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PDBsum entry 2j9f
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Oxidoreductase
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PDB id
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2j9f
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References listed in PDB file
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Key reference
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Title
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The two active sites in human branched-Chain alpha-Keto acid dehydrogenase operate independently without an obligatory alternating-Site mechanism.
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Authors
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J.Li,
M.Machius,
J.L.Chuang,
R.M.Wynn,
D.T.Chuang.
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Ref.
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J Biol Chem, 2007,
282,
11904-11913.
[DOI no: ]
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PubMed id
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Note In the PDB file this reference is
annotated as "TO BE PUBLISHED".
The citation details given above were identified by an automated
search of PubMed on title and author
names, giving a
percentage match of
96%.
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Abstract
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A long standing controversy is whether an alternating activesite mechanism
occurs during catalysis in thiamine diphosphate (ThDP)-dependent enzymes. We
address this question by investigating the ThDP-dependent
decarboxylase/dehydrogenase (E1b) component of the mitochondrial branched-chain
alpha-keto acid dehydrogenase complex (BCKDC). Our crystal structure reveals
that conformations of the two active sites in the human E1b heterotetramer
harboring the reaction intermediate are identical. Acidic residues in the core
of the E1b heterotetramer, which align with the proton-wire residues proposed to
participate in active-site communication in the related pyruvate dehydrogenase
from Bacillus stearothermophilus, are mutated. Enzyme kinetic data show that,
except in a few cases because of protein misfolding, these alterations are
largely without effect on overall activity of BCKDC, ruling out the requirement
of a proton-relay mechanism in E1b. BCKDC overall activity is nullified at 50%
phosphorylation of E1b, but it is restored to nearly half of the
pre-phosphorylation level after dissociation and reconstitution of BCKDC with
the same phosphorylated E1b. The results suggest that the abolition of overall
activity likely results from the specific geometry of the half-phosphorylated
E1b in the BCKDC assembly and not due to a disruption of the alternating
active-site mechanism. Finally, we show that a mutant E1b containing only one
functional active site exhibits half of the wild-type BCKDC activity, which
directly argues against the obligatory communication between active sites. The
above results provide evidence that the two active sites in the E1b
heterotetramer operate independently during the ThDP-dependent decarboxylation
reaction.
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Figure 1.
FIGURE 1. Identical conformation in the two active sites of
the human E1b heterotetramer. Ribbon representation of S320P
 human E1b in the
crystal form containing an entire heterotetramer in the
asymmetric unit. The N and C termini are indicated. All figures
of molecular structures were created with the program PyMol
(DeLano Scientific, San Carlos, CA).
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Figure 4.
FIGURE 4. SDS-PAGE and Western blotting analysis of
affinity-column fractions containing hybrid (S292Q /WT)
E1b. The hybrid E1b contains the S292Q mutation in the
His[6]-tagged subunit, and the
wild-type sequence in the Strep-tagged subunit was prepared,
as described under "Experimental Procedures." A, SDS-PAGE of
peak fractions from a HisTrap column; B, SDS-PAGE of peak
fractions from a consecutive Strep-Tactin Superflow column; C,
Western blotting with probes for the His[6] or Strep tag. Hyb,
hybrid E1b.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2007,
282,
11904-11913)
copyright 2007.
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Secondary reference #1
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Title
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Cross-Talk between thiamin diphosphate binding and phosphorylation loop conformation in human branched-Chain alpha-Keto acid decarboxylase/dehydrogenase.
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Authors
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J.Li,
R.M.Wynn,
M.Machius,
J.L.Chuang,
S.Karthikeyan,
D.R.Tomchick,
D.T.Chuang.
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Ref.
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J Biol Chem, 2004,
279,
32968-32978.
[DOI no: ]
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PubMed id
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Figure 3.
FIG. 3. Substitutions of residues participating in the
hydrogen-bonding network result in markedly decreased reductive
acylation (Reaction 3) activity. Invariant residues (Arg287-
,
Asp295- , Tyr300- , and
Arg301- ) that form the
hydrogen-bonding network were changed to alanine or
phenylalanine in the case of Tyr300- . Reductive acylation
of lip-LBD catalyzed by wild-type or mutant E1b was measured
with [U-14C]KIV as a substrate as described under "Experimental
Procedures." Activity for reductive acylation is expressed as
percent relative to the wild type (2.3 min-1). The nonspecific
radioactivity incorporated into nonlipoylated LBD and the
wild-type or mutant E1b protein served as a blank. Results are
averages of two independent experiments.
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Figure 7.
