NMR studies of heterocyclization and epimerization in yersiniabactin synthesis

耶尔森菌素合成中杂环化和差向异构化的 NMR 研究

• 批准号: 8667485
• 负责人: Dominique Pascal Frueh
• 金额: $ 30.78万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8667485
• 关键词: Anabolism; Antibiotics; Antineoplastic Agents; Bacitracin; Bacteria; Binding; Binding Sites; Biochemical; Biological Assay; Biological Factors; Biological Models; Bleomycin; Bubonic Plague; Carrier Proteins; Catalytic Domain; Chemicals; Cholera; Collaborations; Communication; Complex; Cyclization; Cyclosporine; DNA Sequence Rearrangement; Data; Diagnostic; Drug Design; Engineering; Epothilones; Equilibrium; Food; Goals; Immunosuppressive Agents; Infection; Isotope Labeling; Knowledge; Logic; Methods; Modification; Molecular; Molecular Probes; Monitor; Mutagenesis; Mycobacterium tuberculosis; Nature; Nuclear Magnetic Resonance; Penicillins; Peptides; Pharmacologic Substance; Plague; Production; Property; Protein Dynamics; Proteins; Reaction; Research; Signal Transduction; Sirolimus; Solutions; Specificity; Structure; Substrate Interaction; System; Techniques; Therapeutic; Tuberculosis; Urinary tract infection; Uropathogenic E. coli; Vertebral column; Vibrio cholerae; Virulence; Virulence Factors; Yersinia enterocolitica; Yersinia pestis; antitumor agent; bacterial resistance; base; biological systems; conformer; design; epimerization; frontier; fungus; improved; inhibitor/antagonist; microbial; novel; novel therapeutics; pathogen; pathogenic bacteria; peptide synthase; protein 50 kDa; public health relevance; research study; stereochemistry; success; yersiniabactin

DESCRIPTION (provided by applicant): Non-ribosomal peptide synthetases (NRPSs) are enzymatic assembly lines that produce a wealth of natural products in bacteria and fungi. These products confer virulence to pathogens and often are valuable therapeutics, including antibiotics (penicillin, bacitracin), antitumor agents (bleomycin, epothilone), and immunosuppressants (rapamycin). NRPSs use multiple domains, organized in contiguous modules, to covalently load, modify, and join substrates in an assembly line fashion. This remarkable organization holds the promise of producing novel pharmaceuticals by swapping domains or modules to reprogram the NRPS assembly line. However, most NRPS domain interactions remain uncharacterized, the structure and mechanism of important domains are unknown, and artificially engineered NRPSs are generally unproductive. This proposal aims to reveal the structural basis for heterocycle formation and alteration of their stereochemistry in NRPSs, and unravel domain communication during related synthesis. We will focus on HMWP2, an NRPS that participates in the synthesis of yersiniabactin (Ybt), a virulence factor found in pathogens such as Yersinia pestis, the causative agent of the bubonic plague, Y. enterocolitica, a food pathogen, and uropathogenic E. coli, responsible for urinary tract infections. Our results will contribute to understanding the molecular logic employed by these pathogens during infections. We will primarily use Nuclear Magnetic Resonance (NMR) because of the transient nature of molecular interactions, as well as the existence of multiple conformers in equilibrium. NMR will be used to determine the structures of cyclization and epimerization domains, identify binding sites of chemical and protein substrates, and characterize dynamics within domains during molecular interactions. In a synergistic approach, we will combine mutagenesis and biochemical assays with NMR experiments to provide an atomic level description of reaction mechanisms. The size of the multi-domain complexes reaches 70 kDa and is a challenge for NMR studies, which are typically limited to 20 kDa. In the past we designed methods to solve structures of 50 kDa proteins and obtain useful data from 800kDa complexes. HMWP2 will provide a model system to further develop new NMR methods for large dynamic proteins and to understand conformational rearrangements during protein interactions in general. Our research will simultaneously enable us to push the frontier in NMR studies of larger proteins, help understand the function of protein dynamics in biological systems, reveal the structure of critical domains, and provide a basis for efficient reprogramming of NRPS assembly lines to produce new pharmaceuticals.

描述（由申请人提供）：非核糖体肽合成酶（NRPS）是一种酶促装配线，可在细菌和真菌中产生丰富的天然产物。这些产品赋予病原体毒力，通常是有价值的治疗药物，包括抗生素（青霉素、杆菌肽）、抗肿瘤剂（博来霉素、埃坡霉素）和免疫抑制剂（雷帕霉素）。 NRPS 使用多个域，以连续模块的形式组织，以装配线方式共价加载、修改和连接基底。这个非凡的组织有望通过交换域或模块来重新编程 NRPS 装配线来生产新型药物。然而，大多数 NRPS 结构域相互作用仍未得到表征，重要结构域的结构和机制尚不清楚，人工设计的 NRPS 通常是无效的。该提案旨在揭示 NRPS 中杂环形成及其立体化学改变的结构基础，并阐明相关合成过程中的域通信。我们将重点关注 HMWP2，这是一种参与耶尔森菌素 (Ybt) 合成的 NRPS，Ybt 是一种在病原体中发现的毒力因子，例如鼠疫耶尔森氏菌（黑死病的病原体）、小肠结肠炎耶尔森氏菌（一种食物病原体）和尿路致病性大肠杆菌（导致尿路感染）。我们的结果将有助于理解这些病原体在感染过程中所采用的分子逻辑。由于分子相互作用的瞬态性质以及平衡状态下存在多个构象异构体，我们将主要使用核磁共振 (NMR)。 NMR 将用于确定环化和差向异构化结构域的结构，识别化学和蛋白质底物的结合位点，并表征分子相互作用过程中结构域内的动力学。在协同方法中，我们将诱变和生化测定与核磁共振实验相结合，以提供反应机制的原子水平描述。多域复合物的大小达到 70 kDa，这对 NMR 研究来说是一个挑战，而 NMR 研究通常限制在 20 kDa。过去，我们设计了解决 50 kDa 蛋白质结构并从 800kDa 复合物中获取有用数据的方法。 HMWP2 将提供一个模型系统，以进一步开发大型动态蛋白质的新 NMR 方法，并了解蛋白质相互作用过程中的构象重排。我们的研究同时将使我们能够推动较大蛋白质的核磁共振研究的前沿，帮助了解生物系统中蛋白质动力学的功能，揭示关键域的结构，并为 NRPS 装配线的有效重编程以生产新药物提供基础。