Studies on the Hydrolyzing Mechanism for Cyclodextrins of Thermoactinomyces vulgaris R-47 .ALPHA.-Amylase 2(TVAII). X-Ray Structure of the Mutant E354A Complexed with .BETA.-Cyclodextrin, and Kinetic Analyses on Cyclodextrins.
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概要
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Crystals of the mutant E354A of Thermoactinomyces vulgaris R-47 α-amylase 2 (TVAII) complexed with β-cyclodextrin were prepared by a soaking method, and the diffraction data were collected at 100 K, using Synchrotron radiation (SPring-8). The crystals belong to an orthorhombic system with the space group P212121 and cell dimensions a=111.1 Å, b=117.7 Å, c=113.3 Å, which is almost isomorphous with crystals of the wildtype TVAII, and the structure was refined to an R-factor=0.208 (Rfree=0.252) using 3.0 Å resolution data. The refined structure shows that the interactions between Phe286 and two C6 atoms of β-cyclodextrin at the hydrolyzing site are important for TVAII to recognize cyclodextrins as substrates. This observation from the X-ray structure was supported by kinetic analyses of cyclodextrins using the wild-type TVAII, the mutant F286A and F286L. These studies also suggested that the TVAII-hydrolyzing mechanism for cyclodextrins is slightly different from that for starch.
- 社団法人 日本生化学会の論文
著者
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KAMITORI Shigehiro
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology
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KONDO Shin
Department of Biotechnology & Life Science, Tokyo University of Agriculture & Technology
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OHTAKI Akashi
Department of Biotechnology & Life Science, Tokyo University of Agriculture & Technology
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Tonozuka Takashi
Department Of Applied Biological Science Faculty Of Agriculture Tokyo University Of Agriculture And
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SAKANO Yoshiyuki
Department of Agricultural Chemistry, Faculty of Agriculture, Tokyo Noko University
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Sakano Yoshiyuki
Department of Applied Biological Science, Tokyo University ofAgriculture & Technology
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