The biosynthetic implications of late-stage condensation domain selectivity during glycopeptide antibiotic biosynthesis†,Chemical Science

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The biosynthetic implications of late-stage condensation domain selectivity during glycopeptide antibiotic biosynthesis†
Chemical Science ( IF 7.6 ) Pub Date : 2018-10-10 00:00:00 , DOI: 10.1039/c8sc03530j
Melanie Schoppet _{1,

2} , Madeleine Peschke ₂ , Anja Kirchberg ₁ , Vincent Wiebach ₃ , Roderich D Süssmuth ₃ , Evi Stegmann _{4,

5} , Max J Cryle _{1,

2,

6}

Affiliation

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：

糖肽抗生素生物合成过程中后期缩合结构域选择性的生物合成影响†

非核糖体肽合成是形成许多医学相关次级代谢产物的非常重要的生物合成途径。由于这些装配线的许多产品的化学合成面临挑战，了解非核糖体肽合成酶 (NRPS) 机器的活性和选择性是重新设计此类机器以生产新的生物活性肽的重要一步。虽然 NRPS 合成过程中负责氨基酸激活的腺苷酸化结构域的选择性已得到广泛研究，但必需的肽键形成结构域（称为缩合结构域）的选择性尚不清楚。在这里，我们展示了对产生糖肽抗生素 (GPA) 的 NRPS 机器的最终缩合结构域进行体外和体内研究相结合的结果。我们的结果表明，该缩合结构域能够耐受一系列肽底物，甚至是肽 C 末端具有非天然立体化学的肽底物，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形成鲜明对比。此外，我们表明，与交联肽相比，该缩合结构域对线性肽底物具有显着的偏好，这表明 GPA 交联级联靶向与 NRPS 机器最终模块结合的七肽，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执行的氨基酸激活速率相关。这是一个对 NRPS 重新设计有影响的重要结果，因为它表明必须维持修饰腺苷酸化结构域的氨基酸激活速率，以防止由于氨基酸选择和肽的生产性偶联的丧失而导致 NRPS 发生不需要的肽水解。债券的形成。总而言之，我们的结果表明，评估缩合结构域活性不仅是了解 NRPS 介导的肽组装的生物合成逻辑和时间安排的重要一步，而且也是重新设计工作必须遵守的规则，以便成功产生功能性、修饰的 NRPS 组装线。

更新日期：2018-10-10

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