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

在前一年成功克隆了哺乳动物羊毛甾醇合成酶cDNA和豌豆环蒿烯醇合成酶cDNA后，我们开始尝试从人参毛状根中克隆三萜合成酶cDNA，这些三萜合成酶除了植物甾醇外，还能积累达马烷和齐烷型三萜皂苷。(1)人参毛状根氧化角鲨烯环化酶cDNA克隆。巢式pcr使用两组从已知氧化角鲨烯环化酶中发现的保守序列设计的简并寡核苷酸引物扩增预期大小的片段。利用这些片段的序列，用RACE法分离到三个独立的全长cDNA克隆，分别是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artenol合成酶，PNY的翻译产物为β -amyrin合成酶，而PNZ的功能尚不清楚，但推测其编码dammaarenediol合成酶。PNY是第一个克隆的编码三萜合成酶的cDNA。(2)鹿皮醇与β -amyrin合成酶嵌合克隆的构建及功能分析。利用数据库中已报道的序列，从拟南芥（Atabidopsis thaliana）中克隆了另一种三萜合成酶lupeol合成酶cDNA。这两个克隆有70%的序列同源性，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区域，构建了16个嵌合克隆，并对其功能进行了分析，结果表明，在n端260 ~ 340个氨基酸之间的区域决定了产物特异性。

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.