The sesquiterpene cyclase epi-isozizaene synthase (EIZS) from Streptomyces coelicolor catalyzes the metal-dependent conversion of farnesyl diphosphate (FPP) into the complex tricyclic product epi-isozizaene. This remarkable transformation is governed by an active site contour that serves as a template for catalysis, directing the conformations of multiple carbocation intermediates leading to the final product. Mutagenesis of residues defining the active site contour remolds its three-dimensional shape and reprograms the cyclization cascade to generate alternative cyclization products. In some cases, mutagenesis enables alternative chemistry to quench carbocation intermediates, e.g., through hydroxylation. Here, we combine structural and biochemical data from previously characterized EIZS mutants to design and prepare F95S–F198S EIZS, which converts EIZS into an α-bisabolol synthase with moderate fidelity (65% at 18 °C, 74% at 4 °C). We report the complete biochemical characterization of this double mutant as well as the 1.47 Å resolution X-ray crystal structure of its complex with three Mg²⁺ ions, inorganic pyrophosphate, and the benzyltriethylammonium cation, which partially mimics a carbocation intermediate. Most notably, the two mutations together create an active site contour that stabilizes the bisabolyl carbocation intermediate and positions a water molecule for the hydroxylation reaction. Structural comparison with a naturally occurring α-bisabolol synthase reveals common active site features that direct α-bisabolol generation. In showing that EIZS can be redesigned to generate a sesquiterpene alcohol product instead of a sesquiterpene hydrocarbon product, we have expanded the potential of EIZS as a platform for the development of designer cyclases that could be utilized in synthetic biology applications.

中文翻译：

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基于结构的倍半萜环化酶生成酒精产品：表异异嗪合酶转化为 α-红没药醇合酶

来自天蓝色链霉菌的倍半萜环化酶表异异烯合酶 (EIZS)催化法呢基二磷酸 (FPP) 依赖金属的转化为复杂的三环产物表异异烯。这种显着的转变是由活性位点轮廓控制的，该轮廓充当催化模板，指导多种碳正离子中间体的构象形成最终产品。定义活性位点轮廓的残基的诱变重塑其三维形状并重新编程环化级联以产生替代的环化产物。在某些情况下，诱变使得替代化学能够淬灭碳阳离子中间体，例如通过羟基化。在这里，我们结合先前表征的 EIZS 突变体的结构和生化数据来设计和制备 F95S–F198S EIZS，它将 EIZS 转化为具有中等保真度的 α-红没药醇合酶（18 °C 时为 65%，4 °C 时为 74%）。我们报告了这种双突变体的完整生化特征，以及其与三个 Mg ²⁺离子、无机焦磷酸盐和部分模拟碳阳离子中间体的苄基三乙基铵阳离子复合物的 1.47 Å 分辨率 X 射线晶体结构。最值得注意的是，这两个突变共同创建了一个活性位点轮廓，可稳定红没药基碳阳离子中间体并为羟基化反应定位水分子。与天然存在的 α-红没药醇合酶的结构比较揭示了指导 α-红没药醇生成的常见活性位点特征。在证明 EIZS 可以重新设计以生成倍半萜醇产品而不是倍半萜烃产品时，我们扩展了 EIZS 作为开发可用于合成生物学应用的设计环化酶的平台的潜力。