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PDBsum entry 2owc
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References listed in PDB file
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Key reference
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Title
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Three-Way stabilization of the covalent intermediate in amylomaltase, An alpha-Amylase-Like transglycosylase.
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Authors
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T.R.Barends,
J.B.Bultema,
T.Kaper,
M.J.Van der maarel,
L.Dijkhuizen,
B.W.Dijkstra.
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Ref.
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J Biol Chem, 2007,
282,
17242-17249.
[DOI no: ]
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PubMed id
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Abstract
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Amylomaltases are glycosyl hydrolases belonging to glycoside hydrolase family 77
that are capable of the synthesis of large cyclic glucans and the
disproportionation of oligosaccharides. Using protein crystallography, we have
generated a flip-book movie of the amylomaltase catalytic cycle in atomic
detail. The structures include a covalent glycosyl-enzyme intermediate, and a
covalent intermediate in complex with an analogue of a co-substrate, and show
how the structures of both enzyme and substrate respond to the changes required
by the catalytic cycle as it proceeds. Notably, the catalytic nucleophile
changes conformation dramatically during the reaction. Also, Gln256 on the 250s
loop is involved in orienting the substrate in the +1 site. The absence of a
suitable base in the covalent intermediate structure explains the low hydrolysis
activity.
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Figure 1.
FIGURE 1. Crystallographic analysis of amylomaltase
complexes. A, stereo figure of the F[o] - DF[c] electron density
for acarbose bound to Asp-293, calculated prior to incorporation
of acarbose in the model. The density was contoured at 2.5  and
overlaid on the refined structure. B, F[o] - DF[c] electron
density after refinement with a non-covalently bound acarbose.
To check the density for the covalent bond and to correctly
identify the various acarbose residues, the Asp-293 carboxylate
group as well as the acarbose 6-OH groups were omitted from the
calculations (see "Materials and Methods"). Positive difference
density is shown in green (3.5 ), negative density in
red (-3.5 ). 2mF[o] - DF[c]
density (blue, 1.0 ) (C) F[o] - DF[c]
electron density (green, 2.5 ) (D) for the covalent
intermediate-4-deoxyglucose complex. To avoid model bias, both
maps were calculated prior to the incorporation of
4-deoxyglucose in the model. All figures were produced using
PyMol (37).
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Figure 4.
FIGURE 4. Stereo images of structures representing
different states during the reaction cycle of amylomaltase. A,
empty active site at pH 5.6. The residues Asp-293, Glu-340,
Asp-395, and Gln-256 are indicated. B, acarbose (ACR) complex as
determined by Przylas et al. (22). The subsites +1 and -1 are
indicated. C, acarbose covalent intermediate-4-deoxyglucose
complex. D, covalent intermediate with acarbose. Possible
hydrolytic water molecules are indicated as spheres.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2007,
282,
17242-17249)
copyright 2007.
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