Molecular Determinants of Natural Product Heterocyclization

拟议的研究计划的主要主题是提高我们设计天然产品的能力 产生候选类药物分子的生物合成途径。中国生产的天然产品 自然界显示出广泛的化学功能、形状和大小，注入了其中的许多分子。 具有重要的治疗作用，包括抗癌和抗生素活性。通过增加我们的 在原子水平上理解这些生物合成途径，我们将能够操纵不同的 天然产物系统，用于生产药物发现所需的化学目标化合物。这项建议 目的研究五元杂环形成的分子和生化特征。 非核糖体肽合成酶(NRPS)是一类天然产物，负责产生 多肽和多肽/聚酮杂化分子，如抗癌药物博莱霉素和 埃博西酮类，以及抗生素杆菌肽。 NRPS杂环化(HC或Cy)结构域负责安装五元噻唑啉或 NRPS产品中的恶唑啉环，起源于半胱氨酸或丝氨酸/苏氨酸， 分别进行了分析。这些杂环的存在增加了化合物的稳定性，并改变了化合物的3D形状 因此，天然产物在确定生物活性和能力方面发挥着极其重要的作用 将这些环设计成新的化合物是非常有意义的。目标1需要明确不同的设计 Hc结构域的构建，以便从 埃博西隆和叶尔辛那巴菌素生物合成基因簇。这些结果将提供一个直接的比较 来自相关系统的不同HC结构域被修改以适应不同大小的衬底 分子。该提案的第二个目标是合理地修改底物结合残基。 并表征了由此产生的底物偏好和反应动力学的变化。这 AIM将把有关这些结构的信息转化为工程应用，这是指导 这一系统的工程化，为未来的药物设计。 这一领域提案的总体优势在于确定国家资源保护方案活动的综合办法。 用于生物工程工作的HC领域。这项工作结合了蛋白质结构设计、X射线的使用 用结晶学、诱变和生化活性分析来表征HC的分子功能 域。除了直接让本科生和研究生参与研究外，这些结果还将 影响多个领域，包括天然产物生物合成、生物工程、蛋白质设计和生物催化。

The overarching theme of the proposed research plan is to advance our ability to engineer natural product biosynthetic pathways for the production of candidate drug-like molecules. Natural products produced in Nature display a broad range of chemical functionalities, shapes, and sizes, imbuing many of these molecules with important therapeutic benefits that include anticancer and antibiotic activities. By increasing our understanding of these biosynthetic pathways at the atomic level, we will be able to manipulate different natural product systems to produce target compounds of chemical interest for drug discovery. This proposal aims to characterize the molecular and biochemical features of five-membered heterocycle formation in nonribosomal peptide synthetases (NRPSs), a family of natural products responsible for the generation of peptides and peptide/polyketide hybrid molecules such as the anticancer agents bleomycin and the epothilones, as well as the antibiotic bacitracin. The NRPS heterocyclization (HC or Cy) domain is responsible for installing five-membered thiazoline or oxazoline rings within NRPS products, originating from the amino acids cysteine or serine/threonine, respectively. The presence of these heterocycles increases compound stability and modifies the 3D shape of the natural products, therefore they play extremely important roles in defining biological activity and the ability to engineer these rings into novel compounds is of utmost interest. Aim 1 entails crystallizing different designed HC domain constructs in order to obtain a series of X-ray crystal structures of different HC domains from the epothilone and yersiniabactin biosynthetic gene clusters. These results will provide a direct comparison of how different HC domains from a related system are modified to accommodate differently sized substrate molecules. The second aim of this proposal focuses on rationally modifying substrate-binding residues within the HC domain and characterizing the resulting changes on substrate preferences and reaction kinetics. This aim will translate information regarding these structures to engineering applications, an important step to guide engineering of this system for future drug design. The overall strength of this AREA proposal is in the integrated approach at defining the activity of the NRPS HC domain for bioengineering efforts. This work combines the use of protein construct design, X-ray crystallography, mutagenesis, and biochemical activity assays to characterize the molecular function of the HC domain. In addition to directly involving undergraduate and graduate students in research, these results will impact multiple fields that include natural product biosynthesis, bioengineering, protein design and biocatalysis.