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X-ray structures of Thermoactinomyces vulgaris R-47 α-Amylase 2 with Acarbose
and β-Cyclodextrin
Shigehiro KAMITORI*1, Akashi OHTAKI1, Masahiro MIZUNO2, Takashi TONOZUKA2 1Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan 2Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan Introduction
depending on the structure and size of the ligands. Trp356 α-Amylase (α-(1,4)-D-glucan-4-glucanohydrolase; EC and Tyr374 are thought to be responsible for the multiple 3.2.1.1) belonging to glycoside hydrolase family 13 (1), substrate-recognition mechanism of TVAII, providing the catalyzes the hydrolysis of α-(1,4)-D-glucosidic linkages unique substrate specificity. In the β-CD complex, the β- in starch to release α-anomer products. CD maintains a regular conical structure, making it Cyclomaltooligosaccharides (cyclodextrins, CDs) are difficult for Glu354 to protonate the O4 atom at the cyclic oligosaccharides with six (α-), seven (β-), or eight hydrolyzing site as a previously proposed hydrolyzing (γ-) glucose units, having a rigid conical structure. mechanism of α-amylase, as shown in Figure 1. From the Usually, α-amylases can not hydrolyze CDs, but X-ray structures, it is suggested that the protonation of the Thermoactinomyces vulgaris R-47 α-amylase 2 (TVAII, O4 atom is possibly carried out via a hydrogen atom of 585 amino acids, 67,500 Da) can efficiently hydrolyze the inter-glucose hydrogen bond at the hydrolyzing site. CDs by what is called cyclodextrinase activity (2). To obtain new insights into the CD-hydrolyzing Table 1. Refinement statistics.
mechanism of TVAII, we reported here the X-ray structures of TVAII/acarbose complex, and complexe of inactive mutant TVAII (Asp325-Asn325, Asp421- Asn421; D325N·D421N) with β-CD, at 2.9 Å and 2.8 Å, Materials and Methods
A crystal of TVAII/acarbose complex was obtained by a soaking method using the reservoir solution containing 1 mM acarbose; soaking time was 1 hour. Crystals of D325N·D421N/β-CD were also obtained by a soaking method using reservoir solution containing 20 mM of β- Figure 1. The catalytic site structure of D325N·D421N/β-CD
CD. X-ray diffraction data for TVAII/acarbose and D325N·D421N/β-CD were collected at 100K using an ADSC/CCD detector system on the BL6A beam line in the Photon Factory (Tukuba, Japan), using a reservoir solution containing 20 %(w/v) of 2-methyl-2,4-pentadiol as a cryoprotected solution. Diffraction data were processed using the program MOSFLM and SCALA in the CCP4 program suite. Initial phases were determined by a molecular replacement method using the structure of TVAII as a probe model with the program CNS. Refinement statistics are listed in Table 1. Results and Discussion
In both complexes, the interactions between ligands and enzymes at subsites -1, -2 and -3 were almost the same, but striking differences in the catalytic site References
structure were found at subsites +1 and +2, where Trp356 [1] Ohtaki, A. et al., J. Biol. Chem. (2004), in press. and Tyr374 changed the conformation of the side chain

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