Aminoacyl-SNACs as small-molecule substrates for the condensation domains of nonribosomal peptide synthetases

Background: Nonribosomal peptide synthetases (NRPSs) are large multidomain proteins that catalyze the formation of a wide range of biologically active natural products. These megasynthetases contain condensation (C) domains t hat catalyze peptide bond formation and chain elongation. The natural subst rates for C domains are biosynthetic intermediates that are covalently teth ered to thiolation (T) domains within the synthetase by thioester linkages. Characterizing C domain substrate specificity is important for the enginee red biosynthesis of new compounds. Results: We synthesized a series of aminoacyl-N-acetylcysteamine thioesters (aminoacyl-SNACs) and show that they are small-molecule substrates for NRP S C domains. Comparison of rates of peptide bond formation catalyzed by the C domain from enterobactin synthetase with various aminoacyl-SNACs as down stream (acceptor) substrates revealed high selectivity for the natural subs trate analog L-Ser-SNAC. Comparing L- and D-Phe-SNACs as upstream (donor) s ubstrates for the first C domain from tyrocidine synthetase revealed clear D- versus L-selectivity, Conclusions: Aminoacyl-SNACs are substrates for NRPS C domains and are usef ul for characterizing the substrate specificity of C domain-catalyzed pepti de bond formation.