Department of Microbial Natural Products (MINS), Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) - Helmholtz Centre for Infection Research (HZI) and Institute for Pharmaceutical Biotechnology, Saarland University, 66123 Saarbrücken, Germany; German Center for Infection Research (DZIF), Partner Site Hannover-Braunschweig, 38124 Braunschweig, Germany.
Structure and Function of Proteins, Helmholtz Centre for Infection Research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Cell Chem Biol. 2017 Oct 19;24(10):1216-1227.e8. doi: 10.1016/j.chembiol.2017.08.001. Epub 2017 Sep 7.
In vitro reconstitution and biochemical analysis of natural product biosynthetic pathways remains a challenging endeavor, especially if megaenzymes of the nonribosomal peptide synthetase (NRPS) type are involved. In theory, all biosynthetic steps may be deciphered using mass spectrometry (MS)-based analyses of both the carrier protein-coupled intermediates and the free intermediates. We here report the "total biosynthesis" of the pyrrolo[4,2]benzodiazepine scaffold tomaymycin using an in vitro reconstituted NRPS system. Proteoforms were analyzed by liquid chromatography (LC)-MS to decipher every step of the biosynthesis on its respective megasynthetase with up to 170 kDa in size. To the best of our knowledge, this is the first report of a comprehensive analysis of virtually all chemical steps involved in the biosynthesis of nonribosomally synthesized natural products. The study includes experiments to determine substrate specificities of the corresponding A-domains in competition assays by analyzing the adenylation step as well as the transfer to the respective carrier protein domain.
在体外重建和生化分析天然产物生物合成途径仍然是一项具有挑战性的工作，特别是如果涉及非核糖体肽合酶 (NRPS) 类型的巨型酶。从理论上讲，可以使用基于质谱 (MS) 的分析方法来分析结合载体蛋白的中间产物和游离中间产物，从而破译所有生物合成步骤。在这里，我们使用体外重建的 NRPS 系统报道了吡咯并[4,2]苯并二氮杂卓骨架托马霉素的“全生物合成”。通过液相色谱 (LC)-MS 分析蛋白变体，在各自大小达 170 kDa 的巨型合酶上对生物合成的每一步进行解码。据我们所知，这是首次对非核糖体合成天然产物生物合成中涉及的几乎所有化学步骤进行全面分析的报告。该研究包括通过分析腺嘌呤化步骤以及向相应的载体蛋白结构域的转移，在竞争测定中确定相应 A 结构域的底物特异性的实验。
