Many amino acid-containing natural products are biosynthesized by large, multifunctional enzymes known as non-ribosomal peptide synthetases (NRPSs). Adenylation (A) domains in NRPSs are responsible for the incorporation of amino acid building blocks and can be considered as engineering domains; therefore, advanced techniques are required to not only rapidly verify expression and folding, but also accelerate the functional prediction of the A-domains in lysates from native and heterologous systems. We recently developed activity-based protein profiling (ABPP) of NRPSs that offers a simple and robust analytical platform for A-domains and provides insights into their enzyme–substrate specificity. In this chapter, we describe the design and synthesis of these ABPP probes and provide a summary of our work on the development of a series of protocols for labeling, visualizing, and analyzing endogenous NRPSs in complex biological systems.
CITATION STYLE
Ishikawa, F., & Tanabe, G. (2023). Chemoproteomic Profiling of Adenylation Domain Functions in Gramicidin S-Producing Non-ribosomal Peptide Synthetases. In Methods in Molecular Biology (Vol. 2670, pp. 69–100). Humana Press Inc. https://doi.org/10.1007/978-1-0716-3214-7_4
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