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 是最近从光杆菌中分离出来的一种翻译后修饰的肽抗生素 该分子包含一种新型的内部氧化交联模式。 导致其预组织为β片层模拟物，该化合物与β桶蛋白BamA结合并 Darobactin A 广泛抑制细菌外膜蛋白折叠所必需的伴侣功能。 在体外和感染动物模型中对革兰氏阴性病原体具有活性。 dar 操纵子编码单个自由基 SAM 酶，可催化氧化交联的形成 我们尚不清楚在天然产物中观察到的这一显着结果背后的机制。 当在铁和硫下正确重构时，表达并纯化带有 His 标签的重组 DarE。 厌氧条件下，铁滴定数据表明该酶含有三个铁硫簇，并且它迅速 从 SAM 生成 5dAdo –​​ 表明重构的 DarE 作为 SPASM 酶具有活性。 结合化学合成和生物合成计划来研究 DarE 酶学并设计半 可以大规模生产的天然产物的合成形式具有很大的前景。 自从一类对革兰氏阴性菌感染具有活性的新型抗生素问世以来，已有近 60 年的历史。 发达。