Capturing the key protein and substrate interactions in polyketide synthases using isosteric mimetics

项目摘要。市场上可获得的大多数治疗药物是天然产品或 它们的衍生物，了解生物体和酶是如何发挥作用来产生这些结构复杂的 化合物是必不可少的。聚酮类化合物是由聚酮类化合物生物合成的一类次生代谢物。 合成酶(PKs)，常用作抗菌、抗真菌和抗癌药物。PKS是复杂的 涉及蛋白质和底物相互作用的生物机制，具有高度的特异性 组装聚酮。这些独特的蛋白质-蛋白质和蛋白质-底物相互作用是如何 这些合成酶是受控制的，因此对理解是至关重要的。在所有三种类型的PKS中共有的是 聚酮中间体通过两个碳单元的迭代伸长，但它们各自的伸长酶 功能和稳定底物，同时防止它们发生不必要的副反应继续 保持默默无闻。在这项建议中，我们的目标是首先了解在一个口袋里种植聚酮的适合性 载体蛋白引导的伸长酶开发聚酮中间体的异构化模拟 II型PKS模型。在这里，我们将使用交联来捕获伙伴蛋白质，以阐明关键的相互作用 中间产物是拉长的。然后，我们计划在第二项研究中应用类似的化学生物学工具。 目的确定III型PKS中辅酶A依赖的伸长酶催化的底物相互作用。 系统。在这项研究中，我们将开发聚酮中间体模拟物和丙二酰辅酶A类似物，以便能够 通过X射线结晶学提供天然底物相互作用的快照。这些研究将允许 美国将揭开推动形成核心碳的伸长过程的分子细节 聚酮的主链。对这些蛋白质和底物相互作用的更深入了解使 他们的操作和重新设计，以生产具有不同药效团的新型聚酮。

Project Summary. With the majority of therapeutic drugs available on the market being natural products or derivatives of them, understanding how organisms and enzymes function to produce these structurally complex compounds is essential. Polyketides are a class of secondary metabolites that are biosynthesized by polyketide synthases (PKSs) and often serve as antibacterial, antifungal, and anticancer agents. The PKSs are complex biological machineries that involve proteins and substrates interacting with one another with high specificity to assemble polyketides. These unique protein-protein and protein-substrate interactions are the basis for how these synthases are governed and are therefore critical to understand. Common in all three types of PKSs is the iterative elongation of polyketide intermediates by two-carbon units, but how their respective elongation enzymes function and stabilize the substrates while preventing them from undergoing unwanted side reactions continues to remain unknown. In this proposal, we aim to first understand the fit of growing polyketones in the pocket of a carrier protein-guided elongation enzyme by developing isosteric mimetics of polyketide intermediates from a type II PKS model. Here, we will use crosslinking to trap the partner proteins to elucidate the key interactions as the intermediates are elongated. We then plan to apply similar chemical biology tools in our second research aim to define the substrate interactions catalyzed by a CoA-dependent elongation enzyme in a type III PKS system. In this study, we will develop polyketide intermediate mimetics and malonyl-CoA analogs to be able to provide a snapshot of the natural substrate interactions through x-ray crystallography. These studies will allow us to uncover the molecular details that drive the elongation process responsible for building the core carbon backbone of polyketides. Gaining a deeper understanding of these protein and substrate interactions enables their manipulation and redesign to produce novel polyketides with different pharmacophores.