Evidence for differential folding of farnesyl pyrophosphate in the active site of aristolochene synthase:: A single-point mutation converts aristolochene synthase into an (E)-β-farnesene synthase
Evidence for differential folding of farnesyl pyrophosphate in the active site of aristolochene synthase:: A single-point mutation converts aristolochene synthase into an (E)-β-farnesene synthase
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DOI：
10.1021/bi034410m
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发表时间：
2003-07-01
期刊：
影响因子：
2.9
通讯作者：
Allemann, RK
中科院分区：
文献类型：
--
作者：
Deligeorgopoulou, A;Allemann, RK
倍半萜环化酶，其中许多具有显着的结构相似性，催化通用脂环族前体法呢基焦磷酸的环化反应，产生 300 多种具有高区域和立体特异性的不同烃骨架。这种精细特异性的分子基础尚不清楚，但 FPP 在倍半萜环化酶活性位点所采用的构象被认为是反应途径的重要决定因素。来自青霉的马兜铃合酶 (AS) 催化法尼基焦磷酸环化为双环倍半萜马兜铃烯。 AS的X射线结构表明，残基92的空间体积是FPP以准环构象与AS活性位点结合的中心，从而促进C10-C11双键攻击C1以产生顺式融合的Decalin S-germacrene A。我们在此证明，残基92侧链尺寸的减小导致脂环倍半萜的产生 (E)-β-和(E,E)-α-法呢烯。形成的线性产物的相对量线性依赖于第 92 位残基的大小。ASY92A（其中 Tyr92 已被 Ala 取代）产生了几乎 80% 的脂环倍半萜，表明环状和非环状反应途径之间的能量分离小于 0.8 kcal/mol。提出了 FPP 以扩展构象与突变酶结合的机制来解释选择性的改变。突变体还产生少量分子量为 204 的额外碳氢化合物，即 α-selinene、β-selinene、selina-4,11-diene、(E,Z)-α-farnesene 和 β-红没药烯。 (E)-β-法呢烯和β-红没药烯的产生表明，AS 中 FPP 向吉发烯 A 的初始环化是通过法呢基阳离子逐步进行的。
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.