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PDBsum entry 2c3y
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Oxidoreductase
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PDB id
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2c3y
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References listed in PDB file
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Key reference
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Title
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Flexibility of thiamine diphosphate revealed by kinetic crystallographic studies of the reaction of pyruvate-Ferredoxin oxidoreductase with pyruvate.
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Authors
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C.Cavazza,
C.Contreras-Martel,
L.Pieulle,
E.Chabrière,
E.C.Hatchikian,
J.C.Fontecilla-Camps.
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Ref.
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Structure, 2006,
14,
217-224.
[DOI no: ]
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PubMed id
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Abstract
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Pyruvate-ferredoxin oxidoreductases (PFOR) are unique among thiamine
pyrophosphate (ThDP)-containing enzymes in giving rise to a rather stable
cofactor-based free-radical species upon the decarboxylation of their first
substrate, pyruvate. We have obtained snapshots of unreacted and partially
reacted (probably as a tetrahedral intermediate) pyruvate-PFOR complexes at
different time intervals. We conclude that pyruvate decarboxylation involves
very limited substrate-to-product movements but a significant displacement of
the thiazolium moiety of ThDP. In this respect, PFOR seems to differ
substantially from other ThDP-containing enzymes, such as transketolase and
pyruvate decarboxylase. In addition, exposure of PFOR to oxygen in the presence
of pyruvate results in significant inhibition of catalytic activity, both in
solution and in the crystals. Examination of the crystal structure of inhibited
PFOR suggests that the loss of activity results from oxime formation at the 4'
amino substituent of the pyrimidine moiety of ThDP.
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Figure 2.
Figure 2. Electron Density Map of Oxygen-Inhibited PFOR
Pyruvate was excluded from phase and structure factor
calculations (omit map). The electron density peak next to N4'
of the aminopyrimidine ring may represent the oxygen atom of an
oxime moiety. This and the displacement of a water molecule are
the only differences between inhibited and active PFORs (see
Figure 3A). This figure and Figure 3 and Figure 6 were prepared
with TURBO (Roussel and Cambillaud, 1989).
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The above figure is
reprinted
by permission from Cell Press:
Structure
(2006,
14,
217-224)
copyright 2006.
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Secondary reference #1
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Title
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Crystal structure of the free radical intermediate of pyruvate:ferredoxin oxidoreductase.
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Authors
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E.Chabrière,
X.Vernède,
B.Guigliarelli,
M.H.Charon,
E.C.Hatchikian,
J.C.Fontecilla-Camps.
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Ref.
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Science, 2001,
294,
2559-2563.
[DOI no: ]
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PubMed id
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Figure 3.
Fig. 3. (A) Stereo pair of the acetyl-ThDP moiety and the bound
CO[2 ]molecule of PFOR and their protein environment. (B) Stereo
pair of the superposition of a part of the active site of PFOR
in the uncomplexed (green), and radical forms. The movements of
the thiazole ring and the side chains of Asn996 and Tyr994 are
concerted, and the S1 atom from the thiazole ring keeps its
hydrogen bond to Asn996 in the two conformations. Part (A) was
prepared using Molscript (39) and Raster3d (40); (B) was
prepared with Turbo-Frodo (38).
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Figure 4.
Fig. 4. Postulated mechanism of acetyl-CoA synthesis by PFOR.
Only the thiazolium ring moiety of ThDP is fully depicted (R and
R' as in Fig. 1, B and C). (A) Deprotonated carbanion species
(see Fig. 1A). The proton is putatively bound to
4'-iminopyridimine (not shown). (B) Pyruvate decarboxylation and
hypothetical enamine formation; the CO[2] reaction product stays
in the active site. (C) One electron transfer from the active
site to one of the [4Fe4S] clusters. Hypothetical n cation
radical formation. (D) Observed  /n cation
radical with a long C2-C2  bond (27)
and a bent thiazole ring (Fig. 2). Note that (i) ketonization of
the enamine (B) upon radical formation (C) and (ii)
tautomerization of the C5-C4 double bond to a C4-C4 double
bond, in going from (B) to (C), are required to explain the
observed stereochemistry of the adduct. The net result of these
two rearrangements is a significant reduction in the aromaticity
of the thiazole ring. Because this process is generally
considered to be unfavorable, the protein environment is thought
to play a key role in the stabilization of (C) and (D). The loss
of one electron from the active site and the bending of the
thiazole ring are shown here as a single step because we do not
know the detailed sequence of events. (E) Hypothetical
fragmented C-C bond resulting in carbocation and acetyl radical
species (28, 29). Upon fragmentation, the aromaticity of the
thiazole ring is thought to be restored (A), closing the cycle.
(F) Acetyl-CoA synthesis through condensation of a thiyl CoA
radical with the acetyl radical. Although the reaction is shown
in the direction of acetyl-CoA synthesis, PFORs are capable of
catalyzing the reverse reaction.
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The above figures are
reproduced from the cited reference
with permission from the AAAs
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