Mechanistic Investigation of Copper-Dependent Peptide Cyclases for Macrocycle Engineering

项目摘要 大环肽是抗生素药物发现的有效支架，因为它们可以联合收割机结合口服抗生素。 具有代谢稳定性和靶向性的小分子药物的生物利用度和细胞膜通透性 生物制品的特殊性14元双环达柔菌素是针对革兰氏阴性菌的肽抗生素先导结构。 多重耐药菌阴性。Darobactin由两个侧链-侧链-大环键定义， 由自由基S-腺苷甲硫氨酸（SAM）铁硫簇酶环化。由于合成的挑战 darobactin大环复杂性及其自由基SAM环化酶的厌氧性质，一种生物催化剂， 需要一种替代方法来在需氧环境中生产和多样化14元双环肽。打嗝 结构域蛋白最近已从植物基因组中被表征为铜依赖性自催化蛋白 肽环化酶，其在需氧条件下催化darobactin型大环化合物的形成。 BURP结构域蛋白是植物核糖体编码的前体肽， 修饰肽（RIPPs）。BURP结构域前体肽包括核心肽基序和C-末端BURP结构域。 结构域，其催化铜依赖性的核心肽中的氨基酸侧链的环化。 反应BURP结构域衍生的肽具有不同的大环：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 由于BURP结构域环化酶的结构和作用机制尚不清楚，因此BURP结构域环化酶的结构和作用机制尚不清楚。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，基于自由基的 机制，并需要分子氧进行催化。电子顺磁共振将确定是否存在 自由基种类和Cu（I）在BURP结构域催化中的作用，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表征作为辅因子的双氧。在本提案中， 两个代表性BURP结构域的蛋白质结构将在具体目标1中确定。I型打嗝 结构域环化酶编码BURP结构域内的单个核心肽，由来自 花生，不打嗝。II型BURP结构域环化酶具有一个重复的N-末端核心肽结构域， 的BURP域，并将从非洲石松，肽双环酶SkrBURP进行研究。具体 目的2利用AhyBURP和SkrBURP研究BURP结构域的催化机制。我还预测， BURP结构域环化酶可以被工程化以产生定制的大环。具体目标3是生成模拟 通过SkrBURP的合理设计，并测试这些darobactin模拟物的功效， 抵抗耐药致病菌。BURP结构域环化酶工程的研究 代表了产生新的大环肽文库以解决抗微生物剂抗性的可能性。

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.