Deligeorgopoulou Athina, Allemann Rudolf K
School of Chemical Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Biochemistry. 2003 Jul 1;42(25):7741-7. doi: 10.1021/bi034410m.
Sesquiterpene cyclases, many of which share significant structural similarity, catalyze the cyclization reactions of the universal alicyclic precursor farnesyl pyrophosphate to produce more than 300 different hydrocarbon skeletons with high regio- and stereospecificity. The molecular basis of this exquisite specificity is not well-understood, but the conformation adopted by FPP in the active site of a sesquiterpene cyclase is thought to be an important determinant of the reaction pathway. Aristolochene synthase (AS) from Penicillium roqueforti catalyzes the cyclization of farnesyl pyrophosphate to the bicyclic sesquiterpene aristolochene. The X-ray structure of AS suggested that the steric bulk of residue 92 was central in binding of FPP to the active site of AS in a quasi-cyclic conformation, thereby facilitating attack of C1 by the C10-C11 double bond to produce the cis-fused Decalin S-germacrene A. We demonstrate here that reduction of the size of the side chain of residue 92 leads to the production of the alicyclic sesquiterpenes (E)-beta- and (E,E)-alpha-farnesene. The relative amounts of linear products formed depended linearly on the size of the residues at position 92. ASY92A, in which Tyr92 had been replaced with Ala, produced almost 80% of alicyclic sesquiterpenes, suggesting an energetic separation of less than 0.8 kcal/mol between the cyclic and noncyclic reaction pathways. A mechanism by which FPP binds to the mutant enzymes in an extended conformation is proposed to explain the altered selectivity. The mutants also produced small amounts of additional hydrocarbons with a molecular weight of 204, namely, alpha-selinene, beta-selinene, selina-4,11-diene, (E,Z)-alpha-farnesene, and beta-bisabolene. The production of (E)-beta-farnesene and beta-bisabolene suggested that the initial cyclization of FPP to germacrene A in AS proceeded in a stepwise fashion through farnesyl cation.
倍半萜环化酶中的许多酶具有显著的结构相似性，它们催化通用脂环族前体法呢基焦磷酸的环化反应，以高区域和立体特异性生成300多种不同的烃骨架。这种精确特异性的分子基础尚未得到很好的理解，但法呢基焦磷酸在倍半萜环化酶活性位点所采取的构象被认为是反应途径的一个重要决定因素。来自罗克福特青霉的马兜铃烯合酶（AS）催化法呢基焦磷酸环化生成双环倍半萜马兜铃烯。AS的X射线结构表明，残基92的空间体积对于法呢基焦磷酸以准环状构象结合到AS的活性位点至关重要，从而促进C10 - C11双键对C1的进攻，生成顺式稠合的十氢化萘S - 吉马烯A。我们在此证明，残基92侧链大小的减小会导致脂环族倍半萜（E） - β - 和（E，E） - α - 金合欢烯的生成。形成的线性产物的相对量与92位残基的大小呈线性关系。将Tyr92替换为Ala的ASY92A产生了近80%的脂环族倍半萜，这表明环状和非环状反应途径之间的能量差小于0.8千卡/摩尔。提出了一种法呢基焦磷酸以伸展构象结合到突变酶上的机制来解释选择性的改变。这些突变体还产生了少量分子量为204的额外烃类，即α - 芹子烯、β - 芹子烯、芹子 - 4,11 - 二烯、（E，Z） - α - 金合欢烯和β - 没药烯。（E） - β - 金合欢烯和β - 没药烯的产生表明，AS中FPP最初环化生成吉马烯A是通过法呢基阳离子逐步进行的。
