Structures of Peptide Synthetases and Related Enzymes

描述（由申请人提供）：非核糖体肽合成酶（NRPS）是催化肽抗生素和肽铁载体合成的多结构域酶家族。NRPS的功能相当于模块化装配线，其中氨基酸和肽中间体与蛋白质结合，并从上游结构域转移到下游结构域，在下游结构域中进行延伸和化学修饰。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酶的特征将通过所提出的实验来鉴定。假设140度结构域旋转允许以协调方式将肽引导至不同催化结构域的多个状态。这将通过生物化学分析进行测试，其中该结构域旋转被阻断，并且多结构域NRPS酶在功能上被测定。此外，晶体结构将确定构象捕获的酶。这些结构将解释酶如何采取必要的构象。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，人们一直对工程改造这些酶以生产新型抗生素感兴趣。这些实验已经遇到了有限的成功，因为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。我们的实验将确定高度相关的催化状态的酶，这将解释目前缺乏的催化结构域的活性位点架构的晶体结构。这些信息，连同对构象动力学的见解，将使NRPS系统的工程生产新的肽。这些研究将导致开发新抗生素的第二种方式涉及NRPS参与铁载体合成。这些细菌铁清除化合物在许多病原生物中作为重要的毒力因子。这些途径的抑制是抗生素设计的一个有吸引力的目标。在一个合作的努力，我们将确定细菌铁载体生产NRPS酶绑定到新的亚微摩尔抑制剂的晶体结构。这些结构将为优化化合物以改善结合性能提供基础。 公共卫生相关性：NRPS蛋白是模块化蛋白，以迷人的装配线方式运作。对这些蛋白质如何工作的全面了解将使新抗生素的生产能够以两种方式进行。这些蛋白质工厂可以被改造成新的活性化合物。此外，这些NRPS蛋白质使感染所需的细菌毒力因子，我们将确定这些酶的分子图像结合到铅抑制剂，使第二代化合物的设计具有改善的性能。

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.