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

描述(申请人提供)：基因组学革命一再证明，我们对天然产物(NP)生物合成的理解还远远不完整。鉴于现有和新兴基因组中沉默和神秘的生物合成簇的频率，再加上99%的微生物无法培养，已发现的微生物NPs远远不到1%。该项目建议表征来自两个土壤细菌的新的NP生物合成簇。毫无疑问，来自土壤细菌的NPs是我们最丰富的药物来源。解锁由这些生物编码的新型NPs的化学结构和生物功能具有巨大的潜力，可以扩大我们的药物库。对这一提议感兴趣的生物合成簇是最近描述的、进化上保守的家族成员，被称为噻唑/恶唑修饰微囊素(TOMM)。作为一个新的NP家族，TOMM代表了NP化学空间中一个未被开发的领域--很少有已知的结构或作用机制。所有已知活性的TOMM都具有毒素的功能，这使它们成为现代医学最感兴趣的东西。在两个已知病例中，由人类病原体产生的TOMMS在发病的分子机制中发挥着关键作用。因此，对生物合成途径的更完整的了解可能会导致毒力靶向抗生素的开发，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理解中的几个空白。这个项目分为三个相关但独立的具体目标。对于目标1，将使用体外重组、天然产物分离和高级光谱分析相结合的方法来确定TOMM产品的化学结构。在目标2中，将使用高分辨率质谱学和定点突变来动力学地评估催化噻唑和恶唑形成的第一步的关键酶。这种酶是一种环状脱水酶，负责识别TOMM前体多肽，并将半胱氨酸和丝氨酸/苏氨酸残基转化为噻唑啉和(甲基)恶唑啉。目的3试图揭示使底物识别和下游的噻唑/恶唑形成活性的蛋白质-蛋白质相互作用。通过表征参与TOMM生物合成的酶的特征，将为未来的工作奠定基础，包括从人类病原体中开发TOMMS的生物合成抑制剂，以及利用组合生物合成的力量来进化具有所需生物靶点的TOMMS的策略。该项目的进展将填补我们目前对多肽衍生毒素子集如何生物合成的一个主要空白。所开发的工具将广泛适用于其他TOMM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.