Structural Basis of Substrate Processing in Modular Polyketide Synthases

 描述（由申请人提供）：细菌I型聚酮化合物脱氢酶（PKS）是负责产生多种聚酮化合物产品的大环内酯核心的巨型酶组装线，这些聚酮化合物是具有生物活性的天然化合物（例如，抗微生物、抗真菌、抗病毒、抗癌和免疫抑制剂化合物）。聚酮化合物天然产物目前构成了近三分之一药物的基础，这表明了其重要性。PKS采用模块化多步骤机制来产生聚酮化合物，并且生物工程化这些系统对于创造在药物发现中具有宝贵应用的新化学型具有巨大潜力。然而，这些努力取得了有限的成功，反映了我们对产生聚酮化合物的模块化过程的结构和机理理解不足。我们最近采用冷冻电子显微镜（cryo-EM）显示的第一个亚纳米分辨率结构的全长PikAIII模块从pikromycin PKS生物合成途径，组装线PKS系统的原型。研究结果不仅揭示了一个意想不到的模块架构进行广泛的结构重排，但也表明，连接到一个高度移动的酰基载体蛋白（ACP）结构域的底物类型指定其定位的方式，促进装配线的吞吐量。通过采用低温EM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，以解决有关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，试图揭示这些显着的大分子工厂中底物识别和加工的原理。拟议研究的结果将成为重新开展生物工程工作的基础，以创造能够有效生产具有高药用价值的新型化合物的PKS。

 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.