod
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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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Structure of nitric oxide synthase oxygenase dimer with pterin and substrate.
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Authors
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B.R.Crane,
A.S.Arvai,
D.K.Ghosh,
C.Wu,
E.D.Getzoff,
D.J.Stuehr,
J.A.Tainer.
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Ref.
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Science, 1998,
279,
2121-2126.
[DOI no: ]
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PubMed id
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Abstract
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Crystal structures of the murine cytokine-inducible nitric oxide synthase
oxygenase dimer with active-center water molecules, the substrate L-arginine
(L-Arg), or product analog thiocitrulline reveal how dimerization, cofactor
tetrahydrobiopterin, and L-Arg binding complete the catalytic center for
synthesis of the essential biological signal and cytotoxin nitric oxide. Pterin
binding refolds the central interface region, recruits new structural elements,
creates a 30 angstrom deep active-center channel, and causes a 35 degrees
helical tilt to expose a heme edge and the adjacent residue tryptophan-366 for
likely reductase domain interactions and caveolin inhibition. Heme propionate
interactions with pterin and L-Arg suggest that pterin has electronic influences
on heme-bound oxygen. L-Arginine binds to glutamic acid-371 and stacks with heme
in an otherwise hydrophobic pocket to aid activation of heme-bound oxygen by
direct proton donation and thereby differentiate the two chemical steps of
nitric oxide synthesis.
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Figure 1.
Fig. 1. NOS[ox]  -  fold, dimer
assembly, and likely interaction surface for NOS[red] and
caveolin. (A) The symmetric iNOS[ox] dimer viewed along the
crystallographic twofold axis, showing left (and^ right)
subunits with orange (yellow) winged sheets and
flanking blue (cyan) helices. Ball-and-stick models (white bonds
with red^ oxygen, blue nitrogen, yellow sulfur, and purple iron
atoms) highlight active-center hemes (left-most and right-most),
interchain disulfide^ bonds (center, foreground), pterin
cofactors (white, left-center and right-center), and substrate
L-Arg (green left and magenta^ right). The NH[2]-terminal ends
contribute hairpins
(center top and bottom) to the dimer interface, and the
COOH-termini (lower left and upper right) lie 85 Å apart.
Gray loops (residues 101^ to 107) are disordered. (B) iNOS[ox]
dimer shown rotated^ 90° about a horizontal axis from (A).
Each heme is cupped between the inward-facing palm (webbed sheet) and
thumb (magenta loop in front of left heme and green loop behind
right heme) of the^ "catcher's mitt" subunit fold. (C)
Solvent-accessible surface^ (29) of the iNOS[ox] dimer (one
subunit red, one subunit blue) oriented as in (B) and
color-coded by residue conservation (paler to more saturated
represents less conserved to more conserved) in NOS[ox]
sequences of known species and isozymes. The heme (white^ tubes)
is also solvent-exposed on the side (left subunit) opposite^ the
active-center channel (right subunit) and surrounded by a^
highly conserved hydrophobic surface for NOS[red] and caveolin
binding. (Stereo variations of Figs.
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Figure 5.
Fig. 5. Proposed L-Arg-assisted NOS oxygen activation. First,
substrate L-Arg (only guanidinium shown) donates a proton to
peroxo-iron, facilitating O-O bond cleavage and conversion to a
proposed oxo-iron(IV)  -cation
radical species, which then rapidly hydroxylates the^ neutral
guanidinium to NOH-L-Arg, possibly through a radical-based^
mechanism (3).
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The above figures are
reprinted
by permission from the AAAs:
Science
(1998,
279,
2121-2126)
copyright 1998.
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Secondary reference #1
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Title
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The structure of nitric oxide synthase oxygenase domain and inhibitor complexes.
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Authors
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B.R.Crane,
A.S.Arvai,
R.Gachhui,
C.Wu,
D.K.Ghosh,
E.D.Getzoff,
D.J.Stuehr,
J.A.Tainer.
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Ref.
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Science, 1997,
278,
425-431.
[DOI no: ]
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PubMed id
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Figure 3.
Fig. 3. Mobility, surface properties, and shape. (A) C trace of
NOS[ox]  114 (cubic
crystal form) colored by the crystallographic^ temperature
factor (low to high B factors colored blue to red) and displayed
with heme and mutation sites that affect function. Mutation
sites (side chains displayed and labeled by residue number)
affecting dimerization, L-Arg binding, or H[4]B binding
(defined^ in Fig. 2) cluster to highly mobile (red) projecting
regions. The view is rotated by about 45° from Fig. 1 about
a vertical axis. (B) Solvent-accessible molecular surface of
flattened^ (left) and concave (center) face. The orientation is
the same^ as in (A). The exposed heme edge (gold), residues
contributing to the distal pocket (cyan), and exposed conserved
hydrophobic^ residues (green) (defined in Fig. 2) map to the
same flattened^ face of the molecule and cluster in the regions
of high mobility and mutational sensitivity shown in (A), making
this surface the^ prime candidate for a symmetric dimer
interface. (C) Solvent-accessible^ molecular surface of the
narrow curved face. This face has few conserved exposed
hydrophobic residues. The view is rotated 90° from (A) and
(B) around a vertical axis.
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Figure 5.
Fig. 5. Comparison of the proximal heme-binding regions of
iNOS[ox] and cytochrome P450s. Structural elements contributing
to the proximal heme-binding regions of iNOS[ox] 114 and
P450[cam] (cyan C traces)
are substantially different. Only the proximal Cys ligands
(magenta bonds with yellow sulfur atoms, bound to gold^ hemes)
and immediately COOH-terminal three residues (magenta C traces)
have similar conformations. In iNOS[ox], Cys194 lies at the
COOH-terminal end of a helix and precedes an extended^ strand,
whereas in P450[cam], Cys357 lies at the NH[2]-terminal end of a
helix and follows an extended^ strand. Also, these two cysteine
thiolates bind opposite faces of iron protoporphyrin IX. C positions
for iNOS[ox] 114
residues 194 to 197 were superimposed with P450[cam] residues
357^ to 360 and then separated for clarity.
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The above figures are
reproduced from the cited reference
with permission from the AAAs
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Secondary reference #2
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Title
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Characterization of the inducible nitric oxide synthase oxygenase domain identifies a 49 amino acid segment required for subunit dimerization and tetrahydrobiopterin interaction.
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Authors
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D.K.Ghosh,
C.Wu,
E.Pitters,
M.Moloney,
E.R.Werner,
B.Mayer,
D.J.Stuehr.
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Ref.
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Biochemistry, 1997,
36,
10609-10619.
[DOI no: ]
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PubMed id
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