Nonribosomal peptide synthetases (NRPSs): Understanding functional domain interplay and biosynthetic directionality using FRET spectroscopy

非核糖体多肽合成酶(NRPS)是一类巨型酶，可作为蛋白质模板和催化剂合成许多重要的多肽天然产物，如达托霉素和环孢素。单个氨基酸构建块在ATP的消耗下被腺化结构域(A结构域)激活，以硫代酯的形式共价结合在4‘-磷酸泛氨酸载体蛋白(PCP结构域)上，并通过缩合结构域(C结构域)连接。其他结构域，如异构化结构域(E结构域)，提供了额外的结构多样化。硫酯酶结构域(TE结构域)释放最终产物。在典型的线性NRP中，简单的规则规定了特定多肽产品所需的结构域排列。为了结合一个氨基酸，所有必需的结构域都被整合到一个模块中，多功能酶中模块的顺序与产品中氨基酸的序列相对应。然而，关于分子内结构域-结构域相互作用的时间和空间性质以及这些相互作用如何转化为步进式多肽组装的基本方向性，人们知之甚少。多域NRPS片段的晶体结构提供了重要的洞察力，但仅代表缺乏动态信息的静态快照。最近，我们报道了第一个简化的A-PCP双域FRET传感器，利用时间历程实验实时研究催化活性酶的构象变化。这些研究已经带来了重要的新见解和新模型。在这里，我们建议将这一概念显著扩展到使用FRET来研究NRPS的动力学。我们希望获得关于NRPS生物合成方向的新的机械见解。

Non-ribosomal peptide synthetases (NRPSs) are mega-enzymes that act as protein templates and catalysts for the biosynthesis of many important peptide natural products like daptomycin and cyclosporine. The single amino acid building blocks are activated by adenylation domains (A domains) under the consumption of ATP, covalently bound as thioesters on 4’-phosphopantetheine-equipped peptidyl-carrier proteins (PCP domains) and linked by condensation domains (C domains). Further domains like the epimerization domain (E domain) provide additional structural diversification. A thioesterase domain (TE domain) releases the final product. In a typical linear NRPS simple rules dictate the necessary domain arrangements for a certain peptide product. To incorporate a single amino acid, all necessary domains are integrated into one module and the order of modules in the multifunctional enzyme corresponds to the sequence of the amino acids in the product. However, only very little is known about the temporal and spatial nature of intramolecular domain-domain interactions and how these translate into the essential directionality of the step-wise peptide assembly. Crystal structures of multi-domain NRPS fragments provide important insight, but only represent static snap-shots that lack dynamic information. Very recently, we have reported the first FRET sensor of a simplified A-PCP di-domain to investigate conformational changes of the catalytically active enzyme in real time using time-course experiments. These studies have already led to important new insights and models. Here, we propose to significantly expand the concept to study the dynamics of NRPSs using FRET. We expect to gain novel mechanistic insights into how directional NRPS biosynthesis is driven.