The biosynthesis of the glycopeptide antibiotics (GPAs)—which include teicoplanin and vancomycin—is a complex enzymatic process relying on the interplay of nonribosomal peptide synthesis and a cytochrome P450-mediated cyclization cascade. This unique cyclization cascade generates the highly cross-linked state of these nonribosomal peptides, which is crucial for their antimicrobial activity. Given that these essential oxidative transformations occur while the peptide remains bound to the terminal module of the nonribosomal peptide synthetase (NRPS) machinery, it is important to assess the selectivity of the terminal thioesterase (TE) domain and how this domain contributes to the maintenance of an efficient biosynthetic pathway while at the same time ensuring GPA maturation is completed. In this study, we report the in vitro characterization of the thioesterase domain from teicoplanin biosynthesis, the first GPA thioesterase to be characterized. Our results show that the activity of this TE domain relies on the presence of an unusual extended N-terminal linker region that appears to be unique to the NRPS machineries found in GPA biosynthesis. In addition, we show that the activity of this domain against carrier protein bound substrates is dramatically enhanced for mature GPA aglycones as opposed to linear peptides and partially cyclized intermediates. These results demonstrate how the interplay between NRPS and P450s during late stage GPA biosynthesis is not only maintained but also leads to the efficient production of mature GPA aglycones. Thus, GPA TE domains represent another impressive example of the ability of TE domains to act as logic gates during NRPS biosynthesis, ensuring that essential late-stage peptide modifications are completed before catalyzing the release of the mature, bioactive peptide product.

中文翻译：

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糖肽抗生素生物合成中的硫酯酶结构域对交联的糖苷配基是选择性的。

糖肽抗生素（GPA）的生物合成-包括替考拉宁和万古霉素-是一个复杂的酶促过程，依赖于非核糖体肽合成与细胞色素P450介导的环化级联反应的相互作用。这种独特的环化级联反应产生了这些非核糖体肽的高度交联状态，这对它们的抗菌活性至关重要。鉴于在肽保持与非核糖体肽合成酶（NRPS）机制的末端模块结合的同时发生了这些必要的氧化转化，因此评估末端硫酯酶（TE）域的选择性以及该域如何有助于维持脂质体非常重要。有效的生物合成途径，同时确保GPA成熟。在这项研究中，我们报告了体外teicoplanin生物合成中硫酯酶结构域的特征分析，这是第一个要表征的GPA硫酯酶。我们的结果表明，该TE结构域的活性取决于是否存在异常延伸的N末端接头区域，该区域似乎是GPA生物合成中发现的NRPS机器所特有的。此外，我们显示，相对于线性肽和部分环化的中间体，成熟的GPA糖苷配基可显着增强该结构域针对与载体蛋白结合的底物的活性。这些结果表明，如何在晚期GPA生物合成过程中保持NRPS和P450之间的相互作用，还可以有效地生产成熟的GPA糖苷配基。因此，