1997 Fiscal Year Final Research Report Summary
Mechanistic Studies on Precise Substrate Recognition and Product Specificity of 2,3-Oxidosqualene Cyclases
| Project/Area Number |
08458173
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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 | The University of Tokyo |
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Principal Investigator |
EBIZUKA Yutaka The University of Tokyo, Graduate School of Pharmaceutical Sciences, Professor, 大学院・薬学系研究科, 教授 (90107384)
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| Project Period (FY) |
1996 – 1997
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| Keywords | 2,3-Oxidosqualene / Cyclase / Lanosterol / Cycloartenol / Triterpene / beta-Amyrin / Lupeol / Chimeric Enzyme |
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| Research Abstract |
The successful cloning of lanosterol synthase cDNA from mammals and cycloartenol synthase cDNA from pea in the previous year led us to try cDNA clonig of triterpene synthases from Panax ginseng hairy roots which accumulate dammarane and oleanane type triterpene saponins in addition to phytosterols. (1) cDNA cloning of oxidosqualene cyclases from Panax ginseng hairy roots. Nested PCRs using two sets of degenerate oligonucleotide primers designed from the conserved sequence found in the known oxidosqualene cyclases amplified fragments of expected size. By utilizing the sequence of these fragments, three independent full length cDNA clones, namely PNX,PNY and PNZ,were isolated by RACE method. Expression of these cDNA in yeast established the translation product of PNX to be cycloartenol synthase and that of PNY to be beta-amyrin synthase while the function of PNZ remained unclear although it is presumed to encode dammarenediol synthase. PNY is the first cDNA ever cloned which encodes triterpene synthase. (2) Construction of chimeric clones of lupeol and beta-amyrin synthases and analysis of their functions. Another triterpene synthase, namely lupeol synthase, cDNA was cloned from Atabidopsis thaliana by PCR using the sequence reported in the data base. These two clones share 70% sequence identity each other indicating the difference of 30% amino acid residue determins the product specificity. In order to identify the region responsible for the product specificity, 16 chimeric clones were constructed and analyzed for their functions to reveal that the region between 260 to 340 amino acid from N-terminus is determing the product specificity.
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