In vitroreconstitution 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 anin vitroreconstituted 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.

中文翻译：

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体外重建的NRPS系统上吡咯并[4,2]苯并二氮杂支架托马霉素的总生物合成

天然产物生物合成途径的体外重组和生化分析仍然是一项艰巨的努力，尤其是如果涉及非核糖体肽合成酶（NRPS）类型的大酶时。理论上，可以使用基于质谱（MS）的载体蛋白偶联中间体和游离中间体的分析来解密所有生物合成步骤。我们在这里报告了吡咯并[4,2]苯并二氮杂pine支架到maymycin的“全部生物合成”，使用了体外重组的NRPS系统。通过液相色谱（LC）-MS分析蛋白形式，以解析生物合成的每个步骤，其各自的最大合成酶的大小可达到170 kDa。据我们所知，这是对非核糖体合成天然产物生物合成中几乎所有化学步骤进行全面分析的第一份报告。该研究包括通过分析腺苷酸化步骤以及转移至相应载体蛋白结构域来确定竞争测定中相应A结构域的底物特异性的实验。