Structural Studies of Nonribosomal Peptide Synthesis

微生物生产天然产物，以便在独特的环境中茁壮成长，在那里它们面临着广泛的 各种条件和相互竞争的有机体。这些不同的分子通常是 个体物种并在细胞通讯和信号传递中发挥作用，物种间 竞争、营养获取和毒力。天然产物在体内发挥作用的能力 生物环境使它们成为发现新药的有吸引力的候选者。 事实上，大约三分之二的小分子药物是从天然产品中提取的。 了解不寻常的天然产物的生物合成将有助于分析 数以千计的生物合成基因簇，没有已知的产物，可能导致发现 新的药用活性分子。一类以多肽为基础的天然产品是 由多域非核糖体肽合成酶(NRPS)产生。在数百个人中 其中的NRPS产品是重要的多肽抗生素万古霉素和替考巴坦，β-内酰胺 抗生素，如一些青霉素和头孢菌素，基因毒素Colibactin， 免疫抑制剂环孢素，以及细菌铁载体，如肠杆菌素，吡哆醇， 和霉菌素。NRPS使用非凡的装配线架构；多个蛋白质结构域 都连接在一个长度可以是数千或数万个残基的单一蛋白质中。 在生物合成过程中，氨基酸构建块被共价加载到载体结构域上，并 递送到邻近的催化结构域进行肽的延伸和修饰。对.的使用 核糖体和mRNA非依赖性合成中的非蛋白生成氨基酸，以及 随后通过辅助结构域的化学修饰，使NRPS具有惊人的多样性 产品。我们建议继续我们的生化和结构研究，以了解 NRPS功能的分子基础。我们将确定管理高效的 通过NRPS结构循环进行，以允许结合底物在 协调、高效的流程。这些研究将确定大型、多领域的结构 催化相关构象中的酶来理解使这一点成为可能的特征 结构周期。我们还将研究不常见的NRPS，以确定赋予NRPS的特征 具有执行意想不到的化学反应能力的结构域。我们将审查NRPS 生物合成机制尚未确定，特别关注不寻常的催化 缩合结构域和硫酯结构域的性质，并将决定产物 来自人类病原体的未鉴定的NRPS。

Microbes produce natural products to thrive in unique environments where they face a wide range of conditions and competing organisms. These diverse molecules are often unique to individual species and play roles in cellular communication and signaling, interspecies competition, nutrient acquisition, and virulence. The ability of natural products to function in the biological setting make them attractive candidates for the discovery of new pharmaceuticals. Indeed, roughly two-thirds of small molecule drugs are derived from natural products. Understanding the biosynthesis of unusual natural products will facilitate the analysis of the thousands of biosynthetic gene clusters with no known product and may lead to the discovery of new pharmaceutically active molecules. One family of peptide-based natural products is produced by the multidomain nonribosomal peptide synthetases (NRPSs). Among the hundreds of NRPS products are the important peptide antibiotics vancomycin and teixobactin, β-lactam antibiotics like some penicillins and cephalosporins, the genotoxin colibactin, the immunosuppressant cyclosporin, as well as bacterial siderophores like enterobactin, pyoverdine, and mycobactin. NRPSs use a remarkable assembly line architecture; multiple protein domains are joined in a single protein that can be thousands or tens of thousands of residues in length. During biosynthesis, amino acid building blocks are covalently loaded onto a carrier domain and delivered to neighboring catalytic domains for peptide extension and modification. The use of nonproteinogenic amino acids in a ribosome- and mRNA-independent synthesis, as well as subsequent chemical modification by auxiliary domains, enables the striking diversity of NRPS products. We propose to continue our biochemical and structural studies to understand the molecular basis of NRPS function. We will identify the features that govern the efficient progression through the NRPS structural cycle to allow the delivery of bound substrates in a coordinated, efficient process. These studies will determine structures of large, multidomain enzymes in catalytically relevant conformations to understand features that enable this structural cycle. We will also examine unusual NRPSs to identify the features that endow NRPS domains with the ability to perform unexpected chemical reactions. We will examine NRPSs with undetermined biosynthetic mechanism with a particular focus on unusual catalytic properties of condensation and thioester domains, and will determine the products of uncharacterized NRPSs from human pathogens.