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

在2001年至2002年的研究期间，我们对角鲨烯-Hopene环化酶进行了以下几个方面的研究：(1)针对Tyr495、Tyr612和Tyr609的定点突变实验表明，这些氨基酸的作用归因于Asp377和Phe365在阳离子-π相互作用中的作用增强。这一发现表明，关键氨基酸的空间体积大小干扰了底物的折叠。(3)我们合成了一个乙基位于10位的2，3-氧化橙烯类似物。该类似物被羊毛甾醇合成酶环合，得到具有三甲基环己烷部分的异常环化产物，该环化产物通过船型折叠结构产生，由3R-氧化月桂烯催化产生。羊毛甾醇合成酶对3S-氧化橙烯有严格的专一性，对3R-型具惰性。本研究对羊毛甾醇合成酶对底物的识别有了更深入的了解。(4)我们还合成了C(10)降角鲨烯，并通过角鲨烯-Hopene环化酶进行了酶促反应。该反应得到了新的碳环骨架(S)，即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)比较角鲨烯环化酶和羊毛甾醇环化酶的氨基酸序列，鼓励删除角鲨烯环化酶的Gly600。这个缺失的突变体是通过聚合酶链式反应方法产生的。角鲨烯环化酶催化3种底物，即角鲨烯(原始底物)、3S-和3R-氧化角叉烯。然而，该突变体只对3S-氧化并喹烯有活性。这种行为类似于羊毛甾醇合成酶。因此，我们已经成功地改变了底物的特异性。

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.