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PDBsum entry 2hhf
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References listed in PDB file
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Key reference
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Title
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Human mitochondrial branched chain aminotransferase isozyme: structural role of the cxxc center in catalysis.
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Authors
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N.H.Yennawar,
M.M.Islam,
M.Conway,
R.Wallin,
S.M.Hutson.
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Ref.
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J Biol Chem, 2006,
281,
39660-39671.
[DOI no: ]
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PubMed id
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Abstract
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Mammalian branched chain aminotransferases (BCATs) have a unique CXXC center.
Kinetic and structural studies of three CXXC center mutants (C315A, C318A, and
C315A/C318A) of human mitochondrial (hBCATm) isozyme and the oxidized hBCATm
enzyme (hBCATm-Ox) have been used to elucidate the role of this center in hBCATm
catalysis. X-ray crystallography revealed that the CXXC motif, through its
network of hydrogen bonds, plays a crucial role in orienting the substrate
optimally for catalysis. In all structures, there were changes in the structure
of the beta-turn preceding the CXXC motif when compared with wild type protein.
The N-terminal loop between residues 15 and 32 is flexible in the oxidized and
mutant enzymes, the disorder greater in the oxidized protein. Disordering of the
N-terminal loop disrupts the integrity of the side chain binding pocket,
particularly for the branched chain side chain, less so for the dicarboxylate
substrate side chain. The kinetic studies of the mutant and oxidized enzymes
support the structural analysis. The kinetic results showed that the predominant
effect of oxidation was on the second half-reaction rather than the first
half-reaction. The oxidized enzyme was completely inactive, whereas the mutants
showed limited activity. Model building of the second half-reaction substrate
alpha-ketoisocaproate in the pyridoxamine 5'-phosphate-hBCATm structure suggests
that disruption of the CXXC center results in altered substrate orientation and
deprotonation of the amino group of pyridoxamine 5'-phosphate, which inhibits
catalysis.
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Figure 1.
FIGURE 1. C XX C center of oxidized and mutant hBCATm
proteins. A, oxidized WT-hBCATm; B, C315A hBCATm; C, C315A/C318A
double mutant hBCATm. In the reduced WT protein, the sulfurs of
Cys^315 and Cys^318 form a thiol-thiolate hydrogen bond. Upon
mutation or oxidation, the thiol-thiolate interaction does not
exist. Instead, the Cys^315 and Cys^318 sulfurs form a disulfide
bridge.
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Figure 4.
FIGURE 4. Active site structures of C315A mutant hBCATm in
complex with N-methylleucine (C315A-NML). A and B show the 2F[o]
- F[c] difference Fourier electron density maps of the active
site in monomer A and monomer B, respectively. C, the
superimposed model of the N-methylleucine-bound WT-hBCATm
structure and structure of N-methylleucine-bound C315A mutant
hBCATm. The carbon skeletons are in khaki in WT-hBCATm and in
green in the single mutant C315A-NML structures.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2006,
281,
39660-39671)
copyright 2006.
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Secondary reference #1
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Title
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Overexpression and characterization of the human mitochondrial and cytosolic branched-Chain aminotransferases.
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Authors
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J.Davoodi,
P.M.Drown,
R.K.Bledsoe,
R.Wallin,
G.D.Reinhart,
S.M.Hutson.
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Ref.
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J Biol Chem, 1998,
273,
4982-4989.
[DOI no: ]
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PubMed id
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Figure 2.
Fig. 2. SDS-PAGE of purified human BCATm and BCATc. 3-5
µg of protein were loaded onto the gels, and molecular
mass standards^ are indicated. Panel A shows BCATm after
cleavage of the histidine^ tag by thrombin (41.7 kDa). Panel B
shows BCATc expressed using^ the pTrcHis vector (44.3 kDa), and
panel C shows BCATc expressed^ using the pET-28a vector
following removal of the histidine tag^ (43.4 kDa).
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Figure 4.
Fig. 4. Circular dichroism spectra of BCATc and BCATm in
the PLP and PMP forms. Spectra from the far UV range are shown
in^ panel A, and spectra from the near UV range are shown in
panel^ B. Conditions were as described under "Materials and
Methods."^ Lines used are the same as in Fig.
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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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Cloning of the rat and human mitochondrial branched chain aminotransferases (bcatm).
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Authors
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R.K.Bledsoe,
P.A.Dawson,
S.M.Hutson.
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Ref.
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Biochim Biophys Acta, 1997,
1339,
9.
[DOI no: ]
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PubMed id
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