Heterochiral coupling in non-ribosomal peptide macrolactamization

Heterochiral coupling is favoured in abiotic peptide bond formation, whereas biotic peptide bond formation is dominated by homochiral coupling. Here, we report that heterochiral coupling is a rather general paradigm in the head-to-tail macrolactamization of non-ribosomal peptide biosynthesis. The canonical cis-acting offloading cyclases, such as type I thioesterase (TE) and terminal condensation-like domains, catalyse head-to-tail macrolactamization between N- and C-terminal residues with d- and l-configurations, respectively. In contrast, the penicillin-binding protein-type TEs, a recently identified family of trans-acting cyclases, couple heterochiral residues with complementary stereoselectivity to the canonical one. Thus, a suite of cis- and trans-TE non-ribosomal peptide synthetases could overcome the stereochemical constraints present in heterochiral head-to-tail macrolactam formation in bacterial non-ribosomal peptide biosynthesis. Furthermore, we provide the structural rationale for the C-terminal stereoselectivity of non-canonical offloading cyclases. Penicillin-binding protein-type TEs with broad substrate specificity are potentially applicable as biocatalysts and genetic tools for synthetic biology