Probing enzyme reaction cascades in polyketide biosynthesis at atomic resolution by NMR

许多抗生素和其他潜在的治疗产品是由I型聚酮合成酶产生的。这些系统就像装配线一样工作，以建立分子，这样，通过操纵级联中的酶，最终产品就可以改变。通过提高我们对这些酶的理解，我们甚至可以开始考虑使用它们来设计新的分子。这个项目特别感兴趣的一个系统是bongkrekic酸系统，以它产生的毒素命名。Bongkrekic酸通过抑制腺嘌呤核苷酸转位酶(ANT)、阻断氧化磷酸化来发挥作用。有趣的是，选择性地针对蚂蚁的化合物已经被提出作为可能用于化疗的抗凋亡剂。因此，Bongkrekic酸代表了这种理性开发的一个很好的开始结构，这项工作将为这一过程提供关键信息。它将强调合成和通量是如何由酶控制的，因此可能会被操纵以产生类似物。该项目中的关键问题围绕着酶如何控制哪些贝塔分支事件包括最终脱羧基，以及在没有末端硫代酯酶的情况下合成如何终止。将使用新的核磁共振分析和补充的ESI-MS，以允许监测酶串联中的中间体。这使得可以在原子分辨率下探测bongkrekic酸的合成，而不需要单独分离这些中间体。在适当的情况下，可以使用用于分析蛋白质相互作用的结构技术，例如X射线结晶学和低温电子显微镜。该项目属于EPSRC化学生物学和生物化学研究领域。

Many antibiotics and other potentially therapeutic products are produced by type I polyketide synthases. These systems work like assembly lines in order to build up molecules, such that by manipulating enzymes within the cascade, the end product can be altered. By advancing our understanding of these enzymes, we can even begin to look at using them for the design of novel molecules.One system of particular interest for this project is the bongkrekic acid system, named after the toxin it produces. Bongkrekic acid works by inhibiting the adenine nucleotide translocase (ANT), blocking oxidative phosphorylation. Interestingly, compounds which selectively target the ANT have been put forward as antiapoptotic agents that may be used in chemotherapy. Bongkrekic acid therefore represents an excellent starting structure for this rational development, a process which this work will provide key information for. It will highlight how synthesis and flux is controlled by the enzymes, and therefore how this may be manipulated to produce analogues. Key questions in the project surround how the enzymes control which beta branching events include a final decarboxylation and how synthesis is terminated without the presence of a terminal thioesterase enzyme. Novel NMR assays and supplementary ESI-MS will be used that allow monitoring of intermediates in the enzyme cascade. This allows probing of bongkrekic acid synthesis at atomic resolution, without the need for these intermediates to be individually isolated. Where appropriate, structural techniques for analysis of protein interactions may be used, such as x-ray crystallography and cryo-EM. This project falls within the EPSRC Chemical Biology and Biological Chemistry research area.