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Mechanistic Studies on Precise Substrate Recognition and Product Specificity of 2,3-Oxidosqualene Cyclases

2,3-氧化角鲨烯环化酶精确底物识别和产物特异性的机理研究

基本信息

批准号：
08458173
负责人：
EBIZUKA Yutaka
金额：
$ 4.67万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08458173/
关键词：
2,3-Oxidosqualene Cyclase Lanosterol Cycloartenol Triterpene beta-Amyrin Lupeol Chimeric Enzyme オキシドスクワレン大量発現 cDNA

项目摘要

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.

近年来，由于成功克隆了哺乳动物的羊毛甾醇合成酶cDNA和豌豆的环阿屯醇合成酶cDNA，我们尝试从人参毛状根中克隆三萜皂苷酶的cDNA，这些人参毛状根除植物甾醇外还积累达玛烷和齐墩果烷型三萜皂苷。(1)人参毛状根氧化角鲨烯环化酶的cDNA克隆使用两组简并寡核苷酸引物的巢式PCR从已知氧化角鲨烯环化酶中发现的保守序列设计，扩增预期大小的片段。利用这些片段的序列，通过RACE方法分离得到三个独立的全长cDNA克隆，即PNX、PNY和PNZ。这些cDNA在酵母中的表达确定了PNX的翻译产物为环阿屯醇合酶，PNY的翻译产物为β-香树脂素合酶，而PNZ的功能尚不清楚，尽管推测其编码达玛烯二醇合酶。PNY是第一个克隆到的编码三萜合酶的cDNA。(2)羽扇豆醇和β-香树脂醇脱氢酶嵌合克隆的构建及其功能分析。利用数据库中报道的序列，通过PCR从拟南芥中克隆了另一种三萜合酶，即羽扇豆醇合酶cDNA。这两个克隆彼此共享70%的序列同一性，表明30%氨基酸残基的差异决定了产物的特异性。为了确定产物特异性的区域，构建了16个嵌合克隆，并对其功能进行了分析，发现从N-末端起260至340个氨基酸之间的区域决定产物特异性。