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

• 批准号: 10684663
• 负责人: Lisa Susannah Mydy
• 金额: $ 7.84万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-15 至 2024-04-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10684663
• 关键词: Active Sites; Address; Aerobic; Affinity; African; Amino Acids; Anabolism; Antibiotics; Antimicrobial Resistance; Architecture; Binding; Biological Assay; Biological Availability; Biological Products; C-terminal; Calorimetry; Carbon; Catalysis; Cell Membrane Permeability; Chemicals; Chemistry; Copper; 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; Periodicity; Pharmaceutical Preparations; Plant Genome; Plants; Production; Proteins; Proteolysis; Proteomics; Public Health; Reaction; Recombinants; Research; Resistance; Ribosomes; Role; S-Adenosylhomocysteine; 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; cofactor; crosslink; design; drug discovery; efficacy testing; enzyme activity; improved; member; microbial; pathogenic bacteria; peptide natural products; peptide structure; polypeptide; protein structure; rational design; reconstitution; scaffold; screening; small molecule; Active Sites; Address; Aerobic; Affinity; African; Amino Acids; Anabolism; Antibiotics; Antimicrobial Resistance; Architecture; Binding; Biological Assay; Biological Availability; Biological Products; C-terminal; Calorimetry; Carbon; Catalysis; Cell Membrane Permeability; Chemicals; Chemistry; Copper; 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; Periodicity; Pharmaceutical Preparations; Plant Genome; Plants; Production; Proteins; Proteolysis; Proteomics; Public Health; Reaction; Recombinants; Research; Resistance; Ribosomes; Role; S-Adenosylhomocysteine; 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; cofactor; crosslink; design; drug discovery; efficacy testing; enzyme activity; improved; member; microbial; pathogenic bacteria; peptide natural products; peptide structure; polypeptide; protein structure; rational design; reconstitution; scaffold; screening; small molecule

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.

项目摘要 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 具有代谢稳定性和靶标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元的双环蛋白脱脂蛋白是一种针对革兰氏阴性的肽抗生素铅结构 阴性多药物抗性细菌。 darobactin由两个侧链链链巨环键定义， 由自由基的S-腺苷甲硫代（SAM）铁硫簇酶循环。由于综合挑战 朝向darobactin的大环复杂性和其自由基SAM环酶的厌氧性，一种生物催化 需要替代方法来在有氧环境中产生和多样化14元的双环肽。打bur 域蛋白最近以植物基因组为特征为铜依赖性自催化 肽环化酶在有氧条件下催化了darobactin型大环的形成。 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域前体肽包括核心肽基序和C末端BURP 结构域，它催化核心肽中氨基酸侧链的环化在铜依赖性 反应。 Burp域衍生的肽具有多种大环：单一和双环脚手架，14至21-- 成员环，以及C-O，C-N-和C-C-C-Crocyclic键。尽管其环肽的化学多样性 产物，Burp域循环酶的结构和机制是完全未知的。基于 初步工作，我假设Burp域循环酶使用氧化还原活性铜辅助因子，这是一种基于自由基的辅助因子 机制，需要二氧化物才能催化。电子顺磁共振将确定 BURP域催化中的激进物种和Cu（I），以及重组Burp域的厌氧重建 随后进行自下而上的蛋白质组学分析将表征二恶英作为辅因子。在此提案中， 两个代表性的BURP域的蛋白质结构将在特定的目标1中确定。 域循环酶编码Burp域内的单个核心肽，由Bicyclase表示 花生，艾堡。 II型Burp域循环酶具有附着的重复的N末端核心肽结构域 到Burp域，将从非洲俱乐部蛋白酶型肽施加酶Skrburp进行调查。具体的 AIM 2使用Ahyburp和Skrburp阐明Burp域的催化机制。我也预测 可以设计Burp域循环酶以产生量身定制的大环。特定目标3是生成模仿 Skrburp合理设计的抗生素darobactin，并测试这些darobactin Mimics的功效 针对耐药的致病细菌。拟议的BURP域Cyclase Engineering的研究 代表生成新的大环肽库以解决抗菌耐药性的可能性。