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首页> 外文期刊>Biochemistry >Mechanistic insights from the binding of substrate and carbocation intermediate analogues to aristolochene synthase
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Mechanistic insights from the binding of substrate and carbocation intermediate analogues to aristolochene synthase

机译：从底物和碳正离子中间体类似物与马兜铃合酶的结合机理的见解

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摘要

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 Mg~(2+) 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 Mg~(2+) ions and inorganic pyrophosphate (PP _i). 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 Mg~(2+) _3-PP_i 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 PP_i 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.
机译：马兜铃曲霉属的金属依赖性倍半萜环化酶马兜铃合酶催化二磷酸法呢基酯（FPP）的电离依赖性环化反应，形成具有完美结构和立体化学精密度的双环艾草双环（+）-阿里斯托琴。在这里，我们报告与三个Mg〜（2+）离子和未反应的底物类似物法呢基-S-硫代二磷酸盐（FSPP）络合的马兜铃合酶的X射线晶体结构，表明底物二磷酸基团被金属配位和氢锚定键的相互作用与以前在具有三个Mg〜（2+）离子和无机焦磷酸盐（PP _i）的配合物中观察到的相互作用相同。此外，FSPP的结合构象直接模拟了生产性结合的FPP所期望的构象，除了第一步中形成C1-C10键所需的CS键相对于C10-C11π系统的精确对准外的催化作用。我们还报告了马兜铃合成酶与Mg〜（2+）_3-PP_i和为自然环化级联反应提出的双环碳正离子中间体的铵或亚胺类似物的晶体结构。对于这些双环类似物观察到各种结合取向，并且这些取向似乎是由类似物的带正电荷的铵基团和带负电荷的PP_1阴离子之间的有利的静电相互作用驱动的。令人惊讶地，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。尽管这种结合的允许性是无法预料的，​​但基于催化的立体化学精确度（仅导致（+）-aristolochene立体异构体），它表明了活性位点能够在环化级联过程中实现中间体的受控重取向。总之，这些结构阐明了催化机理的重要方面。

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