Characterization\nof a Dual Function Peptide Cyclase\nin Graspetide Biosynthesis

Graspetides are a diverse family\nof ribosomally synthesized\nand\npost-translationally modified peptides with unique macrocyclic structures\nformed by ATP-grasp enzymes. Group 11 graspetides, including prunipeptin,\nfeature both macrolactone and macrolactam cross-links. Despite the\nknown involvement of a single ATP-grasp cyclase in the dual macrocyclizations\nof groups 5, 7, and 11 graspetides, detailed mechanistic insights\ninto these enzymes remain limited. Here, we reconstructed prunipeptin\nbiosynthesis from Streptomyces coelicolor using recombinant PruA and PruB macrocyclase. PruB exhibited kinetic\nbehavior similar to other characterized graspetide cyclases, with\na notably higher <i>k</i><sub>cat</sub>, likely due to utilization\nof an ATP-regeneration system. The X-ray crystal structure of PruB\nrevealed distinct features as compared to groups 1 and 2 enzymes.\nSite-directed mutagenesis identified critical roles of key residues\nfor the PruB reaction, including the DxR motif conserved in other\ngraspetide cyclases. Additionally, computational modeling of the PruA/PruB\ncocomplex uncovered substrate interactions and suggested that PruB\nfirst catalyzes a macrolactone bond formation on PruA. This study\nenhances our understanding of ATP-grasp enzyme mechanisms in graspetide\nbiosynthesis and provides insights for engineering these enzymes for\nfuture applications.