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.
Keywords: Activity-based protein profiling; Adenylation domain; Aneurinibacillus migulanus ATCC 9999; Gramicidin S-NRPSs; Non-ribosomal peptide synthetase (NRPS); Photoaffinity labeling.
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.