Determining the Architectures and Activities of Polyketide Synthase Modules

模块化聚酮酶领域的复兴已经开始。新的工具和模式正在使 更深入地了解这些酶组装线的结构和活动，并促进我们的 长期目标是将模块化聚酮酶的合成能力应用于开发， 生产新药。使用“模块”的最新定义，我们的实验室设计了多种三- 在体内和体外都有功能的四肽/五肽脱氢酶。虽然这些短装配线 它们在自然界中并不常见，是我们进行结构和功能研究的理想材料。在具体目标1中，我们建议 在这些生产线合成聚酮化合物产品的过程中， 低温电子显微镜由于这种方法使我们能够捕获高分辨率的动态信息， 对于模型三酮化合物合酶的引发酮合酶和酰基转移酶，我们将其应用于 包含其他感兴趣的区域。一个目的是了解酮还原酶，脱氢酶， 烯酰还原酶加工酶相对于彼此和相邻的酶定向， 酮合酶+酰基转移酶双结构域，以了解酰基载体蛋白结构域如何在这些 在聚酮中间体的延伸和加工过程中的酶。因此，我们至少会调查 在我们的实验室中功能性验证的13种工程化三酮/四酮脱氢酶和2种天然脱氢酶， 含有不同的加工酶在具体目标2中，我们建议阐明相互作用 在加工的聚酮中间体和为它们守门的酮基糖苷酶之间。我们有强大 关于底物隧道残基组如何与最接近反应性硫酯的中间体相互作用的假设 以确保它们被上游加工酶适当地修饰。因此，我们将适当地改变 在活性较低的模型酶以及具有以下活性的模型酶中的酮基肽酶的看门残基： 灭活上游加工酶并确定它们的生产率是否如预测的那样提高。 由于我们的数据表明聚酮中间体使酮合酶二聚体和二聚体 酮合酶+酰基转移酶双结构域可以很容易地确定在冷冻电子显微镜研究，我们将 我还进行电子显微镜对停滞的脱氢酶，以解决结构的聚酮结合 酮合酶+酰基转移酶二聚体。在具体目标3中，我们提出确定关键域-域接口。 我们有证据表明，加工酶和下游KSs之间的界面驱动有序的自我- 合成酶多肽的组装比在C-和N-末端之间观察到的小界面更多。 对接结构域并寻找代表性复合物的结构。我们还旨在确定酰基载体如何 蛋白质结构域在转酰反应期间与酮基转移酶对接。如果我们在这些方面取得成功， 项目，这将大大有助于合理设计模块化聚酮酶，合成新的 分子，最终，新药。

A renaissance in the field of modular polyketide synthases has begun. New tools and paradigms are enabling deeper insights into the architectures and activities of these enzymatic assembly lines and are facilitating our long-term goal of applying the synthetic power of modular polyketide synthases to the development and production of new medicines. Using the updated definition of the “module”, our lab has engineered diverse tri- /tetra-/pentaketide synthases that are functional both in vivo and in vitro. While these short assembly lines are uncommon in nature, they are ideal for our structural and functional studies. In Specific Aim 1 we propose to plunge-freeze these assembly lines as they are synthesizing their polyketide products and investigate them by cryo-electron microscopy. Since this approach has enabled us to capture high-resolution, dynamic information of the priming ketosynthase and acyltransferase of a model triketide synthase, we will apply it to synthases that contain other regions of interest. One objective is to learn how the ketoreductase, dehydratase, and enoylreductase processing enzymes are oriented relative to one another and the neighboring ketosynthase+acyltransferase didomains to understand how acyl carrier protein domains move between these enzymes during the extension and processing of polyketide intermediates. Thus, we will investigate at least thirteen engineered tri-/tetraketide synthases and two natural synthases functionally validated in our lab that contain different sets of these processing enzymes. In Specific Aim 2 we propose to elucidate interactions between processed polyketide intermediates and the ketosynthases that gatekeep for them. We have strong hypotheses for how sets of substrate tunnel residues interact with intermediates closest to the reactive thioester to ensure they are properly modified by upstream processing enzymes. Thus, we will appropriately mutate the gatekeeping residues of ketosynthases in less active model synthases as well as model synthases with inactivated upstream processing enzymes and determine whether their productivities improve as predicted. Since our data indicate that polyketide intermediates rigidify the ketosynthase dimer and dimeric ketosynthase+acyltransferase didomains can be readily identified in cryo-electron microscopy studies, we will also perform electron microscopy on stalled synthases to solve structures of polyketide-bound ketosynthase+acyltransferase dimers. In Specific Aim 3 we propose to determine key domain-domain interfaces. We have evidence that interfaces between processing enzymes and downstream KSs drive the ordered self- assembly of synthase polypeptides more than the small interface observed between the C- and N-terminal docking domains and seek structures of representative complexes. We also aim to determine how acyl carrier protein domains dock with ketosynthases during the transacylation reaction. If we are successful in these projects, it will greatly inform the rational engineering of modular polyketide synthases that synthesize new molecules and, ultimately, new medicines.