Engineering Water Capture in Terpene Synthases

酶是一种非凡的生物分子，它能够在生命系统中发现的温和条件下以惊人的精度进行复杂的化学反应。萜烯合成酶是一种酶，它催化少量底物转化为无数种不同的碳氢化合物产物，称为萜类化合物，这种结构构成了最大的天然产物群的化学基础。许多萜类化合物都有有益的应用，例如抗癌药物紫杉醇、抗疟疾药物青蒿素和可以作为驱虫剂的所谓的半化学物质。这些是基于倍半萜烯，萜类化合物家族的一个子集，含有15个碳原子，来自同一分子，法尼酯二磷酸（FDP）。天然倍半萜合成酶可以从FDP中生成300多种不同的碳氢化合物支架。这些支架是否含有水（通过羟基化或“水捕获”）会影响它们的生物活性和使用。在过去几十年中进行的结构和机制研究表明，这些酶具有三维折叠，并且使用类似的化学策略来实现FDP转化为产品。虽然在破译生化细节方面已经取得了很大进展，但我们的知识还不够完整，无法预测，因此可以使用合理的方法以有针对性的方式转换萜烯合酶的“水捕获”行为。拟议的研究是基于先前工作的坚实基础和申请人的互补经验，将汇集酶学家，计算生物化学家和结构生物学家，以深入了解萜烯合酶中“水捕获”的机制。这种理解将被用来合理地改变示例酶的“水捕获”行为，这将导致具有潜在有益特性的新型萜类产品。最先进的计算和实验工作的结合对于实现这一目标至关重要。通过使用实验数据，例如通过x射线晶体学获得的结构，计算模型提供了通过实验室实验无法获得的独特细节，从而可用于预测氨基酸变化的影响。这些预测随后需要实验验证。通过这种结合的方法，我们将非常详细地发现酶的结构和流动性如何影响反应结果。一旦成功地改变了水捕获行为，我们将开发一般的、流线型的方案，可应用于其他萜烯合成酶。因此，这些方案将允许对有效的生物催化剂进行合理修饰，以获得具有所需性能的特定萜类产品，例如开发新药或驱蚊剂。

Enzymes are remarkable biomolecules that are able to make complicated chemical reactions occur under the mild conditions found in living systems, with remarkable precision. Terpene synthases are enzymes that catalyze the conversion of only a small number of substrates to a myriad of different hydrocarbon products called terpenoids, structures that form the chemical basis of the largest group of natural products. Many terpenoids have beneficial applications, such as the anti-cancer drug taxol, the anti-malaria drug artemisinin and so-called semiochemicals that can act as insect repellents. These are based on sesquiterpenes, a subset of the terpenoid family that contain 15 carbon atoms and derive from the same molecule, farnesyl diphosphate (FDP). Natural sesquiterpene synthases can generate more than 300 different hydrocarbon scaffolds from FDP. Whether or not these scaffolds incorporate water (through hydroxylation, or 'water capture') affects their biological activity and use. Structural and mechanistic work performed over the last decades has revealed that these enzymes share a three-dimensional fold and use similar chemical strategies to achieve the transformation of FDP into products. While much progress has been made to decipher the biochemical details, our knowledge is not complete enough to have predictive power so that a rational approach could be used to convert the 'water capture' behaviour of terpene synthases in a targeted fashion. The proposed research, which is based on a solid foundation of previous work and the complimentary experience of the applicants, will bring together enzymologists, computational biochemists and structural biologists to generate in-depth understanding of the mechanisms of 'water capture' in terpene synthases. This understanding will then be used to rationally change 'water capture' behaviour in example enzymes, which will lead to novel terpenoid products with potential beneficial properties. The combination of state-of-the-art computational and experimental work is crucial for achieving this. By using experimental data, such as structures obtained through X-ray crystallography, computational modelling offers unique detail not accessible through laboratory experiments, which in turn can be used to predict the effect of amino acid changes. These predictions subsequently need experimental validation. Through this combined approach we will discover in great detail how enzyme structure and mobility affect the reaction outcome. Once successful alteration of water capture behaviour has been achieved, we will develop general, streamlined protocols that can be applied to other terpene synthases. These protocols will therefore allow rational modification of efficient biocatalysts to obtain specific terpenoid products with desired properties, e.g. to develop new drugs or insect repellents.