Understanding substrate translocation in polyketide synthase (PKS) assembly lines

“装配线”一词通常会让人联想到汽车生产设施：车辆经过几个工位，在这些工位上执行工作步骤。大自然在聚酮化合物天然产物的合成中使用了类似的合成原理。像汽车装配线一样，模块化聚酮酶（PKS）由称为模块的工作站组成，在这些工作站上，最初简单的前体分子（通常是活化的羧酸）被加工。虽然前体分子是简单的，但产物是复杂的生物活性化合物，在医学治疗中具有广泛的应用。正如宏观汽车生产线的机制一样清楚-汽车在皮带或轨道上运输，机器人或人进行，例如，螺丝、焊接或胶合工作-PKS生产线的分子基础直到今天仍然很难理解，并有争议地讨论。不断增长的聚酮化合物是如何通过装配线输送的？模块如何识别聚酮化合物已被处理，现在应该转发到下一个模块？与此相关的是，一个模块如何防止聚酮化合物的重复加工？对这些问题只有模糊的解释。本项目旨在为PKS装配线的矢量合成的分子基础提供明确的答案。它基本上包括两个方面：（i）项目的生物化学部分旨在按照“通过重建理解”的方法，对PKS生产线的矢量合成进行功能理解。为了理解哪些单元负责矢量合成，PKS装配线将被明确的非矢量单元重新建模。具体来说，我们将非载体同源蛋白的单位移植到PKS装配线中，并监测是否保留了载体合成的能力。对于这种方法，我们使用非矢量（但原型迭代）脂肪酸合酶（FAS）和两个良好的特点迭代PKS作为供体蛋白。(ii)在该项目的结构生物学部分，我们的目标是矢量合成的结构表征。仔细选择的PKS生产线的亚基的基础上，并使用低温电子显微镜（cryo-EM），我们特别寻求从结构上描述从一个模块到另一个，所谓的易位步骤的增长聚酮的通道。在这里，智能蛋白质设计旨在锁定矢量合成的关键构象。最先进的单粒子cryo-EM可以处理复杂蛋白质样品的异质性，并且能够以近原子分辨率从构象集合中提取结构信息。结构生物学部分设计为与Timm Maier教授（Biozentrum巴塞尔）的合作项目。他的实验室在PKS和相关多结构域蛋白的结构分析方面具有杰出的专业知识。

The term "assembly line" usually evokes associations with car production facilities: the vehicle passes through several stations at which work steps are carried out. Nature uses a similar synthetic principle in the synthesis of polyketide natural products. Like car assembly lines, modular polyketide synthases (PKSs) consist of stations, named modules, at which an initially simple precursor molecule, often activated carboxylic acid, is processed. While the precursor molecule is simple, the products are complex bioactive compounds with wide applications in medical therapy. As clear as the mechanism of macroscopic car production lines is - cars are transported on belts or tracks, and robots or people carry out, e.g., screwing, welding or gluing work - the molecular basis of PKS production lines remains until today largely elusive and controversially discussed. How is the growing polyketide channeled through the assembly line? How does a module recognize that the polyketide has been processed and should now be forwarded to the next module? And linked to this, how does a module prevent repeated processing of a polyketide? There are only vague attempts to explain these questions. The project presented in this grant proposal seeks to provide clear answers to the molecular basis of the vectorial synthesis of PKS assembly lines. It comprises basically two axes: (i) The biochemical part of the project aims at the functional understanding of the vectorial synthesis of PKS production lines, following an “understanding by remodeling” approach. In order to understand which units are responsible for vectorial synthesis, PKS assembly lines will be remodeled by explicitly non-vectorial units. Specifically, we transplant units of non-vectorial homologous proteins into the PKS assembly line, and monitor whether the ability for vectorial synthesis is retained. For this approach, we use the non-vectorial (but prototypically iterative) fatty acid synthase (FAS) and two well-characterized iterative PKSs as donor proteins. (ii) In the structural biology part of the project, we aim at the structural characterization of vectorial synthesis. On the basis of carefully selected subunits of PKS production lines and using cryogenic electron microscopy (cryo-EM), we particularly seek to structurally describe the passage of the growing polyketide from one module to the other, the so-called translocation step. Here, intelligent protein design aims to lock key conformations of vectorial synthesis. State-of-the-art single-particle cryo-EM can handle heterogeneity of complex protein samples, and is able to extract structural information from ensembles of conformations at near-atomic resolution. The structural biology part is designed as collaborative project with Prof. Timm Maier (Biozentrum Basel). His laboratory has outstanding expertise in the structural analysis of PKSs and related multidomain proteins.