| Project/Area Number |
13306009
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bioproduction chemistry/Bioorganic chemistry
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| Research Institution | Yamagata University |
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Principal Investigator |
SASSA Takeshi Yamagata University, Dept.of Bioresource Engineering, Professor, 農学部, 教授 (80023456)
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| Co-Investigator(Kenkyū-buntansha) |
OIKAWA Hideaki Hokkaido University, Division of Chemistry, Professor, 大学院・理学研究科, 教授 (00185175)
KATO Nobuo Osaka University, The Institute of Scientific and Industrial Research, Professor, 産業科学研究所, 教授 (50150537)
HOSHINO Tsutomu Niigata University, Dept.of Applied Biological Chemistry, Professor, 農学部, 教授 (30165542)
TOYOMASU Tomonobu Yamagata University, Assistant Prof., 農学部, 助教授 (60272085)
DAIRI Tohru Toyama Prefectural University, the Biotechnology Research Center, Assistant Prof., 生物工学研究センター, 助教授 (70264679)
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| Project Period (FY) |
2001 – 2003
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| Keywords | diterpene / terpene cyclase / biosynthesis / cyclization mechanism / biosynthetic intermediate / tetraterpene / squarene |
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| Research Abstract |
(1)We isolated (+)-cyatha-3,12-diene from an erinacine-producing fungus, and confirmed its stereostructure by semi-synthesis from erinacine P. The cyathane hydrocarbon could be obtained from GGDP by the cell-free system. From a fusicoccin-mass-producing fungus we isolated (+)-δ-araneosene as the presumed bicyclic hydrocarbon intermediate. (+)-Fusicocca-3(16),10(14)-diene was also isolated and its identification suggested the presence of a new fusicoccenyl cation in the biosynthetic pathway. (2)We identified two gene clusters responsible for diterpene biosynthesis by chromosome waling from GGDP synthase genes from Phomopsis amygdali. The gene clusters led us to successful isolation and characterization of two cDNAs encoding diterpene cyclases. (3)The gene cluster for aphidicolin biosynthesis has been identified by PCR-based genome walking using reported sequence of cDNA coding the diterpene cyclase. Based on non-enzymatic cyclization under various conditions and molecular orbital calculations, cyclization mechanism for construction of aphidicolane skeleton was proposed. (4)We have cloned two diterpene cyclase genes essential for biosynthesis of terpentecin. By using recombinant enzymes, it was revealed that one enzyme converted GGDP into a cyclized intermediate (terpentedienol diphosphate), and then it was transformed into terpentetriene by the other enzyme. (5)By using the site-specific mutants and the substrate analogs, we clearly demonstrated that the steric bulk size of active site residues has an important role in determining the folding conformation of squalene substrate during the polycyclization cascade.
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