German Cancer Research Center DKFZ and Faculty of Biosciences, University of Heidelberg, 69120, Heidelberg, Germany.
Signalling Research Centers BIOSS and CIBSS, University of Freiburg, 79104, Freiburg, Germany.
Sci Rep. 2019 Mar 26;9(1):5119. doi: 10.1038/s41598-019-41492-8.
Non-ribosomal peptide synthetases (NRPSs) are large, modular enzymes that produce bioactive peptides of tremendous structural and chemical diversity, due to the incorporation, alongside the canonical 20 amino acids, of non-proteinogenic amino acids, fatty acids, sugars and heterocyclic rings. For linear NRPSs, the size and composition of the peptide product is dictated by the number, order and specificity of the individual modules, each made of several domains. Given the size and complexity of NRPSs, most in vitro studies have focused on individual domains, di-domains or single modules extracted from the full-length proteins. However, intermodular interactions could play a critical role and regulate the activity of the domains and modules in unpredictable ways. Here we investigate in vitro substrate activation by three A domains of the tyrocidine synthetase TycC enzyme, systematically comparing their activity when alone (with the respective PCP domain), in pairs (di-modular constructs) or all together (tri-modular construct). Furthermore, we study the impact of mutations in the A or PCP domains in these various constructs. Our results suggest that substrate adenylation and effects of mutations largely depend on the context in which the domains/modules are. Therefore, generalizing properties observed for domains or modules in isolation should be done with caution.
非核糖体肽合成酶（NRPSs）是一类大型的模块化酶，能够产生具有巨大结构和化学多样性的生物活性肽，这是因为除了经典的 20 种氨基酸外，还可以掺入非蛋白质氨基酸、脂肪酸、糖和杂环。对于线性 NRPSs，肽产物的大小和组成取决于各个模块的数量、顺序和特异性，每个模块由几个结构域组成。鉴于 NRPSs 的大小和复杂性，大多数体外研究都集中在单个结构域、双结构域或从全长蛋白中提取的单个模块上。然而，模块间的相互作用可能会发挥关键作用，并以不可预测的方式调节结构域和模块的活性。在这里，我们研究了三种来自 Tyrocidine 合成酶 TycC 酶的 A 结构域的体外底物激活情况，系统比较了它们在单独（与相应的 PCP 结构域一起）、成对（双模块化构建体）或全部一起（三模块化构建体）时的活性。此外，我们还研究了在这些不同构建体中 A 或 PCP 结构域突变的影响。我们的结果表明，底物的腺苷酸化和突变的影响在很大程度上取决于结构域/模块所处的环境。因此，对于在孤立状态下观察到的结构域或模块的性质，应该谨慎推广。
