Cyclization of fungal nonribosomal peptides by a terminal condensation-like domain

Cyclization of linear peptidyl precursors produced by nonribosomal peptide synthetases (NRPSs) is an important step in the biosynthesis of bioactive cyclic peptides. Whereas bacterial NRPSs use thioesterase domains to perform the cyclization, fungal NRPSs have apparently evolved to use a different enzymatic route. In verified fungal NRPSs that produce macrocyclic peptides, each megasynthetase terminates with a condensation-like (C-T) domain that may perform the macrocyclization reaction. To probe the role of such a C-T domain, we reconstituted the activities of the Penicillium aethiopicum trimodular NPRS TqaA in Saccharomyces cerevisiae and in vitro. Together with the reconstituted bimodular NRPS AnaPS, we dissected the cyclization steps of TqaA in transforming the linear anthranilate-(D)-tryptophan-(L)-alanyl tripeptide into fumiquinazoline F. Extensive biochemical and mutational studies confirmed the essential role of the C-T domain in catalyzing cyclization in a thiolation domain-dependent fashion. Our work provides evidence of a likely universal macrocyclization strategy used by fungal NRPSs.