X-domain of peptide synthetases recruits oxygenases crucial for glycopeptide biosynthesis
X-domain of peptide synthetases recruits oxygenases crucial for glycopeptide biosynthesis
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DOI：
10.1038/nature14141
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发表时间：
2015-05-07
期刊：
影响因子：
64.8
通讯作者：
Cryle, Max J.
中科院分区：
文献类型：
--
作者：
Haslinger, Kristina;Peschke, Madeleine;Cryle, Max J.
非核糖体肽合成酶 (NRPS) 大酶复合物是模块化装配线，参与众多独立于核糖体的肽代谢物的生物合成(1)。 NRPS 机制内催化域之间的多重相互作用通过与外部酶的额外相互作用得到进一步补充，特别是最终的肽成熟过程。需要对 NRPS 结合肽进行大量外部修饰的一类重要 NRPS 代谢物是糖肽抗生素 (GPA)，其中包括万古霉素和替考拉宁 (2,3)。这些临床相关的肽类抗生素经过细胞色素 P450 催化的芳香族侧链氧化交联，以实现其最终的活性构象 (4-12)。然而，细胞色素 P450 加氧酶募集至 NRPS 结合肽的机制此前尚不清楚。在这里，我们通过体外研究表明，X 结构域 (13,14) 是所有 GPA NRPS 机器最终模块中存在的未知功能的保守结构域，负责将加氧酶募集到 N RPS 结合肽以执行必要的侧链交联。 X射线晶体学表明，X结构域在结构上与缩合结构域相关，但其氨基酸取代使其失去催化活性。我们发现 X 结构域将细胞色素 P450 加氧酶招募到 NRPS，并通过解析 P450-X 结构域复合物的结构来确定界面。此外，我们证明，体外加氧酶对肽前体的修饰，特别是 GPA 生物合成中第二个交联的安装，仅在 X 结构域存在时才会发生。我们的结果表明，在 GPA 生物合成中，用于氧化的肽基载体蛋白 (PCP) 结合底物的呈递需要 NRPS X 结构域的存在，以确保前体肽转化为成熟的糖苷配基，并且单独的载体蛋白结构域并不总是足以生成外部细胞色素 P450 加氧酶的有效底物。
Non-ribosomal peptide synthetase (NRPS) mega-enzyme complexes are modular assembly lines that are involved in the biosynthesis of numerous peptide metabolites independently of the ribosome(1). The multiple interactions between catalytic domains within the NRPS machinery are further complemented by additional interactions with external enzymes, particularly focused on the final peptide maturation process. An important class of NRPS metabolites that require extensive external modification of the NRPS-bound peptide are the glycopeptide antibiotics (GPAs), which include vancomycin and teicoplanin(2,3). These clinically relevant peptide antibiotics undergo cytochrome P450-catalysed oxidative crosslinking of aromatic side chains to achieve their final, active conformation(4-12). However, the mechanism underlying the recruitment of the cytochrome P450 oxygenases to the NRPS-bound peptide was previously unknown. Here we show, through in vitro studies, that the X-domain(13,14), a conserved domain of unknown function present in the final module of all GPA NRPS machineries, is responsible for the recruitment of oxygenases to theN RPS-bound peptide to perform the essential side-chain crosslinking. X-ray crystallography shows that the X-domain is structurally related to condensation domains, but that its amino acid substitutions render it catalytically inactive. We found that the X-domain recruits cytochrome P450 oxygenases to the NRPS and determined the interface by solving the structure of a P450-X-domain complex. Additionally, we demonstrated that the modification of peptide precursors by oxygenases in vitro-in particular the installation of the second crosslink in GPA biosynthesis-occurs only in the presence of the X-domain. Our results indicate that the presentation of peptidyl carrier protein (PCP)-bound substrates for oxidation in GPA biosynthesis requires the presence of the NRPS X-domain to ensure conversion of the precursor peptide into a mature aglycone, and that the carrier protein domain alone is not always sufficient to generate a competent substrate for external cytochrome P450 oxygenases.