Chemoenzymatic Synthesis of Darobactin Antibiotics

Darobactin抗生素的化学酶法合成

• 批准号: 10592211
• 负责人: Patrick G. Harran
• 金额: $ 23.41万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-11 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10592211
• 关键词: Anabolism; Animal Model; Antibiotics; Bacterial Outer Membrane Proteins; Binding; C-terminal; Catalysis; Chemicals; Cyclic Amino Acids; Dangerousness; Data; Dipeptides; Electronics; Electrons; Engineering; Enzymatic Biochemistry; Enzymes; Esters; Goals; Human; Hydrolysis; In Vitro; Infection; Iron; Knowledge; Ligation; Methodology; Methods; Modification; Molecular Chaperones; Multi-Drug Resistance; Natural Products; Nematoda; Operon; Outcome; Pathway interactions; Pattern; Peptide Antibiotics; Peptides; Photorhabdus; Process; Proline; Proteins; Reaction; Recombinants; Series; Sulfur; Supporting Cell; Titrations; analog; beta barrel; beta pleated sheet; chemical synthesis; commensal bacteria; crosslink; experimental study; mimetics; novel; novel antibiotic class; novel therapeutics; pathogen; pathogenic bacteria; programs; reconstitution; stable plasma protein solution; symbiont

Abstract Darobactin A is a post-translationally modified peptide antibiotic recently isolated from photorhabdus symbionts present in nematodes. The molecule contains a novel pattern of internal oxidative cross links that result in its pre-organization as a beta sheet mimetic. The compound binds to the beta barrel protein BamA and inhibits chaperone functions essential for folding of bacterial outer membrane proteins. Darobactin A is broadly active against gram-negative pathogens in vitro and in animal models of infection. The dar operon encodes a single radical SAM enzyme that catalyzes formation of both oxidative cross links observed in the natural product. The mechanism(s) behind this remarkable outcome is not yet known. We have expressed and purified recombinant His-tagged DarE. When properly reconstituted with iron and sulfur under anaerobic conditions, iron titration data indicates the enzyme contains three iron sulfur clusters and it rapidly generates 5dAdo from SAM – indicating reconstituted DarE is active as a SPASM enzyme. Here we propose a combined chemical synthesis and biosynthesis program to study DarE enzymology and to engineer semi- synthetic forms of the natural product that can be produced on scale. Such studies hold considerable promise. It has been nearly 60 years since a new class of antibiotics active against gram negative infections have been developed.

抽象的 Darobactin A是一种从Photorhabdus分离出来的翻译后修饰的肽抗生素 线虫中存在的共生体。该分子包含一种新型的内部氧化交叉连接模式 导致其作为Beta片模拟物进行预组织。该化合物与β枪管蛋白巴马和 抑制伴侣的功能对于细菌外膜蛋白的折叠至关重要。 darobactin a广泛 在体外和动物感染模型中对革兰氏阴性病原体进行活跃。 DAR Operaon编码一种催化两种氧化物交叉链路形成的单个自由基SAM酶 在天然产物中观察到。这种显着结果背后的机制尚不清楚。我们有 表达和纯化的重组率敢于敢于。正确地用铁和硫在下面 厌氧条件，铁滴定数据表明该酶含有三个铁硫簇，并且迅速 从SAM产生5DADO-表明重构的Dare是痉挛酶的活性。在这里我们提出一个 化学合成和生物合成计划的合并，以研究DARE酶学和工程师半 天然产物的合成形式，可以大规模生产。这样的研究具有可观的承诺。 自从针对革兰氏阴性感染活跃的新型抗生素已经过去了将近60年 发达。