Terpene synthases (TPSs) play a vital role in forming the complex hydrocarbon backbones that underlie terpenoid diversity. Notably, some TPSs can add water prior to terminating the catalyzed reaction, leading to hydroxyl groups, which are critical for biological activity. A particularly intriguing example of this is the maize (Zea mays) sesquiterpene TPS whose major product is eudesmanediol, ZmEDS. This production of dual hydroxyl groups is presumably enabled by protonation of the singly hydroxylated transient stable intermediate hedycaryol. To probe the enzymatic structure–function relationships underlying this unusual reaction, protein modeling and docking were used to direct mutagenesis of ZmEDS. Previously, an F303A mutant was shown to produce only hedycaryol, suggesting a role in protonation. Here this is shown to be dependent on the steric bulk positioning of hedycaryol, including a supporting role played by the nearby F299, rather than π-cation interaction. Among the additional residues investigated here, G411 at the conserved kink in helix G is of particular interest, as substitution of this leads to predominant production of the distinct (−)-valerianol, while substitution for the aliphatic I279 and V306 can lead to significant production of the alternative eudesmane-type diols 2,3-epi-cryptomeridiol and 3-epi-cryptomeridol, respectively. Altogether, nine residues that are important for this unusual reaction were investigated here, with the results not only emphasizing the importance of reactant positioning suggested by the stereospecificity observed among the various product types but also highlighting the potential role of the Mg²⁺–diphosphate complex as the general acid for the protonation-initiated (bi)cyclization of hedycaryol.

中文翻译：

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探索在二羟基化倍半萜烯合酶ZmEDS中的酶结构和功能。

萜烯合酶（TPS）在形成构成萜类多样性基础的复杂烃骨架中起着至关重要的作用。值得注意的是，某些TPS可以在终止催化反应之前加水，形成羟基，这对于生物活性至关重要。玉米（Zea mays）是一个特别有趣的例子。）倍半萜烯TPS，其主要产品为大马士革二醇，ZmEDS。双羟基的这种产生大概是通过单个羟基化的瞬态稳定的中间水杨醇的质子化实现的。为了探测这种异常反应背后的酶结构与功能之间的关系，蛋白质建模和对接用于指导ZmEDS的诱变。以前，显示F303A突变体仅产生Hedycaryol，暗示在质子化中起作用。此处显示这取决于苏糖精的空间体积位置，包括附近F299发挥的辅助作用，而不是π-阳离子相互作用。在这里研究的其他残基中，螺旋G的保守纽结处的G411特别令人关注，因为对其进行取代会导致主要生成不同的（-）-戊醇，表皮-隐二醇和3-表皮-隐烯醇。总共研究了9个对这种异常反应很重要的残基，其结果不仅强调了在各种产品类型中观察到的立体特异性所暗示的反应物定位的重要性，而且还强调了Mg 2 ^+-二磷酸络合物的潜在作用。作为泛醇质子化引发（双）环化的一般酸。