LAUSR

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. Penicillin-binding protein-type thiosterases are recently discovered trans -acting enzymes constructing macrocycles during non-ribosomal peptide biosynthesis. Now, their synthetic potential is explored and a protein crystal structure provides insights into their unusual stereochemical requirement.