FIG. 7. ThDP inhibits the phosphorylation of wild-type but
not mutant E1b. A, the reaction mixture contained human apoE1b
protein, lipoylated E2b, and maltose-binding protein-tagged rat
BCKD kinase in the absence and presence of increasing ThDP
concentrations. The phosphorylation reaction was initiated by
adding 0.4 mM [  -32P]ATP and was
incubated at 25 °C for 1 min. The reaction mixtures were
separated by SDS-PAGE. 32P incorporation into the subunit
of E1b proteins was quantified by PhosphorImaging. The
PhosphorImage counts in wild-type and each mutant E1b in the
absence of ThDP was set as 100% with respect to the
corresponding E1b protein. B, PhosphorImaging of 32P
incorporation into the subunit of the S302A-
E1b
mutant and E1b double mutants containing the S302A- mutation
and a second mutation in the hydrogen-bonding network. The
phosphorylation was carried out in the absence of ThDP.
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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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Roles of active site and novel k+ ion-Binding site residues in human mitochondrial branched-Chain alpha-Ketoacid decarboxylase/dehydrogenase.
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Authors
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R.M.Wynn,
R.Ho,
J.L.Chuang,
D.T.Chuang.
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Ref.
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J Biol Chem, 2001,
276,
4168-4174.
[DOI no: ]
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PubMed id
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Figure 1.
Fig. 1. Residues in the cofactor TDP binding fold of
human BCKD. The inverted V-shaped conformation of cofactor TDP
is stabilized by stacking of the aminopyrimidine ring against
the side chain of Tyr-102-  ' from the
' subunit
(in greenish yellow) and the side chain of Leu-164-  from the
subunit
(in magenta). The invariant Glu-76- ' important
for cofactor activation coordinates to the N-1' atom of the
aminopyrimidine ring (3.4 Å apart). A ketoacid substrate
analog (in gray) labeled isocaproate is covalently modeled into
the side chain of His-146- ', based on
the crystal structure of BCKD from Pseudomonas putida (6). The
carboxylate group of the inhibitor interacts with the N-4' amino
group of TDP (separated by a distance of 4.3 Å). The side
chain of Ser-162- also
coordinates to the N-4' amino group (3.0 Å apart) to
position the cofactor in the correct conformation. Residue
Ser-292- is
phosphorylation site 1 of human BCKD. The diphosphate moiety of
TDP is stabilized, in part, by an octahedral coordination of the
Mg2+ ion. Two of the amino acid ligands Glu-193- and
Asn-222- in this
coordination are shown. Side chains of Arg-114- , Arg-220-
, and
His-291- , are, in
turn, in direct contact with the distal phosphate oxygens,
whereas the side chains of Gln-112- and
Tyr-113- (not shown)
interact with the proximal phosphate oxygens of the diphosphate
moiety of TDP.
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Figure 3.
Fig. 3. The K+ ion-binding site on the subunit of
human BCKD. The metal ion is bound by two main-chain carbonyl
groups and by the side chains of Ser-161- , Thr-166-
, and
Gln-167- . The side
chain of Leu-164- and the
main-chain carbonyl group of Ser-162- make direct
contacts with cofactor TDP. The octahedral coordination of the
metal ion stabilizes the loop structure on the subunit
(residues 161-167) that is essential for the efficient binding
of the cofactor.
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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Secondary reference #3
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Title
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A versatile conformational switch regulates reactivity in human branched-Chain alpha-Ketoacid dehydrogenase.
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Authors
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M.Machius,
R.M.Wynn,
J.L.Chuang,
J.Li,
R.Kluger,
D.Yu,
D.R.Tomchick,
C.A.Brautigam,
D.T.Chuang.
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Ref.
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Structure, 2006,
14,
287-298.
[DOI no: ]
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PubMed id
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Figure 8.
Figure 8. Scheme of hE1b-Catalyzed Reactions Emphasizing
the Role of Tyr113-a as the Central Regulatory Conformational
Switch
(A) Prior to substrate binding, the side chain of
Tyr113-a, the switch turn (indicated by Q112|Y113|R114), and the
adjacent LBD binding region are in the S conformation.
(B)
Substrate binding displaces the Tyr113-a side chain and forces
it into the P conformation, where it establishes a hydrogen bond
to the terminal phosphate group in the ThDP cofactor and
additional interactions with the sulfur in the thiazolium ring
(dashed lines). Simultaneously, the switch turn is remodeled,
whereupon the adjacent LBD binding region adopts a
higher-affinity state for LBD.
(C and D) After, or
concomitant with, (C) decarboxylation, LBD binds to hE1b,
followed by (D) the transfer of an acyl moiety, derived from the
degradation of valine, leucine, or isoleucine to lipoic acid
(LA).
TZ, thiazolium ring; AP, aminopyrimidine ring; B,
general base.
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The above figure is
reproduced from the cited reference
with permission from Cell Press
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