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PDBsum entry 1dm3
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Crystallographic analysis of the reaction pathway of zoogloea ramigera biosynthetic thiolase.
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Authors
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Y.Modis,
R.K.Wierenga.
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Ref.
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J Mol Biol, 2000,
297,
1171-1182.
[DOI no: ]
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PubMed id
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Abstract
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Biosynthetic thiolases catalyze the biological Claisen condensation of two
acetyl-CoA molecules to form acetoacetyl-CoA. This is one of the fundamental
categories of carbon skeletal assembly patterns in biological systems and is the
first step in many biosynthetic pathways including those which generate
cholesterol, steroid hormones and ketone body energy storage molecules. High
resolution crystal structures of the tetrameric biosynthetic thiolase from
Zoogloea ramigera were determined (i) in the absence of active site ligands,
(ii) in the presence of CoA, and (iii) from protein crystals which were flash
frozen after a short soak with acetyl-CoA, the enzyme's substrate in the
biosynthetic reaction. In the latter structure, a reaction intermediate was
trapped: the enzyme was found to be acetylated at Cys89 and a molecule of
acetyl-CoA was bound in the active site pocket. A comparison of the three new
structures and the two previously published thiolase structures reveals that
small adjustments in the conformation of the acetylated Cys89 side-chain allow
CoA and acetyl-CoA to adopt identical modes of binding. The proximity of the
acetyl moiety of acetyl-CoA to the sulfur atom of Cys378 supports the hypothesis
that Cys378 is important for proton exchange in both steps of the reaction. The
thioester oxygen atom of the acetylated enzyme points into an oxyanion hole
formed by the nitrogen atoms of Cys89 and Gly380, thus facilitating the
condensation reaction. The interaction between the thioester oxygen atom of
acetyl-CoA and His348 assists the condensation step of catalysis by stabilizing
a negative charge on the thioester oxygen atom. Our structure of acetyl-CoA
bound to thiolase also highlights the importance in catalysis of a hydrogen
bonding network between Cys89 and Cys378, which includes the thioester oxygen
atom of acetyl-CoA, and extends from the catalytic site through the enzyme to
the opposite molecular surface. This hydrogen bonding network is different in
yeast degradative thiolase, indicating that the catalytic properties of each
enzyme may be modulated by differences in their hydrogen bonding networks.
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Figure 2.
Figure 2. Stereo picture of the biosynthetic thiolase
tetramer with its "tetrahedral cage" tetramerization motif. The
tetramer is colored according to the B-factor of the C^a atom of
each residue. Dark blue corresponds to a B-factor of  vert,
similar 5 Å2; red corresponds to 100 Å2. The A-B
dimer (upper half of the Figure) has significantly lower
B-factors than the C-D dimer (lower half). This is a result of
the layered packing in the crystal lattice [Modis and Wierenga
1999]. Four molecules of acetyl-CoA are shown in ball-and-stick
representation, bound to each of the four subunits near the
interface between the A-B and C-D dimers. The side-chains of
residues located in high B-factor loops and pointing towards the
CoA moiety are also shown. These include residues Lys133,
Arg172, Lys208 and Arg232-Pro233-Ala234-Phe235-Asp236-Lys237,
which are all located in the loop domain. Figure 2, Figure 5 and
Figure 6 were prepared with MOLSCRIPT [Kraulis 1991] and
Raster3D [Merritt and Bacon 1997].
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Figure 7.
Figure 7. Schematic drawing of the hydrogen bonding network
extending from the acitve site of biosynthetic thiolase, through
the enzyme to the molecular surface opposite the active site
pocket. Water molecules are shown as gray circles.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2000,
297,
1171-1182)
copyright 2000.
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Secondary reference #1
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Title
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A biosynthetic thiolase in complex with a reaction intermediate: the crystal structure provides new insights into the catalytic mechanism.
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Authors
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Y.Modis,
R.K.Wierenga.
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Ref.
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Structure, 1999,
7,
1279-1290.
[DOI no: ]
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PubMed id
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Figure 4.
Figure 4. Contact distances between the CoA molecule and
subunit B of biosynthetic thiolase. The catalytic residues
Cys89, His348 and Cys378 are also shown. Water molecules are
represented as gray circles. There is only one direct hydrogen
bond between the protein and the CoA molecule, between Ser247
and the NP2 atom of CoA. Most other contacts are mediated by one
or more water molecules. Distances are given in Ångstroms.
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The above figure is
reproduced from the cited reference
with permission from Cell Press
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Secondary reference #2
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Title
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Mechanistic studies on beta-Ketoacyl thiolase from zoogloea ramigera: identification of the active-Site nucleophile as cys89, Its mutation to ser89, And kinetic and thermodynamic characterization of wild-Type and mutant enzymes.
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Authors
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S.Thompson,
F.Mayerl,
O.P.Peoples,
S.Masamune,
A.J.Sinskey,
C.T.Walsh.
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Ref.
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Biochemistry, 1989,
28,
5735-5742.
[DOI no: ]
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PubMed id
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