Biosynthesis of antifungal nucleoside antibiotics

项目总结/摘要 肽基核苷（PNs）是天然存在的抗真菌剂，对多种病原真菌具有活性 例如“山谷热”的致病因子。它们还表现出与临床批准的 抗真菌药物我们的长期目标是提供一个全面的了解生物合成的 PN及其作用方式。本申请集中于PN的生物合成。了解PN 生物合成途径将为通过工程改造产生结构多样的PN类似物提供基础。 生物合成、半合成和基因组挖掘。在上一个融资周期中，我们发现PN 通过隐蔽磷酸化和通过氧化C-C键断裂的碳水化合物剪裁来生物合成。对 这些发现的基础上，在本申请中，我们将进行功能和机制表征的 生物合成酶为基因组挖掘发现新的天然核苷提供基础 产物和非天然PN的化学酶促合成。在目的1中，研究了去除隐蔽性蛋白的机制。 磷酸化和其他核苷生物合成途径中的隐蔽磷酸化的普遍性将被 研究了在目标2中，将研究核苷天然产物的自由基介导的趋异生物合成。 通过研究氧化C-C键断裂机制和基因组挖掘表征， 同源酶在目标3中，将表征通过NikS/PolG酶的酰胺连接的机制， 并将研究它们在化学酶法制备PNs中的应用潜力。拟议 这项研究意义重大，因为它将为未来的生物合成和化学酶生成提供基础。 新的治疗PN以及新的抗真菌核苷天然产物的基因组挖掘发现。

Project Summary/Abstract Peptidyl nucleosides (PNs) are naturally occurring antifungal agents active against multiple pathogenic fungi such as the causal agent of “Valley Fever”. They also exhibit potent synergistic effects with clinically approved antifungal drugs. Our long-term goal is to provide a comprehensive understanding of both the biosynthesis of PNs and their mode of action. The current application focuses on the biosynthesis of PNs. Understanding PN biosynthetic pathways will provide a basis for creating structurally diverse PN analogs through engineered biosynthesis, semi synthesis and genome mining. In the previous funding cycle, we found that PNs are biosynthesized through cryptic phosphorylation and carbohydrate tailoring by oxidative C-C bond cleavage. On the basis of these findings, in this application, we will perform functional and mechanistic characterization of the biosynthetic enzymes to provide the foundation for genome mining discovery of novel nucleoside natural products and chemoenzymatic synthesis of unnatural PNs. In Aim 1, the mechanism of removal of cryptic phosphorylation and the generality of cryptic phosphorylation in other nucleoside biosynthesis pathways will be investigated. In Aim 2, the radical mediated divergent biosynthesis of nucleoside natural products will be investigated by studying the mechanism of oxidative C-C bond cleavage and genome mining characterization of homologous enzymes. In Aim 3, the mechanism of amide ligation by NikS/PolG enzymes will be characterized, and their potentials for use in the chemoenzymatic preparation of PNs will be investigated. The proposed research is significant because it will provide a basis for the future biosynthetic and chemoenzymatic generation of novel therapeutic PNs as well as genome mining discovery of novel antifungal nucleoside natural products.