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).

项目摘要/摘要 聚客体天然产物在生命的所有领域都广泛，主要发生在 细菌，植物和真菌。它们巨大的结构多样性使近50年的研究成为了一个大型， 以及在寻找潜在有用的生物活性率的聚酮化合物化合物的成长中。确实，很多 已经鉴定出了暴露于抗菌，抗癌，抗真菌，抗病毒，抗炎，抗炎， 免疫抑制和降低胆固醇的特性；因此，强调其价值作为潜在的来源 毒品。方便的是，聚酮化合物合酶（PKS）的子集，该酶在自然界中伪造了聚酮化合物 类似于单个多肽上包含多个催化结构域的模块化装配线。这样 根据观察到的PKS结构域顺序，将聚酮化合物产物结构模板。那，那里 似乎存在一个自然程序，该程序是基于聚酮化合物的生物合成逻辑的基础，这使许多人想知道 是否可以对PKS进行重编程以赋予它们具有不自然的函数。细菌中的模块化PKS （占聚酮底层约50％的占约50％）为非天然PKS组合工程提供令人兴奋的前景 有了新的功能，许多努力试图通过从中取代催化域来创建此类PKS 外源性PK来源。但是，许多这些杂化酶的催化效率受到损害 出于不太了解的原因。因此，PKS可靠且成功的重编程性需要 对管理天然酶功能的结构和机制有透彻的理解。 我们首先旨在揭示两个核心PK的酰基载体蛋白（ACP）识别的分子碱基 来自细菌模块化PK的催化结构域，酮合酶（KS）和酰基转移酶（AT）。这个目标有 聚酮化合物期间ACP/催化域复合物的瞬时和反应性挑战 加工。为了减轻这种障碍，我们计划将不自然的氨基酸（UAA）与亲电的氨基酸（UAAS）结合在一起 官能团进入ACP，以与相互CYS/LYS核致化学交联的域间交联。确定 交联物种将应用于结构和经验建模研究。第二个目标，由高 亲和力抗原结合片段（FABS）试图了解PK的结构功能关系 酮核酶和新的支持结构数据在这里报告。在最后一个目标中，我们建议研究 PKS模块的构象动力学使用Fab，化学交联和其他稳定器合并 与单个颗粒冷冻电子显微镜（Cryo-EM）一起获得高分辨率结构。初步电子 报道了与此目标相关的显微镜和活动数据，改善粒子质量的策略是 概述。最后，拟议的研究有望在UAA领域提供独特的培训机会 掺入和单个粒子冷冻EM；后者是通过与Wah Chiu教授合作准备的 实验室（斯坦福/SLAC），靠近斯坦福/SLAC CRYO-EM中心（S2C2）。