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Title
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Refined crystal structure of the catalytic domain of dihydrolipoyl transacetylase (E2p) from Azotobacter vinelandii at 2.6 A resolution.
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Authors
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A.Mattevi,
G.Obmolova,
K.H.Kalk,
A.H.Westphal,
A.de Kok,
W.G.Hol.
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Ref.
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J Mol Biol, 1993,
230,
1183-1199.
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PubMed id
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Abstract
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Dihydrolipoyl transacetylase (E2p) is both structurally and functionally the
central enzyme of the pyruvate dehydrogenase multienzyme complex. The crystal
structure of the catalytic domain, i.e. residues 382 to 637, of Azotobacter
vinelandii E2p (E2pCD) was solved by multiple isomorphous replacement and
refined by energy minimization procedures. The final model contains 2182 protein
atoms and 37 ordered water molecules. The R-factor is 18.7% for 10,344
reflections between 10.0 and 2.6 A resolution. The root-mean-square shift
deviation from the ideal values is 0.017 A for bond lengths and 3.3 degrees for
bond angles. The N-terminal residues 382 to 394 are disordered and not visible
in the electron density map, otherwise all residues have well-defined density.
The catalytic domain forms an oligomer of 24 subunits, having octahedral 432
symmetry. In the E2pCD crystals, the 24 subunits are related by the
crystallographic symmetry. The cubic arrangement of subunits gives rise to a
large hollow cube with edges of 120 A. The faces of the cube have pores of
diameter of 30 A. The true building block of the cube is the E2p trimer, eight
of which occupy the corners of the cube. Two levels of intermolecular contacts
can be distinguished: (1) the extensive interactions between 3-fold related
subunits leading to a tightly associated trimer; and (2) the interactions along
the 2-fold axis leading to the assembly of the trimers into the cubic 24-mer.
Each subunit has a topology similar to chloramphenicol acetyltransferase (CAT)
and comprises a central beta-sheet surrounded by five alpha-helices. The
comparison of the two proteins indicates a large rotation of the N-terminal
residues 395 to 426 of E2pCD, which reshapes the substrate binding site and
extends the interaction between threefold related subunits. The catalytic centre
consists of a 30 A long channel extending from the "inner" side of the trimer to
the "outer" side, where inner and outer refer to the position in the 24-meric
cubic core of the pyruvate dehydrogenase complex and correspond with CoA and
lipoamide binding sites, respectively. The active site is formed by the residues
with the lowest mobility as indicated by the atomic B-factors. Five proline
residues surround the active site.(ABSTRACT TRUNCATED AT 400 WORDS)
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