Determining the Architectures and Activities of Polyketide Synthase Modules

摘要 我们对聚酮化合物装配线的研究正在帮助实现一种范式转变，即组件如何 酶协同生物合成聚酮化合物天然产物。模块的更新定义， 酮合酶结构域位于其下游末端，影响模块化聚酮合酶的每一个水平 酶学这些层面中的每一个都必须进一步探索，以实现我们重新编程的长期目标 聚酮化合物组装线，以合成设计分子，并加速药物发现过程。我们 最高分辨率的建议是研究酮酶如何把关，使得只有一种类型的聚酮化合物 通过模块选择中间体，以通过下游装配线进一步延长（特定目标 1）。这将通过我们开发的晶体学观察方法来实现 结合在酮合酶活性位点的聚酮化合物，并测量在 疑似看门残基;工程化的三酮化合物脱氢酶也将有助于这一努力。来自结构 由我们的实验室和其他人确定，我们假设存在几个未表征的域界面 在模块中。我们试图从结构上阐明这些接口的背景下，新定义的 模块（具体目标2）。因此，通过晶体学和低温电子显微镜， 具有上游加工酶和下游酮合酶的多结构域片段将被 测定我们还将继续努力，以表征在反应过程中形成的瞬态界面 环作为酰基载体蛋白结构域将聚酮化合物中间体呈递给用于催化的同源酶。我们 实验室已经收集了几个更高阶建筑的结构证据。在杆菌中 聚酮合酶，一个三螺旋元件邻近酮合酶结构域似乎拉链同源二聚体 在枯草芽孢杆菌细胞内可观察到约100 MDa组装片。我们建议 通过在体外重组这些生物合成的巨型复合物并观察它们的结构， 通过电子显微镜（特定目标3）。我们的实验室已经在可视化分枝杆菌的PKS 12 结核病在其“双模块”和其聚合物装配线状态中。我们试图确定模块如何 堆栈以在这样的系统中构造更高阶的体系结构。通过对每个层面的调查， 聚酮化合物装配线的结构和活动的全貌将出现， 这对这些医学相关分子机器的未来工程特别重要。

ABSTRACT Our research on polyketide assembly lines is helping bring about a paradigm shift for how sets of component enzymes cooperate to biosynthesize polyketide natural products. The updated definition of a module, with the ketosynthase domain positioned at its downstream end, affects every level of modular polyketide synthase enzymology. Each of these levels must be further explored to achieve our long-term goal of reprogramming polyketide assembly lines to synthesize designer molecules and accelerate the drug discovery process. Our highest-resolution proposal is to study how ketosynthases gatekeep such that only one type of polyketide intermediate is selected by a module to be further elongated by the downstream assembly line (Specific Aim 1). This will be accomplished through methodology we have developed to crystallographically observe polyketides bound in ketosynthase active sites and measure the activity of ketosynthases mutated at suspected gatekeeping residues; engineered triketide synthases will also aid in this effort. From structures determined by our lab and others, we hypothesize that several uncharacterized domain interfaces are present within modules. We seek to structurally elucidate these interfaces within the context of the newly-defined module (Specific Aim 2). Thus, through crystallography and cryo-electron microscopy the structures of multidomain fragments possessing upstream processing enzymes and a downstream ketosynthase will be determined. We will also continue our efforts to characterize transient interfaces that form during the reaction cycle as acyl carrier protein domains present polyketide intermediates to cognate enzymes for catalysis. Our lab has collected several pieces of structural evidence for higher-order architecture. In the bacillaene polyketide synthase, a three-helix element adjacent to the ketosynthase domain seems to zipper homodimeric assembly lines into ~100 MDa assembly sheets observable within Bacillus subtilis cells. We propose to understand the structures of such biosynthetic megacomplexes by reconstituting them in vitro and observing them through electron microscopy (Specific Aim 3). Our lab is already visualizing Pks12 from Mycobacterium tuberculosis both in its “bimodular” and its polymeric assembly line states. We seek to determine how modules stack to construct higher-order architecture in such systems. Through investigations at each of these levels, an overall picture of the architectures and activities of polyketide assembly lines will emerge that will be particularly significant to the future engineering of these medicinally-relevant molecular machines.