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Creation of unnatural natural products by the chimera type cyclase of squalene and oxidosqualene cyclases

通过角鲨烯和氧化角鲨烯环化酶的嵌合型环化酶产生非天然天然产物

基本信息

批准号：
13660105
负责人：
HOSHINO Tsutomu
金额：
$ 2.56万
依托单位：
NIIGATA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

During the research period (2001-2002), we got some significant achievements for squalene hopene cyclase as follows.(1) Site-directed mutagenesis experiments targeted for Tyr495, Tyr612 and Tyr609 showed that the role of these amino acids was ascribed to the reinforcement of the role of Asp377 and Phe365 which works for the cation-π interaction.(2) Alteration of the bulk size at 420 and 607 into the larger amino acids allowed the creation of unnatural natural product, named neoachillapentaene, which was produced by the boat structure during the cyclization process. This finding indicates that the steric bulk size of crucial amino acids perturbs the substrate folding.(3) We synthesized a 2,3-oxidosqualene analogue which have ethyl group at 10-position. This analog was cyclized by lanosterol synthase to give the abnormal cyclization products with trimethylcyclohexane moiety, which produced through the boat-folding structure and produced by the catalysis of 3R-oxidosqualene. Lanosterol synthase is rigorously specific to 3S-oxidosqualene and inert to the 3R-form. This investigation gave a deeper insight into the substrate recognition by lanosterol synthase.(4) We also synthesized C(10) norsqualene, which was subjected to the enzymic reaction by squalene-hopene cyclase. This enzymic reaction afforded novel carbocyclic skeleton(s), i. e. 6/5+5/5+(6). This finding also indicated that the bulk size of the substrate also significantly influence the cyclization pathway.(5) Comparison of amino acid alignment between squalene and lanosterol cyclases encouraged to delete Gly600 of the squalene cyclase. This deleted mutant was created by the PCR method. Squalene cyclase catalyzes 3 substrates, i. e. squalene (original substrate), 3S- and 3R-oxidosquaene. However, this mutant was only active to 3S-oxidosquaene. This behavior was analogous to lanosterol synthase. Thus, we have succeed in altering the substrate specificity.

在2001-2002年的研究期间，我们对角鲨烯环化酶的研究取得了如下重要成果。(1)针对Tyr 495、Tyr 612和Tyr 609的定点突变实验表明，这些氨基酸的作用归因于Asp 377和Phe 365的作用的增强，Asp 377和Phe 365对阳离子-π相互作用起作用。(2)将420和607的体积大小改变为较大的氨基酸允许产生非天然的天然产物，称为neoachillapentaene，其在环化过程中由船结构产生。这一发现表明，空间体积大小的关键氨基酸扰动底物折叠。(3)我们合成了一种10位上带有乙基的2，3-氧化角鲨烯类似物。该类似物经羊毛甾醇合成酶环化后，在3R-氧化角鲨烯的催化下，通过船式折叠结构生成了含三甲基环己烷的异常环化产物。羊毛甾醇合酶对3S-氧化角鲨烯具有严格的特异性，对3R-形式呈惰性。本研究对羊毛甾醇合酶的底物识别有了更深入的了解。(4)我们还合成了C（10）去甲角鲨烯，它经过角鲨烯-霍烯环化酶的酶促反应。该酶促反应得到了新的碳环骨架，即：e. 6/5+5/5+（6）。这一发现还表明，底物的体积大小也显著影响环化途径。(5)角鲨烯和羊毛甾醇环化酶之间的氨基酸比对的比较鼓励删除角鲨烯环化酶的Gly 600。该缺失突变体通过PCR方法产生。角鲨烯环化酶催化3种底物，即：e.角鲨烯（原始底物）、3S-和3R-氧化角鲨烯。然而，该突变体仅对3S-氧化鲨烯具有活性。这种行为类似于羊毛甾醇合成酶。因此，我们成功地改变了底物特异性。