Cyclization of backbone-substituted peptides catalyzed by the thioesterasedomain from the tyrocidine nonribosomal peptide synthetase

The excised C-terminal thioesterase (TE) domain from the multidomain tyroci dine nonribosomal peptide synthetase (NRPS) was recently shown to catalyze head-to-tail cyclization of a decapeptide thioester to form the cyclic deca peptide antibiotic tyrocidine A [Trauger, J. W., Kohli, R. M., Mootz, H. D. , Marahiel, M. A., and Walsh, C. T. (2000) Nature 407, 215-218]. The peptid e thioester substrate was a mimic of the TE domain's natural, synthetase-bo und substrate. We report here the synthesis of modified peptide thioester s ubstrates in which parts of the peptide backbone are altered either by the replacement of three amino acid blocks with a flexible spacer or by replace ment of individual amide bonds with ester bonds. Rates of TE domain catalyz ed cyclization were determined for these substrates and compared with that of the wild-type substrate, revealing that some parts of the peptide backbo ne are important for cyclization, while other parts can be modified without significantly affecting the cyclization rate. We also report the synthesis of a modified substrate in which the N-terminal amino group of the wild-ty pe substrate, which is the nucleophile in the cyclization reaction, is repl aced with a hydroxyl group and show that this compound is cyclized by the T E domain to form a macrolactone at a rate comparable to that of the wild-ty pe substrate. These results demonstrate that the TE domain from the tyrocid ine NRPS can catalyze cyclization of depsipeptides and other backbone-subst ituted peptides and suggest that during the cyclization reaction the peptid e substrate is preorganized for cyclization in the enzyme active site in pa rt by intramolecular backbone hydrogen bonds analogous to those in the prod uct tyrocidine A.