Mechanistic Investigation of Copper-Dependent Peptide Cyclases for Macrocycle Engineering

用于大环工程的铜依赖性肽环化酶的机理研究

基本信息

批准号：
10464289
负责人：
Lisa Susannah Mydy
金额：
$ 7.39万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-15 至 2024-04-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10464289
关键词：
Active Sites Address Aerobic Affinity African Amino Acids Anaerobic Bacteria Antibiotics Antimicrobial Resistance Architecture Binding Biological Assay Biological Availability Biological Products C-terminal Calorimetry Carbon Catalysis Cell Membrane Permeability Chemicals Chemistry Copper Crystallization Cyclic Peptides Cyclization Dioxygen Drug resistance Electron Spin Resonance Spectroscopy Engineering Environment Enzymatic Biochemistry Enzyme Kinetics Enzymes Head Investigation Iron Knowledge Lead Libraries Life Lycopodiaceae Metabolic Monitor Multi-Drug Resistance Multiple Bacterial Drug Resistance N-terminal Names Natural Products Nature Oral Organism Oxidation-Reduction Oxygen Peptide Antibiotics Peptide Library Peptides Pharmaceutical Preparations Plant Genome Plants Production Proteins Proteolysis Proteomics Public Health Reaction Recombinants Research Resistance Ribosomes Role S-Adenosylmethionine Scheme Side Site-Directed Mutagenesis Source Specificity Structure Substrate Interaction Sulfur System Tail Tertiary Protein Structure Testing Titrations Tryptophan Tyrosine Vancomycin Work World Health Organization X ray spectroscopy base cofactor crosslink design drug discovery efficacy testing enzyme activity improved microbial pathogenic bacteria peptide structure polypeptide protein structure rational design reconstitution scaffold screening small molecule

项目摘要

PROJECT SUMMARY Macrocyclic peptides are effective scaffolds for antibiotic drug discovery as they can combine the oral bioavailability and cell membrane permeability of small molecule drugs with metabolic stability and target specificity of biologics. The 14-membered bicyclic darobactin is a peptide antibiotic lead structure against Gram- negative multi-drug resistant bacteria. Darobactin is defined by two side-chain-to-side-chain-macrocyclic bonds, cyclized by a radical S-adenosylmethionine (SAM) iron-sulfur cluster enzyme. Due to synthetic challenges towards darobactin macrocyclic complexity and the anaerobic nature of its radical SAM cyclase, a biocatalytic alternative is needed to produce and diversify 14-membered bicyclic peptides in an aerobic environment. BURP domain proteins have recently been characterized from plant genomes as copper-dependent autocatalytic peptide cyclases, which catalyze the formation of darobactin-type macrocycles under aerobic conditions. BURP domain proteins constitute precursor peptides of plant ribosomally-encoded and post-translationally modified peptides (RiPPs). BURP domain precursor peptides include core peptide motifs and a C-terminal BURP domain, which catalyzes the cyclization of amino acid side chains in the core peptide in a copper-dependent reaction. BURP domain-derived peptides have diverse macrocycles: mono- and bicyclic scaffolds, 14- to 21- membered rings, and C-O, C-N- and C-C-macrocyclic bonds. Despite the chemical diversity of their cyclopeptide products, the structure and mechanism of BURP domain cyclases are completely unknown. Based on preliminary work, I hypothesize that BURP domain cyclases use a redox active copper cofactor, a radical-based mechanism, and require dioxygen for catalysis. Electron paramagnetic resonance will identify the presence of radical species and Cu(I) in BURP domain catalysis, and anaerobic reconstitution of recombinant BURP domain cyclases followed by bottom-up proteomic analysis will characterize dioxygen as a cofactor. In this proposal, the protein structures of two representative BURP domains will be determined in Specific Aim 1. Type I BURP domain cyclases encode a single core peptide within the BURP domain, represented by the bicyclase from peanut, AhyBURP. Type II BURP domain cyclases have a repetitive N-terminal core peptide domain attached to the BURP domain, and will be investigated from African clubmoss, the peptide bicyclase SkrBURP. Specific Aim 2 uses AhyBURP and SkrBURP to elucidate the catalytic mechanism of BURP domains. I also predict that BURP domain cyclases can be engineered to yield tailored macrocycles. Specific Aim 3 is to generate mimics of the antibiotic darobactin by rational design of SkrBURP, and testing the efficacy of these darobactin mimics against drug-resistant pathogenic bacteria. The proposed research of BURP domain cyclase engineering represents the possibility to generate new macrocyclic peptide libraries to address antimicrobial resistance.

项目概要大环肽是抗生素药物发现的有效支架，因为它们可以结合口服药物具有代谢稳定性和靶点的小分子药物的生物利用度和细胞膜通透性生物制剂的特异性。 14 元双环darobactin 是一种针对革兰氏阴性菌的肽类抗生素先导结构。多重耐药菌阴性。 Darobactin 由两个侧链到侧链大环键定义，被自由基 S-腺苷甲硫氨酸 (SAM) 铁硫簇酶环化。由于合成挑战达罗巴汀大环复杂性及其自由基 SAM 环化酶（一种生物催化酶）的厌氧性质需要替代方案来在有氧环境中生产 14 元双环肽并使其多样化。打嗝最近，植物基因组中的结构域蛋白被定性为铜依赖性自催化肽环化酶，在有氧条件下催化达罗巴汀型大环化合物的形成。 BURP 结构域蛋白构成植物核糖体编码和翻译后的前体肽修饰肽（RiPP）。 BURP 结构域前体肽包括核心肽基序和 C 端 BURP 结构域，催化铜依赖性核心肽中氨基酸侧链的环化反应。 BURP 结构域衍生肽具有多种大环：单环和双环支架、14- 至 21- 元环和C-O、C-N-和C-C-大环键。尽管它们的环肽具有化学多样性产品，BURP结构域环化酶的结构和机制完全未知。基于在初步工作中，我假设 BURP 结构域环化酶使用氧化还原活性铜辅因子，这是一种基于自由基的机理，并且需要分子氧来催化。电子顺磁共振将识别是否存在 BURP 结构域催化中的自由基物种和 Cu(I)，以及重组 BURP 结构域的厌氧重建环化酶，然后自下而上的蛋白质组学分析将双氧表征为辅助因子。在该提案中，两个代表性 BURP 结构域的蛋白质结构将在特定目标 1 中确定。 I 型 BURP 结构域环化酶编码 BURP 结构域内的单核心肽，由来自的双环化酶代表花生，AhyBURP。 II 型 BURP 结构域环化酶具有重复的 N 末端核心肽结构域 BURP 结构域，并将从非洲石松中研究肽双环酶 SkrBURP。具体的目标 2 使用 AhyBURP 和 SkrBURP 阐明 BURP 结构域的催化机制。我也预测 BURP 结构域环化酶可以被设计来产生定制的大环化合物。具体目标 3 是生成模仿通过合理设计 SkrBURP 来制备抗生素 Darobactin，并测试这些 Darobactin 模拟物的功效针对耐药病原菌。 BURP结构域环化酶工程研究建议代表了生成新的大环肽库来解决抗菌素耐药性的可能性。