Non-ribosomal peptide synthesis is a highly important biosynthetic pathway for the formation of many secondary metabolites of medical relevance. Due to the challenges associated with the chemical synthesis of many of the products of these assembly lines, understanding the activity and selectivity of non-ribosomal peptide synthetase (NRPS) machineries is an essential step towards the redesign of such machineries to produce new bioactive peptides. Whilst the selectivity of the adenylation domains responsible for amino acid activation during NRPS synthesis has been widely studied, the selectivity of the essential peptide bond forming domains – known as condensation domains – is not well understood. Here, we present the results of a combination of in vitro and in vivo investigations into the final condensation domain from the NRPS machinery that produces the glycopeptide antibiotics (GPAs). Our results show that this condensation domain is tolerant for a range of peptide substrates and even those with unnatural stereochemistry of the peptide C-terminus, which is in contrast to the widely ascribed role of these domains as a stereochemical gatekeeper during NRPS synthesis. Furthermore, we show that this condensation domain has a significant preference for linear peptide substrates over crosslinked peptides, which indicates that the GPA crosslinking cascade targets the heptapeptide bound to the final module of the NRPS machinery and reinforces the role of the unique GPA X-domain in this process. Finally, we demonstrate that the peptide bond forming activity of this condensation domain is coupled to the rate of amino acid activation performed by the subsequent adenylation domain. This is a significant result with implications for NRPS redesign, as it indicates that the rate of amino acid activation of modified adenylation domains must be maintained to prevent unwanted peptide hydrolysis from the NRPS due to a loss of the productive coupling of amino acid selection and peptide bond formation. Taken together, our results indicate that assessing condensation domain activity is a vital step in not only understanding the biosynthetic logic and timing of NRPS-mediated peptide assembly, but also the rules which redesign efforts must obey in order to successfully produce functional, modified NRPS assembly lines.

中文翻译：

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糖肽抗生素生物合成过程中后期缩合域选择性的生物合成意义†

非核糖体肽合成是形成许多具有医学意义的次级代谢产物的重要生物合成途径。由于与这些组装生产线许多产品的化学合成相关的挑战，了解非核糖体肽合成酶（NRPS）机械的活性和选择性是重新设计此类机械以生产新的生物活性肽的重要步骤。尽管已经广泛研究了在NRPS合成过程中负责氨基酸活化的腺苷酸化域的选择性，但人们对基本的肽键形成域（称为缩合域）的选择性却知之甚少。在这里，我们介绍了体外和体内结合的结果从产生糖肽抗生素（GPA）的NRPS机制研究了最终的缩合结构域。我们的结果表明，该缩合结构域可耐受多种肽底物，甚至具有肽C末端非自然立体化学的那些，这与这些结构域在NRPS合成过程中作为立体化学看门人的广泛作用相反。此外，我们显示该缩合结构域比线性交联的肽对线性肽底物具有显着的偏好，这表明GPA交联级联靶向靶向结合到NRPS机械最终模块的七肽，并增强了独特的GPA X结构域的作用在这个过程中。最后，我们证明该缩合结构域的肽键形成活性与随后的腺苷酸化结构域进行的氨基酸活化速率有关。这是对NRPS重新设计有重要意义的重要结果，因为它表明必须保持修饰的腺苷酸化域的氨基酸活化速率，以防止由于氨基酸选择和肽的生产性偶联损失而导致不必要的肽从NRPS水解。键的形成。综上所述，我们的结果表明，评估缩合域活性不仅是理解NRPS介导的肽组装的生物合成逻辑和时机的关键步骤，而且也是成功设计功能性修饰NRPS组装必须遵循的重新设计工作的规则。线。