Abstract
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.
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Acknowledgments
This work was partly supported by Grants-in-Aid for Research (C) (19 K05722 to F.I.) from JSPS and by grants from the Noda Institute for Scientific Research (F.I.), Research Foundation for Pharmaceutical Sciences (F.I.), Japan Foundation for Applied Enzymology (F.I.), and Institute Foundation for Science, Osaka (F.I.). We are also thankful for the financial support provided by the Antiaging Project for Private Universities.
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Ishikawa, F., Tanabe, G. (2023). Chemoproteomic Profiling of Adenylation Domain Functions in Gramicidin S-Producing Non-ribosomal Peptide Synthetases. In: Burkart, M., Ishikawa, F. (eds) Non-Ribosomal Peptide Biosynthesis and Engineering. Methods in Molecular Biology, vol 2670. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3214-7_4
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DOI: https://doi.org/10.1007/978-1-0716-3214-7_4
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