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PDBsum entry 2d0h
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References listed in PDB file
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Key reference
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Title
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Complexes of thermoactinomyces vulgaris r-47 alpha-Amylase 1 and pullulan model oligossacharides provide new insight into the mechanism for recognizing substrates with alpha-(1,6) glycosidic linkages.
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Authors
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A.Abe,
H.Yoshida,
T.Tonozuka,
Y.Sakano,
S.Kamitori.
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Ref.
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FEBS J, 2005,
272,
6145-6153.
[DOI no: ]
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PubMed id
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Abstract
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Thermoactinomyces vulgaris R-47 alpha-amylase 1 (TVAI) has unique hydrolyzing
activities for pullulan with sequence repeats of alpha-(1,4), alpha-(1,4), and
alpha-(1,6) glycosidic linkages, as well as for starch. TVAI mainly hydrolyzes
alpha-(1,4) glycosidic linkages to produce a panose, but it also hydrolyzes
alpha-(1,6) glycosidic linkages with a lesser efficiency. X-ray structures of
three complexes comprising an inactive mutant TVAI (D356N or D356N/E396Q) and a
pullulan model oligosaccharide (P2;
[Glc-alpha-(1,6)-Glc-alpha-(1,4)-Glc-alpha-(1,4)]2 or P5;
[Glc-alpha-(1,6)-Glc-alpha-(1,4)-Glc-alpha-(1,4)]5) were determined. The complex
D356N/P2 is a mimic of the enzyme/product complex in the main catalytic reaction
of TVAI, and a structural comparison with Aspergillus oryzaealpha-amylase showed
that the (-) subsites of TVAI are responsible for recognizing both starch and
pullulan. D356N/E396Q/P2 and D356N/E396Q/P5 provided models of the
enzyme/substrate complex recognizing the alpha-(1,6) glycosidic linkage at the
hydrolyzing site. They showed that only subsites -1 and -2 at the nonreducing
end of TVAI are effective in the hydrolysis of alpha-(1,6) glycosidic linkages,
leading to weak interactions between substrates and the enzyme. Domain N of TVAI
is a starch-binding domain acting as an anchor in the catalytic reaction of the
enzyme. In this study, additional substrates were also found to bind to domain
N, suggesting that domain N also functions as a pullulan-binding domain.
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Figure 1.
Fig. 1. Chemical structures of the repeat units of
pullulan, P2 and P5. A solid arrow indicates the main
hydrolysing site, and a dashed arrow indicates the minor site of
pullulan hydrolysed by TVAI.
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Figure 2.
Fig. 2. Overall structure of TVAI (D356N/E396Q/P5).
Domains N, A, B, and C are drawn in blue, green, yellow, and
pink, respectively. The binding substrates and Ca^2+ are shown
by a wire-style model and as orange spheres. The catalytic site,
site-N, site-NA, site-A, and site-C, are labelled. The loop
region from Ser282 to Gln284 in D356N/E396Q/P2 with poor
electron density is shown in red.
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The above figures are
reprinted
by permission from the Federation of European Biochemical Societies:
FEBS J
(2005,
272,
6145-6153)
copyright 2005.
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