Cyclic tetrapeptides (CTPs) are a diverse class of natural products with a broad range of biological activities. However, they are challenging to synthesize due to the ring strain associated with their small ring size. While chemical methods have been developed to access CTPs, they generally require the presence of certain amino acids, limiting their substrate scopes. Herein, we report the first bioinformatics-guided discovery of a thioesterase from a cryptic biosynthetic gene cluster for peptide cyclization. Specifically, we hypothesized that predicted penicillin-binding type thioesterases (PBP-TEs) from cryptic nonribosomal peptide synthetase (NRPS) gene clusters containing four adenylation domains would catalyze tetrapeptide cyclization. We found that one of the predicted PBP-TEs, WP516, efficiently cyclizes a wide variety of tetrapeptide substrates. To date, it is only the second stand-alone enzyme capable of efficiently cyclizing tetrapeptides, and its substrate scope greatly surpasses that of the only other reported tetrapeptide cyclase Ulm16. AlphaFold modeling, covalent docking, molecular dynamics, and mutational analyses were used to rationalize the broad substrate scope of WP516. Overall, the bioinformatics-guided workflow outlined in this paper and the discovery of WP516 represent promising tools for the biocatalytic production of head-to-tail CTPs, as well as a more general strategy for the discovery of enzymes for peptide cyclization.
Jeannette et al. (Thu,) studied this question.