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 (TE) 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 (CT) domain that may perform the macrocyclization reaction. To probe the role of such a CT domain, we reconstituted the activities of the Penicillium aethiopicum trimodular NPRS TqaA in Saccharomyces cerevisiae and in vitro. Together with a 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 CT domain in catalyzing cyclization in a thiolation domain-dependent fashion. Our work provided evidence of a likely universal macrocyclization strategy employed by fungal NRPSs.

Oxidative cyclizations, exemplified by the biosynthetic assembly of the penicillin nucleus from a tripeptide precursor, are arguably the most synthetically-powerful implementation of C-H activation reactions in Nature. Here we show that Rieske oxygenase-like enzymes mediate regio and stereodivergent oxidative cyclizations to form 10- and 12-membered carbocyclic rings in the key steps of the biosynthesis of the antibiotics streptorubin B and metacycloprodigiosin, respectively. These reactions represent the first examples of oxidative carbocyclizations catalyzed by non-heme iron-dependent oxidases and define a novel type of catalytic activity for Rieske enzymes. A better understanding of how these enzymes achieve such remarkable regio and stereocontrol in the functionalization of unactivated hydrocarbon chains will greatly facilitate the development of selective manmade C-H activation catalysts.