Structural basis of substrate processing in modular polyketide synthases

 描述(申请人提供)：细菌I型聚酮合成酶(PKS)是巨型酶装配线，负责产生多种聚酮产品的大内酯核心，这些产品是具有生物活性的天然化合物(例如，抗菌、抗真菌、抗病毒、抗癌和免疫抑制化合物)。聚酮类天然产物目前构成了近三分之一药物的基础，这表明了它们的重要性。PKSS采用模块化的多步机制来生产聚酮，而生物工程这些系统在创造新的化学类型方面具有巨大的潜力，在药物发现中具有无价的应用。然而，这种努力取得的成功有限，反映出我们对产生聚酮的模块化过程缺乏结构性和机械性的理解。我们最近利用低温电子显微镜(Cryo-EM)展示了来自匹克罗霉素PKS生物合成途径的全长PikAIII模块的第一个亚纳米分辨率结构，这是装配线PKS系统的原型。这些发现不仅揭示了经历广泛结构重组的意想不到的模块体系结构，还表明与高流动性酰基载体蛋白(ACP)结构域连接的底物类型以一种促进装配线吞吐量的方式指定了其定位。通过利用低温电磁技术的最新突破，我们现在的目标是获得PikAIII和终端PikAIV模块的高分辨率低温EM结构，以努力解决关于功能接口和模块动力学的几个挥之不去的问题。我们的研究将包括天然和非天然底物，试图揭示这些非凡的高分子工厂中底物识别和处理的原理。拟议研究的结果将为新的生物工程努力创造能够有效地生产具有高药用价值的新化合物的PKS奠定基础。

 DESCRIPTION (provided by applicant): Bacterial type I polyketide synthases (PKSs) are mega-enzyme assembly lines responsible for generating the macrolactone core of a wide range of polyketide products that are biologically active natural compounds (e.g. antimicrobial, antifungal, antiviral, anticancer, and immunosuppressant compounds). Indicative of their significance, polyketide natural products currently form the basis for nearly one-third of pharmaceuticals. PKSs employ a modular multi-step mechanism to produce polyketides, and bioengineering these systems has immense potential for the creation of new chemotypes with invaluable applications in drug discovery. However, such efforts have met with limited success, reflecting our poor structural and mechanistic understanding of the modular process to generate polyketides. We recently employed cryo-electron microscopy (cryo-EM) to show the first subnanometer resolution structures of the full length PikAIII module from the pikromycin PKS biosynthetic pathway, a prototype for assembly-line PKS systems. The findings not only revealed an unexpected module architecture undergoing extensive structural rearrangements, but also showed that the type of substrate linked to a highly mobile acyl carrier protein (ACP) domain specifies its positioning in a way that facilitates assembly-line throughput. By employing recent breakthroughs in cryo-EM technologies, we now aim to obtain high resolution cryo-EM structures of PikAIII and of the terminal PikAIV module, in an effort to resolve several lingering question regarding functional interfaces and module dynamics. Our studies will include both natural and unnatural substrates, seeking to reveal the principles of substrate recognition and processing in these remarkable macromolecular factories. The findings from the proposed studies will for the basis for renewed bioengineering efforts towards the creation of PKSs that can efficiently produce novel compounds of high medicinal value.