Biosynthesis of peptidyl nucleoside antifungal antibiotics

 描述（由申请人提供）：肽基核苷（PN）是天然存在的抗真菌剂，对多种病原性真菌具有活性，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的有效协同作用。我们 长期目标是提供对PNs生物合成及其作用模式的全面了解。本申请集中于PN的生物合成。了解PN的生物合成途径将提供一个基础，通过工程生物合成，半合成和基因组挖掘创造结构多样的PN类似物。在这个应用程序中，我们将建立PN生物合成的共同步骤，通过测试的中心假设，即共同的结构特征，所有PN（氨基己糖醛酸及其酰胺键的氨基酸）是通过7个保守的酶的作用生物合成。在目标1中，NikJ，一种新发现的自由基依赖性酶，负责合成一个关键的环核苷酸中间体，将机械特性。在目标2中，NikS（负责将N末端氨基酸连接至核苷（氨基己糖醛酸）的酶）将表征其底物特异性及其用于化学酶法制备PN的潜力。在目标3中，将通过基因敲除实验鉴定负责氨基己糖醛酸形成的酶。这项研究意义重大，因为它将为未来生物合成和化学酶促生成新型治疗性PN提供基础。

 DESCRIPTION (provided by applicant): 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 the PN biosynthetic pathways will provide a basis for creating structurally diverse PN analogs through engineered biosynthesis, semi synthesis and genome mining. In this application, we will establish the common steps in PN biosynthesis by testing the central hypothesis that the structural features common to all PNs (aminohexuronic acid and its amide linkage to an amino acid) are biosynthesized through the action of seven conserved enzymes. In Aim 1, NikJ, a newly identified free radical dependent enzyme responsible for the synthesis of a key cyclic nucleotide intermediate, will be mechanistically characterized. In Aim 2, NikS, the enzyme responsible for the ligation of the N-terminal amino acid to nucleoside (aminohexuronic acid), will be characterized for both its substrate specificity and its potential for use in the chemoenzymatic preparation of PNs. In Aim 3, the enzymes responsible for aminohexuronic acid formation will be identified through gene knockout experiments. The proposed research is significant because it will provide a basis for the future biosynthetic and chemoenzymatic generation of novel therapeutic PNs.