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

• 批准号: 10227676
• 负责人: Dillon Cogan
• 金额: $ 6.64万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10227676
• 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; Antiviral 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

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).

项目总结/摘要 聚酮化合物天然产物广泛存在于生活的所有领域，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， 并不断增长的聚酮型化合物的库，以寻找潜在有用的生物活性。事实上许多 已经鉴定了聚酮化合物，其表现出抗菌、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的 毒品方便地，聚酮化合物脱氢酶（PKS）的一个子集，在自然界中形成聚酮化合物的酶， 类似于具有包含在单个多肽上的多个催化结构域的模块化装配线。以这种方式 根据观察到的PKS结构域顺序模板化聚酮化合物产物结构。因此 似乎存在一个支持聚酮化合物生物合成逻辑的自然程序， PKS是否可以被重新编程以赋予它们非自然的功能。细菌中的模块化PKS （占聚酮化合物的约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取代催化结构域来产生这样的PKS， 外源PKS源。然而，许多这些杂合酶的催化效率受到损害 原因尚不清楚因此，PKS的可靠和成功的可重新编程性要求 对天然酶功能的结构和机制的透彻理解。 我们的目的首先是揭示两个核心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），从细菌模块PKS。这一目标 受到ACP/催化结构域复合物在聚酮化合物合成过程中的瞬时和反应性质的挑战。 处理.为了缓解这一障碍，我们计划将非天然氨基酸（UAA）与亲电氨基酸（EAA）结合。 将官能团引入ACP中，用于与相互的Cys/Lys亲核试剂进行域间交联。识别 交联物种将被应用于结构和经验建模研究。第二个目标，以高- 亲和抗原结合片段（Fab），旨在了解PKS的结构-功能关系 酮还原酶，和新的，支持的结构数据在这里报道。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 用单粒子低温电子显微镜（cryo-EM）来获得高分辨率结构。初级电子 显微镜和活性数据相关的这一目标的报告，和战略，以提高颗粒的质量， 概述。最后，预计拟议的研究将在UAA领域提供独特的培训机会 掺入和单颗粒冷冻EM;后者是通过与教授的合作促进。 实验室（斯坦福大学/SLAC），并靠近斯坦福大学/SLAC冷冻EM中心（S2 C2）。