Mechanistic Studies of Polyketide Synthases Enabled by Unnatural Amino Acids and Antibody Fragment Structural Tools

项目摘要/摘要 聚酮类天然产物广泛存在于生命的各个领域，主要存在于 细菌、植物和真菌。它们巨大的结构多样性使近50年的研究成为可能， 以及不断增长的聚酮类化合物系列，以寻找潜在的有用生物活性。事实上，有很多人 已鉴定出具有抗菌、抗癌、抗真菌、抗病毒、抗炎、 免疫抑制和降低胆固醇的特性；因此，强调了它们作为潜在来源的价值 毒品。方便的是，聚酮合成酶(PKS)的子集，在自然界中锻造多酮的酶， 类似于模块化装配线，在单个多肽上包含多个催化结构域。以这种方式 根据观察到的PKS结构域顺序，以聚酮产物结构为模板。因此，在那里 似乎存在一个支持聚酮生物合成逻辑的自然程序，这让许多人怀疑 PKS能否被重新编程以赋予它们非自然的功能。细菌中的模块化PKSS (约占聚酮的50%)为非天然PKS的组合工程提供了令人兴奋的前景 具有新的功能，并且许多努力试图通过将催化结构域从 外源PKS来源。然而，许多这些杂交酶的催化效率受到了影响。 原因还不太清楚。因此，PKSS的可靠和成功的可重新编程性需要 对控制天然酶功能的结构和机制有透彻的了解。 我们首先旨在揭示两个核心PKS识别酰基载体蛋白(ACP)的分子基础 来自细菌模块化PKS的催化域，酮合成酶(KS)和酰基转移酶(AT)。这个目标有 被聚酮过程中ACP/催化结构域复合体的瞬时和反应性所挑战 正在处理。为了缓解这一障碍，我们计划将非天然氨基酸(UAA)与亲电性结合起来 在ACP中引入官能团，与相互作用的半胱氨酸/赖氨酸亲核试剂进行结构域间交联。已确定 交联物种将被应用于结构和经验模型研究。第二个目标，在高- 亲和抗原结合片段(Fabs)，试图了解PKS的结构-功能关系 以及新的支持结构的数据在这里报道。在最后一个目标中，我们建议研究 使用Fabs、化学交联剂和其他稳定剂组合使用的PKS模块的构象动力学 用单粒子冷冻电子显微镜(Cryo-EM)获得高分辨率结构。初步电子 报道了与此目标相关的显微镜和活度数据，并提出了改善颗粒质量的策略 概述。最后，拟议的研究预计将在UAA领域提供独特的培训机会 联合和单粒子冷冻-EM；后者是通过与华超教授的合作而促进的 实验室(斯坦福/SLAC)，靠近斯坦福/SLAC冷冻-EM中心(S2C2)。

Project Summary/Abstract Polyketide natural products are widespread across all domains of life, occurring predominantly in bacteria, plants, and fungi. Their vast structural diversity has enabled nearly 50 years of research into a large, and growing, repertoire of polyketide-type compounds in search for potentially useful bioactivities. Indeed, many polyketides have been identified which exhibit antibacterial, anticancer, antifungal, antiviral, anti-inflammatory, immunosuppressive, and cholesterol-lowering properties; thus, underscoring their value as a source of potential drugs. Conveniently, a subset of polyketide synthases (PKSs), the enzymes that forge polyketides in nature, resemble modular assembly lines with multiple catalytic domains contained on a single polypeptide. In this way the polyketide product structures are templated according to the observed PKS domain order. Thus, there appears to exist a natural program underpinning the biosynthetic logic of polyketides, leading many to wonder whether PKSs can be reprogrammed to endow them with unnatural functions. The modular PKSs in bacteria (accounting for ~50% of polyketides) offer exciting prospects for combinatorial engineering of unnatural PKSs with novel function, and many efforts have attempted to create such PKSs by substituting catalytic domains from exogenous PKS sources. However, the catalytic efficiencies of many of these hybrid enzymes are compromised for reasons that are not well understood. Reliable and successful reprogrammability of PKSs therefore requires a thorough understanding of the structures and mechanisms governing natural enzyme function. We first aim to uncover the molecular bases of acyl carrier protein (ACP) recognition by two core PKS catalytic domains, the ketosynthase (KS) and acyltransferase (AT), from a bacterial modular PKS. This goal has been challenged by the transient and reactive nature of ACP/catalytic-domain complexes during polyketide processing. To mitigate this roadblock, we plan to incorporate unnatural amino acids (UAAs) with electrophilic functional groups into the ACPs for interdomain crosslinking with reciprocal Cys/Lys nucleophiles. Identified crosslinked species will be applied to structural and empirical modeling studies. A second Aim, aided by high- affinity antigen-binding fragments (Fabs), seeks to understand the structure-function relationships of a PKS ketoreductase, and new, supporting structural data are reported here. In the last Aim, we propose to study the conformational dynamics of a PKS module using Fabs, chemical crosslinking, and other stabilizers combined with single particle cryo-electron microscopy (cryo-EM) to obtain a high-resolution structure. Preliminary electron microscopy and activity data relevant to this Aim are reported, and strategies to improve the particle quality are outlined. Finally, the proposed research is expected to offer unique training opportunities in the areas of UAA incorporation and single particle cryo-EM; the latter of which is facilitated by collaboration with Prof. Wah Chiu’s lab (Stanford/SLAC) and close proximity to the Stanford/SLAC Cryo-EM Center (S2C2).