Mechanistic Investigation of Copper-Dependent Peptide Cyclases for Macrocycle Engineering

项目总结 大环肽可以与口服药物结合，是抗生素药物开发的有效支架。 具有代谢稳定性和靶向性的小分子药物生物利用度和细胞膜通透性 生物制品的专一性。14元双环Darobactin是一种抗革兰氏杆菌的多肽抗生素先导结构。 阴性多重耐药菌。Darobactin由两个侧链到侧链的大环键定义， 由自由基S-腺苷甲硫氨酸铁硫簇酶环化。由于合成方面的挑战 论Darobactin大环的复杂性及其生物催化剂自由基SAM环酶的厌氧性 在有氧环境中生产和多样化14元双环肽需要替代物。打嗝 最近，植物基因组中的结构域蛋白被表征为依赖铜的自催化 多肽环化酶，在有氧条件下催化形成Darobactin类型的大环。 Burp结构域蛋白构成植物核糖体编码和翻译后的前体多肽 修饰多肽(RIPPS)。Burp结构域前体多肽包括核心肽基序和C-末端Burp 结构域，它催化铜依赖的核心肽中氨基酸侧链的环化。 反应。BURP结构域衍生的多肽具有不同的大环：单环和双环支架，14-到21- 成员环，以及C-O、C-N-和C-C-大环键。尽管它们的环肽具有化学多样性 目前，Burp结构域环化酶的结构和作用机制还完全不清楚。基于 初步工作，我假设打嗝结构域环化酶使用氧化还原活性铜辅因子，一种基于自由基的 机理，并且需要氧气来催化。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铜(I)及重组BURP结构域的厌氧重组 循环酶和自下而上的蛋白质组学分析将把氧气定性为辅因子。在这项提案中， 两个有代表性的打嗝结构域的蛋白质结构将在特定的目标中确定：1.I型打嗝 结构域环化酶编码Burp结构域中的单个核心肽，由来自 花生，AhyBURP。II型Burp结构域环化酶连接有重复的N-末端核心多肽结构域 到BURP结构域，并将从非洲丛生苔藓中进行研究，这种多肽自行车酶SkrBURP。特定的 目的2利用AhyBURP和SkrBURP来阐明BURP结构域的催化机制。我还预测， BUMP结构域环化酶可以被设计成产生定制的大环。具体目标3是生成模拟 通过合理设计SkrBURP，筛选出抗菌药物Darobactin，并测试这些Darobactin模拟物的疗效 对抗耐药病原菌。BUMP结构域环化酶工程的研究进展 代表了产生新的大环肽文库以解决抗菌素耐药性的可能性。

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.