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

项目摘要。由于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，了解生物体和酶如何发挥作用，以产生这些结构复杂的 化合物是必要的。聚酮化合物是由聚酮化合物生物合成的一类次级代谢产物 酶（PKS），通常用作抗菌剂，抗真菌剂和抗癌剂。PKS很复杂 涉及蛋白质和底物以高度特异性相互作用的生物机制， 组装聚酮。这些独特的蛋白质-蛋白质和蛋白质-底物相互作用的基础上， 这些事件是受控制的，因此理解它们至关重要。在所有三种类型的PKS中， 聚酮中间体通过两个碳单元的迭代延伸，但是它们各自的延伸酶如何 在防止底物发生不希望的副反应的同时， 不为人知在这个建议中，我们的目标是首先了解在一个口袋里生长聚酮的合适性。 载体蛋白引导的延伸酶， II型PKS模型。在这里，我们将使用交联来捕获伴侣蛋白，以阐明关键的相互作用， 中间体被拉长。然后我们计划在我们的第二项研究中应用类似的化学生物学工具 目的是确定III型PKS中CoA依赖性延伸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， 通过X射线晶体学提供自然基质相互作用的快照。这些研究将使 我们要揭示的分子细节，驱动伸长过程负责建设核心碳 聚酮化合物的主链。深入了解这些蛋白质和底物的相互作用， 它们的操作和重新设计以产生具有不同药效团的新型聚酮化合物。

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.