Expanding the chemical space of synthetic cyclic peptides using a promiscuous macrocyclase from prenylagaramide biosynthesis
Cyclic peptides are excellent drug candidates, placing macrocyclization reactions at the apex of drug development. PatG and related dual-action proteases from cyanobactin biosynthesis are responsible for cleaving off the C-terminal recognition se-quence and macrocyclizing the substrate to provide cyclic peptides. This reaction has found use in the enzymatic synthesis of diverse macrocycles. However, these enzymes function best on substrates that terminate with the non-proteinogenic
... ogenic thia-zole/thiazoline residue, complicating synthetic strategies. Here, we biochemically characterize a new class of PatG-like macrocy-clases that natively use proline, obviating the necessity of additional chemical or biochemical steps. We experimentally define the biochemical steps involved in synthesizing the widespread prenylagaramide-like natural products, including macrocycliza-tion and prenylation. Using saturation mutagenesis, we show that macrocyclase PagG and prenyltransferase PagF are highly promiscuous, producing a library of more than 100 cyclic peptides and their prenylated derivatives in vitro. By comparing our results to known cyanobactin macrocyclase enzymes, we catalog a series of enzymes that collectively should synthesize most small macrocycles. Collectively, these data reveal that, by selecting the right cyanobactin macrocyclase, a large array of enzy-matically synthesized macrocycles are accessible.