从现代 (+)-Germacrene A 合酶重建 α-Humulene 合酶的进化和机制见解,Journal of the American Chemical Society

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从现代 (+)-Germacrene A 合酶重建 α-Humulene 合酶的进化和机制见解
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2014-10-02 , DOI: 10.1021/ja5066366
Veronica Gonzalez ₁ , Sabrina Touchet , Daniel J Grundy , Juan A Faraldos , Rudolf K Allemann

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来自加拿大一枝黄花的 Germacrene A 合酶 (GAS) 催化法呢基二磷酸 (FDP) 转化为植物倍半萜烯 (+)-germacrene A。二磷酸酯排出后，法呢基阳离子与远端 10,11-双键反应生成 Germacrene A ( >96%) 和 <2% α-葎草烯，这是由 FDP 的 1,11-环化产生的。通过1,10-和1,11-合酶的氨基酸序列比对以及X射线晶体结构与GAS同源模型的比较，研究了GAS 1,11-活性的起源；确定了可能导致 GAS 的主要 1,10-环化活性的三元组 [Thr 401-Gly 402-Gly 403]。用尺寸增加的残基替换 Gly 402 导致 1,11-环化逐渐增加。这些 1,10- /1 的催化稳定性，11-GAS 变体指出 Gly 402 是具有进化意义的功能转换，并表明具有严格功能的酶是通过少量替换从不太特定的祖先进化而来的。在替换同源活性位点残基 Gly 404 后，用germacrene D 合酶 (GDS) 获得了类似的结果：GDS-G404V 产生了大约 20% 双环germacrene，这是一种具有环丙烷环的烃，强调了双 1,10-/1,11 -该突变体的环化活性。这表明，到geracrenes 和humulenes 的反应途径可能通过桥接的1,10,11-碳正离子中间体或过渡态连接起来，类似于双环germacrene。使用 [1-(3)H1]-10-fluorofarnesyl diphosphate 的机理研究和使用 [12, 13-(2)H6]-FDP 支持连接 1,10-和 1,11-通路的germacrene-humulene 重排。这些结果支持现代 1,10-合酶可能从混杂的 1,11-倍半萜合酶进化而来的生物信息学提议。

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Evolutionary and Mechanistic Insights from the Reconstruction of α-Humulene Synthases from a Modern (+)-Germacrene A Synthase

Germacrene A synthase (GAS) from Solidago canadensis catalyzes the conversion of farnesyl diphosphate (FDP) to the plant sesquiterpene (+)-germacrene A. After diphosphate expulsion, farnesyl cation reacts with the distal 10,11-double bond to afford germacrene A (>96%) and <2% α-humulene, which arises from 1,11-cyclization of FDP. The origin of the 1,11-activity of GAS was investigated by amino acid sequence alignments of 1,10- and 1,11-synthases and comparisons of X-ray crystal structures with the homology model of GAS; a triad [Thr 401-Gly 402-Gly 403] that might be responsible for the predominant 1,10-cyclization activity of GAS was identified. Replacement of Gly 402 with residues of increasing size led to a progressive increase of 1,11-cyclization. The catalytic robustness of these 1,10- /1,11-GAS variants point to Gly 402 as a functional switch of evolutionary significance and suggests that enzymes with strict functionalities have evolved from less specific ancestors through a small number of substitutions. Similar results were obtained with germacrene D synthase (GDS) upon replacement of the homologous active-site residue Gly 404: GDS-G404V generated approximately 20% bicyclogermacrene, a hydrocarbon with a cyclopropane ring that underlines the dual 1,10-/1,11-cyclization activity of this mutant. This suggests that the reaction pathways to germacrenes and humulenes might be connected through a bridged 1,10,11-carbocation intermediate or transition state that resembles bicyclogermacrene. Mechanistic studies using [1-(3)H1]-10-fluorofarnesyl diphosphate and deuterium-labeling experiments with [12,13-(2)H6]-FDP support a germacrene-humulene rearrangement linking 1,10- and 1,11-pathways. These results support the bioinformatics proposal that modern 1,10-synthases could have evolved from promiscuous 1,11-sesquiterpene synthases.

更新日期：2014-10-02

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