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

硫代酸生物合成机制的表征及新型硫代烯二炔天然产物的分离

• 批准号: 10189667
• 负责人: Robert Edward Kalkreuter
• 金额: $ 4.73万
• 依托单位: SCRIPPS FLORIDA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-16 至 2022-04-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10189667
• 关键词: Active Sites; Address; Anabolism; Binding; Biochemical; Bioinformatics; Biological Assay; Carboxylic Acids; Carrier Proteins; Catalysis; Chemicals; Chemistry; Clinical; Crystallization; Crystallography; DNA Damage; DNA cassette; Data; Engineering; Enzymatic Biochemistry; Enzymes; Family; Fermentation; Future; Gene Cluster; Genetic; Genome; Genomics; Goals; Kinetics; Knowledge; Laboratories; Lead; Mining; Natural Products; Nature; Organism; Outcome; Pathway interactions; Peptides; Pharmaceutical Preparations; Prevalence; Process; Production; Property; Proteins; Research; Resistance; Resources; Source; Streptomyces; Structure; Sulfur; System; Terpenes; Therapeutic Agents; Time; Variant; Work; analog; anti-cancer; base; clinically relevant; drug discovery; functional group; genomic data; improved; innovation; insight; microbial; novel; novel therapeutics; prototype; scaffold; tool

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.

项目概要 天然产物一直处于新药发现的最前沿，并且随着后药物时代的出现 基因组时代，它们将在未来保持如此良好的状态，在此过程中，数以千计的天然产品已经出现。 已被表征，但其中很少有被表征为含有硫代酸官能团。 目前，迫切需要发现具有新颖且有用的支架或生物活性的天然产物。 由于其极其稀有，含硫代酸的天然产物迄今为止大多尚未被开发。 最近，我们的实验室证明了硫代酸官能团是由一对酶引入的， 由双基因盒编码，催化硫从硫载体蛋白转移到羧基 该基因盒现已在 250 多个基因簇中被发现，其中还有更多。 超过 98% 未被表征并编码不同天然产物家族的生物合成。 更好地表征这些新的硫代酸天然产物，并更好地了解所涉及的化学， 由于已知硫代酸的新颖性和生物活性，相关的生物合成机制需要进一步研究。 含有天然产物，预计对该化学空间的进一步探索将产生新的支架 该项目的长期目标是充分表征硫代酸部分及其酶活性。 生物合成显着提高对稀有含硫天然产物的基本了解 该提案包含两个目标：（i）详细的酶学机制。 （包括动力学、结构表征、残基图谱和底物范围的扩展） 来自硫铂霉素 (tPTM) 和硫铂霉素 (tPTN) 途径的硫代酸生物合成机制以及 (ii) 一种新型且具有药用价值的烯二炔硫代酸的异源生产和结构阐明 与同源硫代酸机制的生化分析相结合，这是该中心的假设。 建议是 (i) tPTM/tPTN 硫代酸机制的结构表征将有助于改进研究 其他硫代酸途径的研究和（ii）另一种含硫代酸的天然物质的发现和表征 具有新型支架的产品将提供有关包含硫代酸官能团的重要信息 这一假设得到了生物信息学分析的支持，揭示了丰富的多样性。 自然界中含硫代酸的天然产物生物合成基因簇——均含有相同且保守的基因簇 硫代酸生物合成基因盒的研究成果将是获得结构和 掺硫酶的工程变体和新型含硫代烯二炔的发现 天然产品。