Kittilä Tiia, Schoppet Melanie, Cryle Max J
Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany.
EMBL Australia, Monash University, Clayton, Victoria, 3800, Australia.
Chembiochem. 2016 Apr 1;17(7):576-84. doi: 10.1002/cbic.201500555. Epub 2016 Feb 23.
Nonribosomal peptide synthetases (NRPSs) produce many important and structurally complex natural products. Because of their architectures, reprogramming NRPSs has long been attempted to access new bioactive compounds. However, detailed characterization of NRPS catalysis and substrate selectivity by adenylation (A) domains is needed to support such efforts. We present a simple coupled NADH/pyrophosphate (PPi ) detection assay for analyzing A domain catalysis in vitro. PPi formation is coupled to the consumption of NADH by four enzymatic steps and is detected spectroscopically (λ=340 nm) for simple analysis. We demonstrate the effectiveness of this assay with several adenylation domains, including a stand-alone A domain (DltA, cell wall biosynthesis) and an embedded A domain (Tcp10, teicoplanin biosynthesis). Substrate acceptance of the Tcp10 A domain was explored for the first time, thus demonstrating the applicability of the assay for complex, multi-domain NRPSs.
非核糖体肽合成酶（NRPSs）可产生许多重要且结构复杂的天然产物。由于其结构特点，长期以来人们一直尝试对NRPSs进行重新编程以获得新的生物活性化合物。然而，需要通过腺苷化（A）结构域对NRPS催化作用和底物选择性进行详细表征，以支持此类研究工作。我们提出了一种简单的偶联NADH/焦磷酸（PPi）检测方法，用于体外分析A结构域的催化作用。PPi的形成通过四个酶促步骤与NADH的消耗相偶联，并通过光谱法（λ=340 nm）进行检测，以便进行简单分析。我们用几个腺苷化结构域证明了该检测方法的有效性，包括一个独立的A结构域（DltA，细胞壁生物合成）和一个嵌入的A结构域（Tcp10，替考拉宁生物合成）。首次探索了Tcp10 A结构域的底物接受情况，从而证明了该检测方法对复杂的多结构域NRPSs的适用性。
