Characterization of the Thioacid Biosynthetic Machinery and Isolation of a Novel Thioacid Enediyne Natural Product

项目总结 天然产物一直走在新药发现的前沿，随着后现代药物时代的到来， 基因组时代，它们将如此美好地保留到未来。在这个过程中，数以千计的天然产品 已被表征，但在这些化合物中，很少被表征为含有硫代酸官能团。 目前，迫切需要发现具有新颖和有用的支架或生物活性的天然产品。 由于其非常稀有，因此，含有硫代酸的天然产物到目前为止大多还没有被开发。 最近，我们实验室证明了硫代酸官能团是由一对酶引入的， 由两个基因盒编码，催化硫从硫磺载体蛋白转移到羧酸 含酸的天然产物。这个基因盒现在已经在超过250个基因簇中被识别，还有更多 超过98%的天然产物没有特征和编码的不同家族的生物合成。为了 为了更好地描述这些新的硫代酸天然产物，并更好地了解其中涉及的化学成分， 相关的生物合成机械还有待进一步研究。由于已知的硫代酸的新颖性和生物活性- 由于含有天然产物，预计对这个化学空间的进一步探索将产生新的支架。 和酶活性。该项目的长期目标是充分表征硫代酸部分及其 生物合成显著提高了对稀有含硫天然产物的基本认识 和含硫的酶。这项建议包含两个目标：(I)详细的机械酶学 (包括动力学、结构表征、残基映射和底物范围的扩展) 硫代铂(TPTM)和硫代铂(TPTN)途径的硫酸生物合成机制和(Ii) 一种新的药用二炔硫代酸的异源生产和结构鉴定 结合同系硫酸机的生化分析。这其中的中心假设是 建议是：(1)tPTM/tPTN硫代酸机械的结构特征将有助于改进研究 其他硫代酸途径的发现和(Ii)另一种含硫代酸天然硫代酸的发现和表征 具有新型支架的产品将提供有关硫代酸官能团的包含的关键信息 在天然产品中。这一假说得到了揭示生物多样性的生物信息学分析的支持 自然界中含有硫代酸的天然产物生物合成基因簇-都含有相同的保守的 硫代酸生物合成基因盒。拟议研究的成果将是接触结构和 含硫酶的工程化变体及一种新的含硫酸烯二炔的发现 天然产物。

PROJECT SUMMARY Natural products have always been at the forefront of new drug discovery, and with the advent of the post- genomic era, they will remain so well into the future. During this process, thousands of natural products have been characterized, but of these, very few have been characterized to contain the thioacid functional group. Currently, there is a critical need to discover natural products that have novel and useful scaffolds or bioactivities. Due to its extraordinary rarity, the thioacid-containing natural products therefore are mostly unexplored to date. Recently, our lab has demonstrated that the thioacid functional group is introduced by a pair of enzymes, encoded by a two-gene cassette, that catalyze the transfer of sulfur from a sulfur carrier protein to a carboxylic acid-containing natural product. This gene cassette has now been identified in over 250 gene clusters, with more than 98% uncharacterized and encoding the biosynthesis of diverse families of natural products. In order to better characterize these new thioacid natural products, and to better understand the chemistry involved, the relevant biosynthetic machinery needs further study. Due to the novelty and bioactivities of the known thioacid- containing natural products, it is expected that further exploration of this chemical space will yield new scaffolds and enzymatic activities. The long-term goal of this project is to fully characterize the thioacid moiety and its biosynthesis for a significantly improved fundamental understanding of rare sulfur-containing natural products and sulfur-incorporating enzymes. This proposal contains two aims: (i) detailed mechanistic enzymology (including kinetics, structural characterization, residue mapping, and expansion of substrate scope) of the thioacid biosynthetic machinery from the thioplatensimycin (tPTM) and thioplatencin (tPTN) pathway and (ii) the heterologous production and structural elucidation of a novel and medicinally relevant enediyne thioacid in combination with biochemical analysis of the cognate thioacid machinery. The central hypotheses of this proposal are that (i) structural characterization of the tPTM/tPTN thioacid machinery will enable improved study of other thioacid pathways and (ii) discovery and characterization of an additional thioacid-containing natural product with a novel scaffold will provide crucial information about the inclusion of the thioacid functional group in natural products. This hypothesis is supported by a bioinformatic analysis unveiling the rich diversity of thioacid-containing natural product biosynthetic gene clusters in Nature—all containing the same well-conserved thioacid biosynthesis gene cassette. The outcomes of the proposed research will be access to structures and engineered variants of sulfur-incorporating enzymes and discovery of a novel thioacid-containing enediyne natural product.