Type II thioesterase restores activity of a NRPS module stalled with an aminoacyl-S-enzyme that cannot be elongated.

Nonribosomal peptide synthetases (NRPSs) carry out the biosynthesis of numerous peptide natural products, including many with important clinical applications. The NRPS, organized into a series of modules, is an efficient, high-fidelity assembly line for the production of a particular peptide. Each module consists of domains, whose activities contribute to the accuracy of these assembly-line systems. The activation (A) domain uses ATP to selectively load an amino acid onto the module through formation of a thioester bond to the pantetheine arm of the thiolation (T) domain. Peptide-bond formation, catalyzed by the condensation (C) domain, is stringent for both sidechain identity and stereochemistry. The C domain accepts an aminoacylor peptidylthioester from the preceding module for nucleophilic addition by the amine of the loaded amino acid; this generates the elongated peptide attached to the downstream module. The peptide product is synthesized one amino acid at a time until it reaches the final module. There, the fully synthesized chain is released by a type I thioesterase (TEI), the terminal domain of the NRPS assembly. Despite the high fidelity of this process, an error in any step of the assembly-line synthesis severely impacts the efficiency of the system and creates a bottleneck that results in a buildup of unprocessed intermediates. For example, an error by the A domain, which can load amino acids other than that normally accepted by the C domain, would prevent peptide-bond formation. The loaded module would be blocked until the incorrect amino acid was hydrolyzed (Figure 1). A type II thioesterase (TEII), whose gene is associated with the gene cluster of many NRPSs and related polyketide synthases (PKSs), improves the efficiency of product formation in these systems and has been proposed to edit modules through hydrolysis of acyl groups. In the surfactin NRPS, TEII was shown to regenerate misacylated modules resulting from priming of the apomodule with acyl-CoA groups. In this study we provide evidence to expand the editing function of TEIIs to include restoring the activity of modules stalled by loaded amino acids that cannot be processed. N-acetylcysteamine (SNAC) thioesters have been used previously to assay NRPS domain activities. 13–15] Hydrolysis of SNAC substrates was used here to explore the specificity of the TEII from the tyrocidine biosynthetic operon, TycF. TycF accepted a broad variety of aminoacyl-SNACs of different sidechain identity and stereochemistry with a 20-fold kcat/Km range between the mostand least-active substrate (Table 1). A series of peptidyl-SNACs derived from the tyrocidine sequence was