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

非天然氨基酸和抗体片段结构工具实现的聚酮化合物合成酶的机理研究

基本信息

批准号：
10448409
负责人：
Dillon Cogan
金额：
$ 6.5万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10448409
关键词：
3-Dimensional Accounting Acyl Carrier Protein Acyltransferase Affinity Amber Amino Acids Amino Acyl-tRNA Synthetases Anti-Bacterial Agents Anti-Inflammatory Agents Antifungal Agents Area Attenuated Bacteria Binding Binding Sites Biochemical Biological Models Catalytic Domain Chemicals Cholesterol Classification Clinical Coenzyme A Collaborations Complement Complex Cryoelectron Microscopy Crystallization Data Data Reporting Dependence Development Electron Microscopy Electrons Engineering Enzymatic Biochemistry Enzyme-Linked Immunosorbent Assay Enzymes Exhibits Fab Immunoglobulins Family Genetic Code Glean Goals Human Immunoglobulin Fragments Label Life Logic Maps Mediating Medicine Modeling Molecular Molecular Conformation Mutation Analysis NADP Natural Products Nature Pharmaceutical Preparations Phase Play Property Protein Engineering Proteins Reaction Reporting Research Resolution Role Scanning Site Source Structure Structure-Activity Relationship Study models Surface Surveys Techniques Terminator Codon Work X-Ray Crystallography anti-cancer base cofactor combinatorial crosslink functional group hybrid enzyme improved inorganic phosphate lead optimization mutant nanomolar novel particle picromycin plant fungi polyketide synthase polypeptide programs reconstruction screening tool training opportunity unnatural amino acids

项目摘要

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).

项目概要/摘要聚酮化合物天然产物广泛存在于生活的各个领域，主要存在于细菌、植物和真菌的巨大结构多样性使得近 50 年的研究得以进行。事实上，许多聚酮类化合物都在寻找潜在有用的生物活性。聚酮化合物已被鉴定具有抗菌、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方便地，聚酮合酶（PKS）的一个子集，即在自然界中形成聚酮化合物的酶，以这种方式，在单个多肽上包含多个催化结构域的类似模块化装配线。聚酮化合物产物结构是根据观察到的 PKS 结构域顺序进行模板化的。似乎存在一个支持聚酮化合物生物合成逻辑的自然程序，这让许多人感到好奇是否可以对 PKS 进行重新编程，赋予它们非自然的功能。（约占聚酮化合物的 50%）为非天然 PKS 的组合工程提供了令人兴奋的前景具有新颖的功能，并且许多努力尝试通过替换催化结构域来创建此类 PKS 然而，许多这些杂合酶的催化效率受到影响。由于目前尚不清楚的原因，因此需要对 PKS 进行可靠且成功的重新编程。全面了解控制天然酶功能的结构和机制。我们首先的目标是揭示两个核心 PKS 识别酰基载体蛋白 (ACP) 的分子基础催化结构域，酮合酶 (KS) 和酰基转移酶 (AT)，来自细菌模块化 PKS。在聚酮化合物过程中受到 ACP/催化域复合物的瞬态和反应性质的挑战为了缓解这一障碍，我们计划将非天然氨基酸（UAA）与亲电子结合。 ACP 中的官能团与已鉴定的 Cys/Lys 亲核试剂进行域间交联。交联物种将应用于结构和经验模型研究，并得到高强度的帮助。亲和抗原结合片段 (Fab)，旨在了解 PKS 的结构与功能关系这里报告了酮还原酶和新的支持结构数据。在最后一个目标中，我们建议研究使用 Fab、化学交联和其他稳定剂组合的 PKS 模块的构象动力学使用单粒子冷冻电子显微镜（cryo-EM）获得高分辨率电子结构。报告了与该目标相关的显微镜和活动数据，并提出了提高颗粒质量的策略最后，拟议的研究预计将为 UAA 领域提供独特的培训机会。掺入和单粒子冷冻电镜；后者是通过与 Wah Chiu 教授的合作促进的实验室（斯坦福/SLAC），靠近斯坦福/SLAC 冷冻电镜中心 (S2C2)。