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PDBsum entry 3d8c
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Transcription regulator, oxidoreductase
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PDB id
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3d8c
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References listed in PDB file
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Key reference
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Title
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Evidence that two enzyme-Derived histidine ligands are sufficient for iron binding and catalysis by factor inhibiting hif (fih).
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Authors
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K.S.Hewitson,
S.L.Holmes,
D.Ehrismann,
A.P.Hardy,
R.Chowdhury,
C.J.Schofield,
M.A.Mcdonough.
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Ref.
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J Biol Chem, 2008,
283,
25971-25978.
[DOI no: ]
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PubMed id
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Abstract
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A 2-His-1-carboxylate triad of iron binding residues is present in many non-heme
iron oxygenases including the Fe(II) and 2-oxoglutarate (2OG)-dependent
dioxygenases. Three variants (D201A, D201E, and D201G) of the iron binding
Asp-201 residue of an asparaginyl hydroxylase, factor inhibiting HIF (FIH), were
made and analyzed. FIH-D201A and FIH-D201E did not catalyze asparaginyl
hydroxylation, but in the presence of a reducing agent, they displayed enhanced
2OG turnover when compared with wild-type FIH. Turnover of 2OG by FIH-D201A was
significantly stimulated by the addition of HIF-1alpha(786-826) peptide. Like
FIH-D201A and D201E, the D201G variant enhanced 2OG turnover but rather
unexpectedly catalyzed asparaginyl hydroxylation. Crystal structures of the
FIH-D201A and D201G variants in complex with Fe(II)/Zn(II), 2OG, and
HIF-1alpha(786-826/788-806) implied that only two FIH-based residues (His-199
and His-279) are required for metal binding. The results indicate that variation
of 2OG-dependent dioxygenase iron-ligating residues as a means of functional
assignment should be treated with caution. The results are of mechanistic
interest in the light of recent biochemical and structural analyses of non-heme
iron and 2OG-dependent halogenases that are similar to the FIH-D201A/G variants
in that they use only two His-residues to ligate iron.
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Figure 1.
The reaction catalyzed by wild-type FIH (and FIH-D201G).
hydroxylation of the β-carbon of Asn-803 within the C-terminal
transactivation domain of HIF-1α. The reaction requires Fe(II)
as a cofactor and dioxygen and 2OG as co-substrates that are
converted to succinate and carbon dioxide concomitant with
hydroxylation of HIF-1α substrate. For the FIH-D201A/E
variants, 2OG decarboxylation is uncoupled from the
hydroxylation of HIF-1α substrate and is stimulated by the
presence of a reducing agent (ascorbate or DTT).
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Figure 2.
Insights from crystal structures of FIH-D201A and FIH-D201G.
a, stereo view of the iron binding site of the
FIH-D201A·Fe(II)·2OG·HIF-1α[786–826]
complex. Experimental electron density OMIT map (F[o] - F[c])
contoured to 5σ represented as blue mesh (electron density is
carved out around the residues and ligands displayed for
clarity). The unanticipated electron density adjacent to the
iron was provisionally modeled as (bi)carbonate (see
“Results” for discussion). b, comparison of the wild-type
FIH·Fe(II)·2OG·HIF-1α[786–826] complex
(PDB ID 1H2L) with the
FIH-D201A·Fe(II)·2OG·HIF-1α[786–826]
complex (wild-type FIH (blue) in complex with HIF substrate
(cyan) and FIH-D201A (yellow) in complex with HIF substrate
(magenta)). This figure emphasizes several important
interactions between wild-type FIH and HIF-1α that are lost in
the FIH-D201A complex: wild-type FIH Asp-201 and HIF-1α Asn-803
main chain nitrogen (yellow dash), wild-type FIH Gln-239 and
HIF-1α Asn-803 side chain, and wild-type FIH Trp-296 and
HIF-1α Val-802. Note the presence of the assigned sulfate ion
(orange and red) in the FIH-D201A structure apparently replacing
the carboxylate of the HIF-1α Glu-801 in the wild-type
FIH·Fe(II)·2OG·HIF-1α[786–826] complex,
which is also observed in uncomplexed wild-type FIH structure
(PDB 1H2N, not shown). c, stereo views from the crystal
structures of 2OG-dependent halogenase, SyrB2 (pink)
superimposed on the FIH-D201A variant (yellow). The FIH-D201A
variant shares the same HXA... H motif as SyrB2 (PDB ID 2FCT);
however, the FIH-D201A apparently does not provide enough space
for a chloride ion to complete octahedral coordination to the
Fe(II), which could explain why FIH-D201A does not have
halogenase activity toward HIF-1α under our assay conditions.
Distances between the FIH-D201A Ala-201 Cβ methyl group and
Fe(II) in each of the structures are shown as black dashed lines
to emphasize this point. d, stereo view ball-and-stick
representation of the
FIH-D201G·Zn(II)·2OG·HIF-1α[786–826]
complex metal binding site. Experimental electron density 2F[o]
- F[c] contoured to 1.0σ represented as blue mesh (electron
density is carved out around the residues and ligands displayed
for clarity). FIH and 2OG are colored green, HIF-1α[786–826]
is colored yellow, Zn(II) is colored as a gray sphere, and the
Zn(II) bound water is colored as a red sphere. Wat, water.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2008,
283,
25971-25978)
copyright 2008.
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Secondary reference #1
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Title
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Structure of factor-Inhibiting hypoxia-Inducible factor (hif) reveals mechanism of oxidative modification of hif-1 alpha.
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Authors
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J.M.Elkins,
K.S.Hewitson,
L.A.Mcneill,
J.F.Seibel,
I.Schlemminger,
C.W.Pugh,
P.J.Ratcliffe,
C.J.Schofield.
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Ref.
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J Biol Chem, 2003,
278,
1802-1806.
[DOI no: ]
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PubMed id
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Figure 1.
Fig. 1. The FIH-CAD complex (a-c, structure 1; d,
structure 2). a, FIH monomer. The CAD peptide is shown as a
ball-and-stick representation in red and the DSBH motif in
green. b, FIH dimer. The two molecules of FIH are in dark and
light blue, the DSBH motif is in green, and the CAD peptide is
in red. c, the 2OG binding site with bound NOG is shown in
yellow. The Fe^(II) is colored pink, and the 2mF[o]  DF[c]
electron density map is contoured at 1.5  . d,
orientation of CAD Asn803 at the FIH active site. The 2OG and
CAD peptide are shown in yellow.
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Figure 2.
Fig. 2. FIH-CAD interactions. a, CAD fragments are shown
as stick models in yellow above a van der Waals surface of FIH.
FIH residues beneath the surface are colored green. Dotted red
lines represent electrostatic bonds. b, alternative view of site
1. Note Asn803 is deeply buried. c, electron density for the
bound CAD peptide (structure 1). CAD residues 795-806 (site 1,
left) and 812-823 (site 2, right) are shown as ball-and-stick
representations in yellow. The difference electron density,
contoured at 2.2 (left) and
1.5 (right),
was calculated after random model perturbation and refinement
with CAD omitted to remove model bias.
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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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Selective inhibition of factor inhibiting hypoxia-Inducible factor.
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Authors
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M.A.Mcdonough,
L.A.Mcneill,
M.Tilliet,
C.A.Papamicaël,
Q.Y.Chen,
B.Banerji,
K.S.Hewitson,
C.J.Schofield.
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Ref.
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J Am Chem Soc, 2005,
127,
7680-7681.
[DOI no: ]
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PubMed id
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