Chemical and biological exploration of a new natural product family, the thiazole

描述（申请人提供）：基因组学革命一再表明，我们对天然产物（NP）生物合成的理解远未完成。考虑到现有和新兴基因组中沉默和隐蔽的生物合成簇的频率，再加上无法培养>99%的微生物，发现的微生物NP远低于1%。该项目提出从两种土壤细菌中表征新的NP生物合成簇。毫无疑问，来自土壤细菌的纳米颗粒是我们最丰富的药物来源。解开由这些生物体编码的新型NP的化学结构和生物学功能对于扩大我们的药物库具有巨大的潜力。该提议感兴趣的生物合成簇是最近描述的进化保守家族的成员，该家族被称为噻唑/恶唑修饰的微菌素（TOMM）。作为一个新的NP家族，TOR 4代表了NP化学空间的一个未被探索的领域-很少有已知的结构或作用机制。所有具有已知活性的毒素都具有毒素功能，使它们成为现代医学的最高兴趣。在两个已知的情况下，由人类病原体产生的TOE 2在发病的分子机制中起关键作用。因此，对生物合成途径的更全面了解可能会导致开发毒性靶向抗生素，这是我们研究计划的长期目标。为了有效地挖掘这一潜力，必须解决我们目前对这些分子了解中的一些差距。该项目分为三个相关但独立的具体目标。对于目标1，将采用体外复溶、天然产物分离和高级光谱学的组合来确定TOMM产物的化学结构。在目标2中，高分辨率质谱和定点诱变将用于动力学评估催化噻唑和恶唑形成的第一步的关键酶。这种酶是一种环化酶，负责识别TOMM前体肽并将Cys和Ser/Thr残基转化为噻唑啉和（甲基）恶唑啉。目的3旨在揭示蛋白质-蛋白质相互作用，使底物识别和下游噻唑/恶唑形成活性。通过表征参与TOMM生物合成的酶，将为未来的工作奠定基础，包括开发来自人类病原体的TOR 4的生物合成抑制剂，以及利用组合生物合成的力量来发展具有所需生物靶标的TOR 4的策略。这个项目的进展将填补我们目前对肽衍生毒素的一个子集是如何生物合成的理解中的一个主要空白。所开发的工具将广泛适用于其他TOEFL的研究。

DESCRIPTION (provided by applicant): The genomics revolution has repeatedly demonstrated that our understanding of natural product (NP) biosynthesis is far from complete. Given the frequency of silent and cryptic biosynthetic clusters in existing and emerging genomes, compounded with the inability to culture >99% of microbes, far less than 1% of microbial NPs have been discovered. This project proposes to characterize novel NP biosynthetic clusters from two soil-dwelling bacteria. Without question, NPs from soil bacteria are our most prolific source of medicine. Unlocking the chemical structure and biological function of novel NPs encoded by these organisms holds enormous potential for expanding our pharmaceutical repertoire. The biosynthetic clusters of interest to this proposal are members of a recently described, evolutionarily conserved family dubbed the thiazole/oxazole-modified microcins (TOMM). As a new NP family, TOMMs represent an underexplored area of NP chemical space - few have a known structure or mechanism of action. All TOMMs with a known activity function as toxins, making them of paramount interest to modern medicine. In two known cases, TOMMs produced by human pathogens play a critical role in the molecular mechanism of pathogenesis. Therefore, a more complete knowledge of the biosynthetic pathway could lead to the development of virulence-targeting antibiotics, which represents a longer-term objective for our research program. To effectively tap into this potential, several gaps in our current understanding of these molecules must be addressed. This project is divided into three related, but independent specific aims. For Aim 1, a combination of in vitro reconstitution, natural product isolation, and advanced spectroscopy will be employed to determine the chemical structure of the TOMM product. In Aim 2, high-resolution mass spectrometry and site-directed mutagenesis will be used to kinetically evaluate a key enzyme that catalyzes the first step in the formation of thiazoles and oxazoles. This enzyme, a cyclodehydratase, is responsible for recognizing the TOMM precursor peptide and converting Cys and Ser/Thr residues into thiazolines and (methyl) oxazolines. Aim 3 seeks to reveal the protein-protein interactions that enable substrate recognition and the downstream thiazole/oxazole forming activity. By characterizing the enzymes involved in TOMM biosynthesis, the foundation for future work will be laid, including the development of biosynthetic inhibitors of TOMMs from human pathogens and strategies to harness the power of combinatorial biosynthesis to evolve TOMMs with desired biological targets. Progress on this project will fill a major void in our current understanding of how a subset of peptide-derived toxins is biosynthesized. The tools developed will be broadly applicable to the study of other TOMMs.