Iterative Polyketide Synthase Function, Structure and Pathways

迭代聚酮合酶功能、结构和途径

• 批准号: 10319955
• 负责人: CRAIG ARTHUR TOWNSEND
• 金额: $ 45.83万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-02-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10319955
• 关键词: 3-hydroxybutanal; Acyl Carrier Protein; Agriculture; Anabolism; Animals; Anthraquinones; Antibodies; Antibody-drug conjugates; Architecture; Behavior; Biochemical; Biosynthetic Proteins; CRISPR/Cas technology; Canis familiaris; Carboxylic Acids; Catalysis; Catalytic Domain; Client; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Cryoelectron Microscopy; Cyclization; DNA; Dehydration; Dynemicin; Environmental Carcinogens; Enzymes; Event; Family; Fatty-acid synthase; Food; Food Supply; Gene Cluster; Gene Deletion; Genes; Goals; Health; Higher Order Chromatin Structure; Human; Individual; Investigation; Ketones; Laboratories; Laccase; Lead; Length; Medicine; Methods; Methylation; Molecular Conformation; Molecular Machines; Mutation; Natural Products; Pathogenicity; Pathway interactions; Pharmacologic Substance; Photosensitizing Agents; Plants; Production; Protein Family; Reactive Oxygen Species; Roentgen Rays; Role; Scientist; Structure; System; Technology; Testing; Toxic Environmental Substances; Toxin; Transferase; United States Department of Agriculture; X-Ray Crystallography; cancer therapy; cercosporin; combat; cost; crosslink; fasciclin; fascinate; grasp; insight; pathogen; plant poison; polyketide synthase; polymerization; protein structure; success; welfare

The importance of polyketides to human health and welfare is recognized both by major pharmaceuticals and important environmental carcinogens and mammalian toxins, as well as phytotoxins that impose heavy costs on agriculture and endanger the food supply. In three major Aims, we propose to undertake fundamental biochemical and structural studies of representatives from two major families of iterative polyketide synthases, which exemplify some of the most sophisticated catalytic systems known and pose many unanswered questions about how the coordinated function of their individual catalytic domains is achieved. Unequivocal determination of the programmed product of the enediyne highly-reducing (HR)-PKSs will be extended to investigation of how this simple, shared intermediate is converted to the enediyne and anthraquinone “halves” of dynemicin A and other enediyne architectures. With advances in antibody technology, conjugates of enediyne natural products are coming again as valuable anti-cancer therapies. The biosynthesis of these structurally intriguing DNA-cleaving molecules remains one of the principal unsolved problems in natural product biosynthesis. Application of precise CRISPR/Cas9 gene deletions in the dynemicin A pathway has brought exciting experimental advances to isolate and characterize the first post-PKS intermediates in any enediyne biosynthetic pathway. Additional mutational studies will be carried out, the structures of other possible intermediates will be elucidated and a strategy of paired CRISPR mutations will be developed to finally crack how these fascinating structures are made. Having first detected and functionally characterized “starter-unit acyl transferase” (SAT) domains and “product template” (PT) domains in NR-PKSs, we are now poised to build from static structures of individual domains, stepwise to tridomains and tetradomains to, finally, full-length structures. In this Aim collaboration with the laboratory of Timm Maier (Biozentrum, Univ. of Basel) will couple biochemical studies, ACP–client crosslinking, x-ray crystallography and cryo-electron microscopy to achieve the next level of understanding to visualize how these separate components integrate their actions into fully functional molecular machines. Cercospora sp. cause immense damage to a range of vital food crops through the production of cercosporin, a diabolically efficient photosensitizer of reactive oxygen species and pathogenic to plants (and animals). The mostly unexplored biosynthesis of this perylenequinone will be undertaken and previous biogenetic proposals will be corrected. The roles of a laccase/fasciclin-family protein and other newly discovered biosynthetic proteins encoded in an expanded biosynthetic gene cluster will be studied in collaboration with scientists at the USDA with the added goal to find “green” ways to combat toxin production by this pathogen. !

多酮类化合物对人类健康和福利的重要性得到了主要药物的认可。 和重要的环境致癌物质和哺乳动物毒素，以及对人体造成严重危害的植物毒素 这会增加农业成本，并危及粮食供应。在三个主要目标中，我们建议从根本上 两个主要迭代聚酮合成酶家族代表的生化和结构研究 它们展示了一些已知的最复杂的催化系统，并提出了许多悬而未决的问题 关于它们各自的催化域的协调功能是如何实现的。毫不含糊 烯二炔高还原(HR)-PKSS程序产物的测定将扩展到 关于这种简单的共享中间体如何转化为烯二炔和蒽醌“一半”的研究 达尼米星A和其他烯二炔构型。 随着抗体技术的进步，烯二炔天然产物的偶联物又出现了 有价值的抗癌疗法。这些结构耐人寻味的DNA裂解分子的生物合成 仍然是天然产物生物合成中尚未解决的主要问题之一。PRECISTION的应用 动力霉素A途径中CRISPR/Cas9基因缺失为分离株带来了令人兴奋的实验进展 并表征了任何烯二炔生物合成途径中的第一个后PKS中间体。附加突变 将进行研究，阐明其他可能的中间体的结构，并制定一项战略 成对的CRISPR突变将被开发出来，最终破解这些迷人的结构是如何形成的。 首先检测到起始单位酰基转移酶(SAT)结构域并对其进行功能表征，以及 “产品模板”(PT)结构域在NR-PKSS中，我们现在准备从个体的静态结构中构建 结构域，逐步到三结构域和四结构域，最后是全长结构。在这个目标中，合作 与Timm Maier的实验室(Biozentum，University.巴塞尔)将结合生化研究，ACP-客户 交联剂，x射线结晶学和低温电子显微镜，以实现对 想象一下这些独立的组件如何将它们的行为整合到功能齐全的分子机器中。 尾孢霉Cercospora sp.通过生产对一系列重要粮食作物造成巨大损害 小孢子素，一种对活性氧和植物致病的极具杀伤力的光敏剂(和 动物)。这是一种尚未被探索的二苯二酮的生物合成方法，将在 生物遗传提议将得到纠正。漆酶/Fasciclin家族蛋白和其他新发现的蛋白质的作用 在扩展的生物合成基因簇中编码的生物合成蛋白质将与 美国农业部的科学家们还有一个额外的目标，那就是找到“绿色”的方法来对抗这种病原体产生的毒素。 好了！