Iterative Polyketide Synthase Function, Structure and Pathways

聚酮化合物对人类健康和福祉的重要性已被主要制药商和制药商所认识。 和重要的环境致癌物和哺乳动物毒素，以及植物毒素， 农业成本，并危及粮食供应。在三个主要目标中，我们建议开展基本的 来自迭代聚酮化合物脱氢酶的两个主要家族的代表的生物化学和结构研究， 这是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 它们各自的催化区域是如何协调功能的。明确 烯二炔高度还原（HR）-PKS的程序化产物的测定将扩展到 研究这种简单的共用中间体如何转化为烯二炔和蒽醌“半” dynemicin A和其他烯二炔结构。 随着抗体技术的进步，烯二炔天然产物的缀合物再次出现， 有价值的抗癌疗法这些结构有趣的DNA切割分子的生物合成 仍然是天然产物生物合成中未解决的主要问题之一。精确的应用 dynemicin A途径中的CRISPR/Cas9基因缺失带来了令人兴奋的实验进展， 并表征任何烯二炔生物合成途径中的第一个PKS后中间体。额外突变 将进行研究，将阐明其他可能的中间体的结构，并提出一种 将开发成对的CRISPR突变，以最终破解这些迷人的结构是如何形成的。 首次检测和功能表征了“起始单元酰基转移酶”（SAT）结构域， “产品模板”（PT）领域的NR-PKS，我们现在准备建立从静态结构的个人 结构域，逐步到三结构域和四结构域，最后，全长结构。在这次合作中， 与Timm Maier（Biozentrum，巴塞尔大学）的实验室将结合生化研究，ACP客户端 交联，X射线晶体学和低温电子显微镜，以达到下一个层次的理解， 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。 尾孢菌通过生产下列物质对一系列重要的粮食作物造成巨大损害： 尾孢菌素，一种活性氧类的恶魔般有效的光敏剂和植物病原体（和 动物）。大多数未探索的生物合成，这perylenequinone将进行和以前 生物遗传学的建议将得到纠正。漆酶/成束蛋白家族蛋白和其他新发现的 生物合成蛋白质编码的扩大生物合成基因簇将研究合作， 美国农业部的科学家们的另一个目标是找到“绿色”方法来对抗这种病原体产生的毒素。 !

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. !