Structures of Peptide Synthetases and Related Enzymes

描述（由申请人提供）：非核糖体肽合成酶（NRPSs）是一个多结构域酶家族，催化肽抗生素和肽铁载体的合成。NRPSs的功能就像模块化的装配线，氨基酸和肽中间体结合在蛋白质上，从上游结构域转移到下游结构域，在那里它们被扩展和化学修饰。这种模块化策略需要大规模的蛋白质重排，以允许新生肽在结构域之间转移。允许这种构象灵活性的NRPS酶的特征将通过提出的实验来确定。假设140度结构域旋转允许多种状态以协调的方式将肽引导到不同的催化结构域。这将通过生化分析进行测试，其中该结构域旋转被阻断，并对多结构域NRPS酶进行功能分析。此外，将确定构象捕获酶的晶体结构。这些结构将解释酶如何采用必要的构象。该项目的长期目标是开发新的抗生素。这将通过两种具体方式实现。首先，许多NRPS产品是肽抗生素。长期以来，人们一直对改造这些酶来生产新型抗生素感兴趣。由于催化的结构基础的不确定性，更重要的是，下游结构域的结合特异性，这些实验的成功有限。我们的实验将确定高度相关的酶催化状态的晶体结构，这将解释目前缺乏的催化结构域的活性位点结构。这些信息，连同对构象动力学的见解，将使NRPS系统的工程用于生产新型肽。这些研究将导致新型抗生素开发的第二种方式与NRPSs参与铁载体合成有关。这些细菌清除铁的化合物在许多致病生物中是重要的毒力因子。抑制这些途径是抗生素设计的一个有吸引力的目标。在合作努力中，我们将确定与新型亚微摩尔抑制剂结合的细菌产铁载体NRPS酶的晶体结构。这些结构将为优化化合物的结合性能提供基础。

DESCRIPTION (provided by applicant): The Non-Ribosomal Peptide Synthetases (NRPSs) are a family of multi-domain enzymes that catalyze the synthesis of peptide antibiotics and peptide siderophores. The NRPSs function as modular assembly lines with amino acids and peptide intermediates bound to the protein and transferred from upstream domains to downstream domains where they are extended and chemically modified. This modular strategy requires large-scale protein rearrangements to allow the nascent peptide to be transferred between domains. The features of the NRPS enzymes that allow this conformational flexibility will be identified through the proposed experiments. A 140-degree domain rotation is hypothesized to allow the multiple states that direct the peptide to different catalytic domains in a coordinated manner. This will be tested through biochemical analyses in which this domain rotation is blocked and multi-domain NRPS enzymes are assayed functionally. Additionally, crystal structures will be determined of conformationally trapped enzymes. These structures will explain how the enzymes adopt the necessary conformations. The long-term objective of this project is the development of new antibiotics. This will be accomplished in two specific ways. First, many NRPS products are peptide antibiotics. There has long been interest in engineering these enzymes to produce novel antibiotics. These experiments have met with limited success because of the uncertainty of the structural basis for catalysis and, more importantly, for the binding specificity of downstream domains. Our experiments will identify crystal structures of highly relevant catalytic states of the enzymes that will explain the active site architecture of catalytic domains that are currently lacking. This information, together with the insights into the conformational dynamics, will enable the engineering of NRPS systems for the production of novel peptides. The second way in which these studies will lead to the development of novel antibiotics relates to the involvement of the NRPSs in siderophore synthesis. These bacterial iron-scavenging compounds serve as important virulence factors in many pathogenic organisms. The inhibition of these pathways is an attractive target for antibiotic design. In a collaborative effort, we will determine the crystal structures of bacterial siderophore-producing NRPS enzymes bound to novel sub micromolar inhibitors. These structures will provide the foundation for optimization of the compounds for improved binding properties. PUBLIC HEALTH RELEVANCE: The NRPS proteins are modular proteins that operate in a fascinating assembly line fashion. A complete understanding of the how these proteins work will enable the production of new antibiotics in two ways. These protein factories can be engineered to make new active compounds. Additionally, these NRPS proteins make bacterial virulence factors needed for infections and we will determine molecular pictures of these enzymes bound to lead inhibitors to allow the design of second generation compounds with improved properties.