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.

非生物肽键的形成有利于异源偶联，而生物多肽键的形成则以同手性偶联为主。在这里，我们报告异手性偶联是非核糖体肽生物合成的头尾大内酰胺化中一个相当普遍的范例。规范的顺式卸载环化酶，例如I型硫酯酶（TE）和末端缩合样结构域，分别催化具有d-和l-构型的N-末端和C-末端残基之间的头尾尾大内酰胺化。相反，青霉素结合蛋白型TEs是最近发现的反式家族-作用环化酶，将具有互补立体选择性的杂手性残基与经典残基偶联。因此，一套顺式和反式TE非核糖体肽合成酶可以克服细菌非核糖体肽生物合成中杂手性头尾尾内酰胺形成中存在的立体化学限制。此外，我们提供了非典型卸载环化酶的C端立体选择性的结构原理。具有广泛的底物特异性的青霉素结合蛋白型TEs可能用作合成生物学的生物催化剂和遗传工具。