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PDBsum entry 1vb9
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References listed in PDB file
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Key reference
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Title
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The crystal structure of thermoactinomyces vulgaris r-47 alpha-Amylase ii (tva ii) complexed with transglycosylated product.
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Authors
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M.Mizuno,
T.Tonozuka,
A.Uechi,
A.Ohtaki,
K.Ichikawa,
S.Kamitori,
A.Nishikawa,
Y.Sakano.
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Ref.
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Eur J Biochem, 2004,
271,
2530-2538.
[DOI no: ]
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PubMed id
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Abstract
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Alphan alpha-amylase (TVA II) from Thermoactinomyces vulgaris R-47 efficiently
hydrolyzes alpha-1,4-glucosidic linkages of pullulan to produce panose in
addition to hydrolyzing starch. TVA II also hydrolyzes alpha-1,4-glucosidic
linkages of cyclodextrins and alpha-1,6-glucosidic linkages of isopanose. To
clarify the basis for this wide substrate specificity of TVA II, we soaked
4(3)-alpha-panosylpanose (4(3)-P2) (a pullulan hydrolysate composed of two
panosyl units) into crystals of D325N inactive mutated TVA II. We then
determined the crystal structure of TVA II complexed with
4(2)-alpha-panosylpanose (4(2)-P2), which was produced by transglycosylation
from 4(3)-P2, at 2.2-A resolution. The shape of the active cleft of TVA II is
unique among those of alpha-amylase family enzymes due to a loop (residues
193-218) that is located at the end of the cleft around the nonreducing region
and forms a 'dam'-like bank. Because this loop is short in TVA II, the active
cleft is wide and shallow around the nonreducing region. It is assumed that this
short loop is one of the reasons for the wide substrate specificity of TVA II.
While Trp356 is involved in the binding of Glc +2 of the substrate, it appears
that Tyr374 in proximity to Trp356 plays two roles: one is fixing the
orientation of Trp356 in the substrate-liganded state and the other is supplying
the water that is necessary for substrate hydrolysis.
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Figure 3.
Fig. 3. Stereo-view of the active site with 4^2-P2.(A) The
whole shape of the active cleft formed collaboratively with
domain N of MOL-2 (green surface model) is shown in the
molecular surface model. The surface model was produced using
PYMOL (http://www.pymol.org). (B) Unliganded TVA II (green)
superimposed into the complex structure (magenta) around the
nonreducing region. 4^2-P2, separated between –1 and +1, is
displayed as dark gray sticks. The residues with asterisks are
located in domain N of the MOL-2 molecule. (C) Reducing region.
The explanation is the same as for (B).
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Figure 4.
Fig. 4. Schematic drawing of the interactions of 4^2-P2
bound to the active site. Hydrogen bonds of less than 3.5
Å are shown as dashed lines. Water molecules are shown as
spheres. The residues with asterisks are located in domain N of
the MOL-2 molecule. Three catalytic residues, except for Asn325,
which is aspartic acid in native TVA II, are surrounded by an
elliptical box.
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The above figures are
reprinted
by permission from the Federation of European Biochemical Societies:
Eur J Biochem
(2004,
271,
2530-2538)
copyright 2004.
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