Online Pyrophosphate Assay for Analyzing Adenylation Domains of Nonribosomal Peptide Synthetases

作者：Kittila Tiia; Schoppet Melanie; Cryle Max J^*

来源：ChemBioChem, 2016, 17(7): 576-584.

DOI：10.1002/cbic.201500555

• adenylation domain
• antibiotics
• enzymes
• non-ribosomal peptide synthetase
• pyrophosphate detection
• teicoplanin

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.

• 出版日期2016-4-1
• 单位河北医科大学