Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase

In the biosynthesis of many macrocyclic natural products by multidomain meg asynthases, a carboxy-terminal thioesterase (TE) domain is involved in cycl ization and product release(1,2); however, it has not been determined wheth er 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(1). Here we report that the e xcised TE domain from tyrocidine synthetase efficiently catalyses cyclizati on of a decapeptide-thioester to form the antibiotic tyrocidine A, and can catalyse pentapeptide-thioester dimerization followed by cyclization to for m the antibiotic gramicidin S. By systematically varying the decapeptide-th ioester substrate and comparing cyclization rates, we also show that only t wo residues (one near each end of the decapeptide) are critical for cycliza tion. 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 enzymat ic assembly lines.