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Online Pyrophosphate Assay for Analyzing Adenylation Domains of Nonribosomal Peptide Synthetases
Author(s) -
Kittilä Tiia,
Schoppet Melanie,
Cryle Max J.
Publication year - 2016
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201500555
Subject(s) - adenylylation , nonribosomal peptide , pyrophosphate , combinatorial chemistry , biosynthesis , chemistry , enzyme , substrate (aquarium) , biochemistry , biology , computational biology , ecology
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 (PP i ) detection assay for analyzing A domain catalysis in vitro. PP i 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.