Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase

Abstract

In the biosynthesis of many macrocyclic natural products by multidomain megasynthases, a carboxy-terminal thioesterase (TE) domain is involved in cyclization and product release; however, it has not been determined whether TE domains can catalyse macrocyclization (and elongation in the case of symmetric cyclic peptides) independently of upstream domains. The inability to decouple the TE cyclization step from earlier chain assembly steps has precluded determination of TE substrate specificity, which is important for the engineered biosynthesis of new compounds. Here we report that the excised TE domain from tyrocidine synthetase efficiently catalyses cyclization of a decapeptide-thioester to form the antibiotic tyrocidine A, and can catalyse pentapeptide-thioester dimerization followed by cyclization to form the antibiotic gramicidin S. By systematically varying the decapeptide-thioester substrate and comparing cyclization rates, we also show that only two residues (one near each end of the decapeptide) are critical for cyclization. This specificity profile indicates that the tyrocidine synthetase TE, and by analogy many other TE domains, will be able to cyclize and release a broad range of new substrates and products produced by engineered enzymatic assembly lines.

Patellamide A and C biosynthesis by a microcin-like pathway inProchloron didemni, the cyanobacterial symbiont ofLissoclinum patella

“…Trunkamide and related molecules often contain proline, thiazolines, and prenylated serine and threonine derivatives. These features could easily result from either a ribosomal or nonribosomal peptide biosynthetic pathway, because precedents exist for heterocyclization and cyclization in both cases (18)(19)(20)(21). We initially chose to investigate the nonribosomal hypothesis of patellamide biosynthesis using a homology-based approach (22).…”

Selective interaction between nonribosomal peptide synthetases is facilitated by short communication-mediating domains

“…In addition to these so-called core domains, optional domains catalyze the modification of incorporated residues, i.e., by epimerization (E) or N-methylation (MT) domains (7). Product release is normally effected by a thioesterase (Te) domain, catalyzing the formation of linear, cyclic, or branched cyclic products, representative for the class of NRPs (8).…”

Mapping and Molecular Modeling ofS-Adenosyl-l-methionine Binding Sites inN-Methyltransferase Domains of the Multifunctional Polypeptide Cyclosporin Synthetase

“…In the case of cyclosporin synthesis, release of the full-length peptide ensues via cycliza-tion. Prokaryotic NRPSs usually employ a thioesterase domain (TE-domain) situated at the extreme COOH terminus of the last module (24,25), whereas most fungal NRPSs, such as CySyn, utilize a unique COOH-terminal C-domain for cyclization and release of the mature peptide (26).…”