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References listed in PDB file
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Key reference
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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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Abstract
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The dehydrogenase/decarboxylase (E1b) component of the 4 MD human branched-chain
alpha-ketoacid dehydrogenase complex (BCKDC) is a thiamin diphosphate
(ThDP)-dependent enzyme. We have determined the crystal structures of E1b with
ThDP bound intermediates after decarboxylation of alpha-ketoacids. We show that
a key tyrosine residue in the E1b active site functions as a conformational
switch to reduce the reactivity of the ThDP cofactor through interactions with
its thiazolium ring. The intermediates do not assume the often-postulated
enamine state, but likely a carbanion state. The carbanion presumably
facilitates the second E1b-catalyzed reaction, involving the transfer of an acyl
moiety from the intermediate to a lipoic acid prosthetic group in the
transacylase (E2b) component of the BCKDC. The tyrosine switch further remodels
an E1b loop region to promote E1b binding to E2b. Our results illustrate the
versatility of the tyrosine switch in coordinating the catalytic events in E1b
by modulating the reactivity of reaction intermediates.
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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
reprinted
by permission from Cell Press:
Structure
(2006,
14,
287-298)
copyright 2006.
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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 his291-Alpha and his146-Beta' In the reductive acylation reaction catalyzed by human branched-Chain alpha-Ketoacid dehydrogenase: refined phosphorylation loop structure in the active site.
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Authors
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R.M.Wynn,
M.Machius,
J.L.Chuang,
J.Li,
D.R.Tomchick,
D.T.Chuang.
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Ref.
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J Biol Chem, 2003,
278,
43402-43410.
[DOI no: ]
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PubMed id
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Figure 4.
FIG. 4. ITC measurements for lip-LBD binding to wild-type,
H146A-  and H291A- human
E1b. ITC experiments were carried out in a MicroCal VP-ITC
microcalorimeter by consecutively injecting aliquots of 1.5 mM
lip-LBD or unlipoylated LBD into the reaction cell containing 25
µM wild-type or mutant human E1b. Binding isotherms for
wild-type (  ), H146A- ' ( o ),
and H291A- (  ) were obtained by
plotting heat changes against the molar ratio of lip-LBD, as
derived from the integrated raw data. The data were fit using
the ORIGIN software supplied by the manufacturer. Wild-type E1b
and the His146- ' variant show similar
affinity for lip-LBD with dissociation constants (K[d]) of 2.52
x 10^-5 M and 1.56 x 10^-5 M, respectively. The binding of the
H291A- mutant to lip-LBD
cannot be detected by ITC as indicated by the absence of heat
changes. Binding of unlipoylated LBD (  ) to wild-type E1b also
cannot be detected.
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Figure 5.
FIG. 5. Refined structure of the human E1b active site at
the interface between - and '-subunits. 2F[o] - F[c]
electron densities (in green) are contoured at 1  . Only
two histidine residues are within 5-Å distance from the C2
atom of the bound ThDP. His146- is hydrogenbonded to the
O4 water molecular, whereas His291- forms hydrogen bonds to
the O1 and O2 water molecules (in red spheres); the former in
turn coordinates to the terminal phosphate oxygen of ThDP. The
channel leading to the activated C2 atom of ThDP lies at the
interface between the - and '-subunits, such that
these two histidine residues flank opposite sides of the
channel. A Mn2+ ion is bound at the metal ion binding site in
place of the common Mg2+ ion. Good electron density is present
for Ser292- (phosphorylation site
1), which is positioned at the opening of the channel. Carbon
atoms are in gold, ThDP in green, oxygen atoms in red, nitrogen
atoms in blue, phosphorous atoms in magenta, and sulfur atoms in
yellow. Graphics were generated with the programs BobScript (24)
and PovRay (Persistence of Vision, v3.02, POV-Team,
www.povray.org).
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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Author's comment:
The crystal structure for the E1 protein shows a dimer
(one alpha plus one beta subuint); the functional unit, however,
is a tetramer (a2b2).
David Chuang
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Secondary reference #3
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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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