| Project/Area Number |
13480185
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bioorganic chemistry
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| Research Institution | Tokyo Institute of Technology |
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Principal Investigator |
KAKINUMA Katsumi Tokyo Institute of Technology, Department of Chemistry, Professor, 大学院・理工学研究科, 教授 (90092543)
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| Project Period (FY) |
2001 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥15,000,000 (Direct Cost: ¥15,000,000)
Fiscal Year 2003: ¥4,500,000 (Direct Cost: ¥4,500,000)
Fiscal Year 2002: ¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 2001: ¥6,200,000 (Direct Cost: ¥6,200,000)
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| Keywords | secondary metabolites / polyketides / polyketide synthases / antitumor antibiotics / aminoglycoside antibiotics / glycosyltransferase / mechanism-based inhibitor / 2-deoxystreptamine / マクロリド / 基質特異性 / 糖質環化酵素 / ビセニスタチン / ラクタム配糖体 / イソプレノイド / 重水素化 / カナマイシン / PKS / メバロン酸 / カロテノイド |
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| Research Abstract |
Major feature of secondary metabolites is found in their diversity in chemical structure and biological activity. Molecular dissection and application studies of the biosynthetic machinery that is responsible for the diversity were pursued. The gene cluster spanning ca. 64 kbp for the biosynthesis of macrolactam polyketide glycoside vicenistatin was cloned and functionally confirmed. The most significant finding was found that the loading domain acquiring the polyketide starter is among the simplest PKSs. The glycosyltransferase VinC functioning at the final step was heterologously expressed and was applied for the preparation of various hydrophobic glycosides having the vicenisamine aminosugar. During the search of PKS genes, an additional PKS gene cluster was found and a partial chemical structure of its metabolite was proposed on the bases of homology search of the predicted ORFs. Two new related-metabolites were in fact isolated and the deduced chemical structures were completely c
… More
ompatible to the predicted structure, that clearly demonstrated significant potential of the genome-based search for new metabolites. The biosynthetic systems of the 2-deoxystreptamine-containing aminoglycoside antibiotics were also studied in detail. Genetic analyses of some Actinomycetes strains showed significant difference in the arrangement of ORFs from that of butirosin-producing Bacillus circulans. Active-site mapping was carried out for 2-deoxy-scyllo-inosose synthase (BtrC) by the use of mechanism-based inhibitor (carbaglucose phosphate) and extensive LC-MS analysis of proteolytic digests of the covalently-modified enzyme, and Lys-141 was elucidated to be a crucial base in the over-all reaction. Further, BtrS was confirmed as the doubly-functional aminotransferase for the biosynthesis of 2-deoxystreptamine, BtrM was proposed to be a glycosyntransferase giving rise to paromamine, and BtrD appeared to be a novel dTDP-glucosamine synthase. The mechanism of a diterpene cyclase derived from a Streptomyces was precisely determined by our methodology featuring pathway switch, hyperdeuterium labeling and 1H-NMR spectroscopy. Less
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