Mechanisms of activity of Non-Ribosomal Peptide Synthases.

鉴于世界范围内人口的增加和对普遍使用的抗生素的耐药性日益增加，寻找新的有效的抗菌药物以及以具有成本效益的方式合成它们的能力是必不可少的。非核糖体合成肽（nrp）是一类重要的天然产物，具有广泛的抗菌和抗肿瘤活性。它们不同的结构为开发新的有效的治疗方法提供了有希望的支架。这些肽是由称为非核糖体肽合成酶（NRPS）的专用分子机器合成的。NRPSs按照“劳动分工”的概念，以装配线的方式工作。每个NRPS被组织成独立的模块，每个模块由一组独立折叠的结构域组成，这些结构域催化通路中的特定反应/步骤。模块分为起始模块、延伸模块和终止模块，分别启动肽合成、延伸肽链和释放最终产物。NRPS合成具有不同结构框架的新型和非典型肽的能力可以被利用为我们的优势；NRPS模块可以设计成结构和化学上优化的治疗方法。显然，在计算机上设计高效的NRPS机器来合成非天然的NRPS需要详细的酶的机制和结构知识。NRPS模块的核心催化结构域是腺苷酸化、缩合和硫酯酶结构域，分别催化底物活化、肽键形成和产物释放。肽基载体（PCP）结构域在不同的反应中心之间穿梭生长的肽链，这些反应中心位于一个模块内或在单独的模块中。虽然腺苷酸化结构域的功能和选择性已经得到了广泛的研究，但迄今为止，还没有与供体和受体PCP模块结合的缩合结构域的结构信息。因此，缩合反应的机理和调控仍然是模糊的。在这个项目中，我们将研究最简单的天然NRPS——托马霉素合成酶。Tomaymycin是由起始模块TomA和延伸/终止模块TomB合成的。我们将研究TomB的C结构域与负载底物的TomA-PCP和TomB- pcp结构域进行缩合反应机理的结构基础。为此，我们将使用核磁共振光谱学，它在处理瞬态相互作用方面具有独特的能力，例如发生在NRPSs中的相互作用。

The search for new potent anti-bacterials as well as the ability to synthesize them in a cost effective way is imperative in view of the world-wide rise of population and of increasing resistance to the prevalent antibiotics. Non-ribosomally synthesized peptides (NRPs) are an important class of natural products that show a wide range of pharmaceutical activity, both as antibacterial and antitumor agents. Their diverse structures form promising scaffolds for the development of new and potent therapeutics. These peptides are synthesized by dedicated molecular machines called Non-ribosomal peptide synthases (NRPS). NRPSs work in an assembly line-like fashion, following the concept of "division of labour". Each NRPS is organized as separate modules, each of which is composed of a set of independently folded domains that catalyse a particular reaction/step in the pathway. The modules are classified as initiation, elongation and termination modules, which respectively start the peptide synthesis, extend the peptide chain and release the final product. The ability of NRPS to synthesize such novel and atypical peptides with varied structural frameworks can be harnessed to our advantage; NRPS modules could be engineered to yield structurally and chemically optimized therapeutics. Clearly, the in silico design of efficient NRPS machinery to synthesize non-natural NRPs requires detailed mechanistic and structural knowledge of the enzyme. The core catalytic domains of the NRPS modules are the adenylation, condensation, and thioesterase domains, which catalyse the substrate activation, peptide bond formation and product release, respectively. The peptidyl carrier (PCP) domain shuttles the growing peptide chain between different reaction centres located either within a module or in separate modules. While the function and selectivity of the adenylation domain has been studied extensively, to date there is no structural information for the condensation domain bound to both the donor and acceptor PCP modules. Consequently, the mechanism and regulation of the condensation reaction remain nebulous. In this project, we will study the simplest natural NRPS, the Tomaymycin synthase. Tomaymycin is synthesized by an initiation module, TomA, and by an elongation/termination module, TomB. We will study the structural basis for the mechanism of the condensation reaction carried out by the C domain of TomB together with the TomA-PCP and TomB-PCP domains loaded with their substrates. To this end, we will use NMR spectroscopy, which is unique in its ability to deal with transient interactions, such as those occurring in the NRPSs.