Mechanistic insights from the binding of substrate and carbocation intermediate analogues to aristolochene synthase.
Mechanistic insights from the binding of substrate and carbocation intermediate analogues to aristolochene synthase.
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DOI：
10.1021/bi400691v
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发表时间：
2013-08-13
期刊：
影响因子：
2.9
通讯作者：
Christianson, David W.
中科院分区：
文献类型：
--
作者：
Chen, Mengbin;Al-lami, Naeemah;Janvier, Marine;D'Antonio, Edward L.;Faraldos, Juan A.;Cane, David E.;Allemann, Rudolf K.;Christianson, David W.
马兜铃烯合酶（Aristolochene synthase）是来源于土曲霉（Aspergillus terreus）的一种金属依赖性倍半萜环化酶，催化法呢基二磷酸（FPP）的电离依赖性环化反应，生成具有完美结构和立体化学精确性的双环艾瑞莫菲烷（+）-马兜铃烯。在这里，我们报告的X-射线晶体结构的马兜铃合成酶与三个镁离子和无反应的底物类似物法呢基-S-硫代二磷酸（FSPP），显示该底物二磷酸基团锚定金属配位和氢键相互作用与以前观察到的那些在复杂的三个镁离子和无机焦磷酸盐（PPi）。此外，FSPP的结合构象直接模拟了有效结合FPP的预期构象，除了C-S键相对于C10-C11 π系统的精确对齐，这是在催化的第一步中C1-C10键形成所需的。我们还报告了马兜铃烯合酶的晶体结构与镁2 +3-PPi和铵或亚胺类似物的双环碳阳离子中间体提出的天然环化级联。观察到这些双环类似物的各种结合方向，这些方向似乎是由带正电的铵基团的类似物和带负电的PPi阴离子之间的有利的静电相互作用驱动的。令人惊讶的是，活性位点足够灵活，以适应具有部分或完全不正确的立体化学的类似物。尽管这种结合的容许性是未预料到的，但基于催化的立体化学精确性仅导致（+）-马兜铃烯立体异构体，它表明活性位点能够在环化级联过程中实现中间体的受控重定向的能力。总之，这些结构阐明了催化机制的重要方面。
Aristolochene synthase, a metal-dependent sesquiterpene cyclase from Aspergillus terreus, catalyzes the ionization-dependent cyclization of farnesyl diphosphate (FPP) to form the bicyclic eremophilane (+)-aristolochene with perfect structural and stereochemical precision. Here, we report the X-ray crystal structure of aristolochene synthase complexed with three Mg2+ ions and the unreactive substrate analogue farnesyl-S-thiolodiphosphate (FSPP), showing that the substrate diphosphate group is anchored by metal coordination and hydrogen bond interactions identical to those previously observed in the complex with three Mg2+ ions and inorganic pyrophosphate (PPi). Moreover, the binding conformation of FSPP directly mimics that expected for productively bound FPP, with the exception of the precise alignment of the C-S bond with regard to the C10-C11 π system that would be required for C1-C10 bond formation in the first step of catalysis. We also report crystal structures of aristolochene synthase complexed with Mg2+3-PPi and ammonium or iminium analogues of bicyclic carbocation intermediates proposed for the natural cyclization cascade. Various binding orientations are observed for these bicyclic analogues, and these orientations appear to be driven by favorable electrostatic interactions between the positively charged ammonium group of the analogue and the negatively charged PPi anion. Surprisingly, the active site is sufficiently flexible to accommodate analogues with partially or completely incorrect stereochemistry. Although this permissiveness in binding is unanticipated, based on the stereochemical precision of catalysis that leads exclusively to the (+)-aristolochene stereoisomer, it suggests the ability of the active site to enable controlled reorientation of intermediates during the cyclization cascade. Taken together, these structures illuminate important aspects of the catalytic mechanism.
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DOI：
10.1351/pac-con-09-09-37
发表时间：
2010
期刊：
Pure and applied chemistry. Chimie pure et appliquee
影响因子：
--
作者：
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通讯作者：
Christianson DW
DOI：
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影响因子：
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通讯作者：
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影响因子：
15
作者：
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通讯作者：
Allemann, RK
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
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DOI：
10.1107/s0907444909042073
发表时间：
2010-01
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
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作者：
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通讯作者：
Richardson DC