Online Pyrophosphate Assay for Analyzing Adenylation Domains of Nonribosomal Peptide Synthetases
Online Pyrophosphate Assay for Analyzing Adenylation Domains of Nonribosomal Peptide Synthetases
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DOI：
10.1002/cbic.201500555
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发表时间：
2016-04-01
期刊：
影响因子：
3.2
通讯作者：
Cryle, Max J.
中科院分区：
文献类型：
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作者：
Kittila, Tiia;Schoppet, Melanie;Cryle, Max J.
非核糖体肽合成酶(NRPS)能产生许多重要的、结构复杂的天然产物。由于NRPS的结构，长期以来，人们一直试图对NRPS进行重新编程以获得新的生物活性化合物。然而，需要通过腺基化(A)结构域详细表征NRPS的催化和底物选择性来支持这些努力。我们提出了一种简单的NADH/焦磷酸(PPI)联用检测方法，用于体外分析Adomain催化。PPI的形成与NADH的消耗通过四个酶步骤耦合，并可通过光谱(=340 nm)检测到，以便于简单分析。我们用几个腺化结构域证明了这种方法的有效性，包括独立的Adomain(DltA，细胞壁生物合成)和嵌入的Adomain(Tcp10，替考拉宁生物合成)。首次探索了Tcp10A结构域的底物接受性，从而证明了该方法对复杂的多结构域NRPS的适用性。
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 Adomain catalysis in vitro. PPi formation is coupled to the consumption of NADH by four enzymatic steps and is detected spectroscopically (=340nm) for simple analysis. We demonstrate the effectiveness of this assay with several adenylation domains, including a stand-alone Adomain (DltA, cell wall biosynthesis) and an embedded Adomain (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.