Study of the cyclodextrin hydrolyzing mechanism of Thermoactinomyces vulgaris R47 α-amylases based on X-ray structures
| Project/Area Number |
14580621
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | Tokyo University Agriculture Arid Technology |
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Principal Investigator |
KAMITORI Shigehiro Tokyo University of Agriculture and Technology, Faculty of Technology, Associate Professor, 工学部, 助教授 (00262246)
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| Co-Investigator(Kenkyū-buntansha) |
SAKANO Yoshiyuki Tokyo University of Agriculture and Technology, Faculty of Agricultre, Professor, 農学部, 教授 (70014959)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2003: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2002: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | X-ray structure / α-amylase / cyclodextrin / hydrolysis / mutant enzyme / Thermoactinomyces vulgaris R47 / glucoside linkage / kinetic parameters / 放射光 |
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| Research Abstract |
Thermoactinomyces vulgaris R-47 produces two α-amylases. TVAI and TVAII. In addition to acting on starch, they have unique hydrolyzing activities for pullulan with the structure [α-(1,6)-Glc-α-(1,4)-Glc-α-(1,4)-Glc], and cyclic-oligosaccharides (cyclodextrins. CDs)which are scarcely hydrolyzed by other α-amylases. TVAI as an extracellular enzyme favors high molecular weight substrates like starch and pullulan. and can hydrolyze γ-CD (eight glucose units), but can not hydrolyze α-and β-CDs (six and seven glucose units) with a small cavity. On the other hand, TVAII as an intracellular enzyme favors low molecular weight substrates and can efficiently hydrolyze small malto-oligosaccharides including α-and β-CDs, by what is called cyclodextrinase activity. To investigate this interesting enzyme property of TVAI and TVAII. we carroed out the following studies
X-ray structures of a TVAII/acarbose complex and inactive mutant TVAII (D325N-D421N)/α-, β-and γ-CDs complexes e determined at resoluti
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ons of 2.9Å, 2.9Å, 2.8Å and 3.1Å, respectively. In all complexes, the interactions between ligands and enzymes at subsites -1 -2 and -3 were almost the same. but striking differences in the catalytic site structure were found at subsites +1 and +2. where Trp356 and Tyr374 changed the conformation of the side chain depending on the structure and size of the ligands. Trp356 and Tyr374 are thought to be responsible for the multiple substrate-recognition mechanism of TVAII. providin~ the unique substrate specificity.
The X-ray structures of complexes of TVAI with an inhibitor acarbose and an inactive mutant TVAI with malto-hexaose and malto-tridecaose have been determined at 2.6. 2.0 and 1.8Å resolutions. Besides the catalytic site. four sugar binding sites on the molecular surface are found in these X-ray structures. Two sugar binding sites in domain N hold the oligosaccharides with a regular helical structure of amylose, which suggests the domain N is a starch binding domain acting as an anchor to starch in the catalytic reaction of the enzyme. An assay of hydrolyzing activity for the raw starches confirmed that TVAI can efficiently hydrolyze raw starch.
According to X-ray structures; it is expected that Phe313 located in the active center inhibited α.-β-cyclodextrin to bind the active site. while Trp398 located at the subsite +1 in the active site play in the recognizing for starch. The replacement of Phe313 by Ala (F313A ) increased the enzymatic activity for α.-β-cyclodextrin. and the replacement of Trp398 by Ala(W398A ) and Val (W398V) decreased that for starch. The enzymatic activity of TVA2 mutant AB76, which has an active cleft of TVA I -type. changed to TVA1 -type activity. These results showed that the difference of substrate specificity between TVA I and TVA2 is related to the shape of active cleft and dimerization. Less
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Report
(3 results)
Research Products
(11 results)
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[Publications] Ohtaki, A., Iguchi, A., Mizuno, M., Tonozuka, T., Sakano, Y., Kamitori, S.: "Mutual conversion of substrate specificities of Thermoactinomyces vulgarius R-47 α-amylase TVAI and TVAII by site-directed mutagenesis"Carbohydrate Research. 338. 1553-1558 (2003)
Description
「研究成果報告書概要(和文)」より
Related Report
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[Publications] Mizuno, M., Tonozuka, T., Uechi, A., Ohtaki, A., Ichikawa, K., Kamitori, S., Nishikawa, A., Sakano, Y.: "The Crystal Structure of Thermoactinomyces vulgaris R-47 α-Amylase II (TVA II) Complexed with Transglycosylated Product"European Journal of Biochemistry. (2004/04/17受理).
Description
「研究成果報告書概要(和文)」より
Related Report
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[Publications] Mizuno.M., Tonozuka.T., Uechi.A., Ohtaki.A., Ichikawa.K., Kamitori, S., Nishikawa, A., Sakano, Y: "The Crystal Structure of The Thermoactinomyces vulgaris R-47-Amylase II (TX/A II) Complexed with Transglvcosvlated Product"European Journal of Biochemistry. (in press.). (2004)
Description
「研究成果報告書概要(欧文)」より
Related Report
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[Publications] Ohtaki, A., Iguchi, A., Mizuno, M., Tonozuka, T., Sakano, Y., Kamitori.S.: "Mutual conversion of substrate specificities of Thermoactinomyces vulgarius R-47 α-amylase TVAI and TVAII by site-directed mutagenesis"Carbohydrate Research. 338. 1553-1558 (2003)
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[Publications] Tonozuka, T., Yokota, T., Ichikawa, K., Mizino, M., Kondo, S., Nishikawa, A., Kamitori, S., Sakano, Y.: "Crystal structures and substrate specificities of two α-amylases hydrolyzing cyclodextrins and pullulan from Thermoactinomyces vulgaris R-47"Biologia Bratislava. 57,Suppl.11. 71-76 (2002)
