Chemoenzymatic Synthesis of Darobactin Antibiotics

摘要 Darobactin A是最近从光杆状菌中分离出的一种后修饰肽抗生素 线虫体内的共生体。该分子含有一种新的内部氧化交联模式， 导致其作为β折叠模拟物的预组织。该化合物与β桶蛋白BamA结合， 抑制细菌外膜蛋白折叠所必需的伴侣蛋白功能。Darobactin A广泛用于 在体外和动物感染模型中对革兰氏阴性病原体有活性。 dar操纵子编码一种单自由基SAM酶，该酶催化氧化交联的形成 在天然产品中观察到。这一显著结果背后的机制尚不清楚。我们有 表达和纯化的重组His标记的DarE。当适当地与铁和硫重组时， 在厌氧条件下，铁滴定数据表明该酶含有三个铁硫簇， 从SAM产生5dAdo-表明重构的DarE作为SPASM酶是有活性的。在这里我们提出了一个 结合化学合成和生物合成程序，研究DarE酶学和工程半 可以大规模生产的天然产物的合成形式。这样的研究有相当大的希望。 自从一种对革兰氏阴性菌感染有活性的新型抗生素被发现以来，已经有近60年的时间了。 开发

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.