| Co-Investigator(Kenkyū-buntansha) |
SAKANO Yoshiyuki Tokyo University of Agriculture & Technology, Institute of Symbiotic Science and Technology, Professor, 大学院・共生科学技術研究部, 教授 (70014959)
YOSHIDA Hiromi Kagawa University, Information Technology Center, Associate Professor, 総合情報基盤センター, 助教授 (10313305)
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| Research Abstract |
Thermoactinomyces vulgaris R-47 produces two α-amylases, TVA1 and TVA2. TVA1 as an extracellular enzyme favors high molecular weight substrates like starch and pullulan, while TVAII as an intracellular enzyme favors low molecular weight substrates and can efficiently hydrolyze cyclodextrins, by what is called cyclodextrinase activity. The gene mapping showed that the genes of ABC transporter (CDK), cyclodextrin binding protein (CDE), transport membrane proteins (CDF, CDG), TVA2, glucoamylase (TGA), and a repressor protein (CDI) are sequentially located. Therefore, it is expected that T.vulgarius has the gene cluster encoding proteins related to the novel cyclodextrin ransport and metabolic pathway. The final goal of this proposal research project is to understand the T.vulgarius cyclodextrin ransport and metabolic pathway based on the structure information derived from protein X-ray crystallographic methods.
(1)The X-ray structures of TVA1/oligosaccharides complexes were successfully determined, showing the unique substrate recognition mechanism of TVA1 (FEBS J. 272, 6145-6153, (2005)).
(2)The X-ray structures of TVA2/cyclodexrins and oligosaccharides complexes were successfully determined, showing the multiple substrate recognition mechanism of TVA2 (J.Biol.Chem. 279, 31033-31040, (2004), J.Appl.Glycosci., 52, 225-231, (2005), Carbohydr.Res., 341, 1041-1046, (2006)).
(3)Crystallization and preliminary X-ray analysis of TGA were reported (Acta Crystallogr., F61, 302-304, (2005)).
(4)The X-ray structure of CDE/γ-CD complex at 2.4Å resolution was successfully determined. The coordinates have been deposited to Protein Data Bank (2DFZ).
(5)The overexpression of CDF, CDG and CDK were successfully done as soluble trigger-factor fused proteins, using pColdTF (Takara).
(6)Crystallization and preliminary X-ray analysis of CDI were successfully done.
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