Enzymatic peptide macrocyclization indole--acylation.

Indole -acylation is chemically challenging, due to the low nucleophilicity of the indole nitrogen. Although a few similar transformations have been proposed in the biosynthesis of indole-containing natural products, their enzymatic basis remains elusive. Here, we show that BulbE TE is an -acylindole-forming macrocyclase involved in the biosynthesis of the non-ribosomal cyclopeptide bulbiferamide. BulbE catalyzed macrocyclization not only the indole nitrogen, but also a primary amine and an alcohol. The uncommon catalytic residue Cys731 in BulbE TE was indispensable for the nucleophilic attack from the indole nitrogen. While the C731S variant failed to utilize the indole nitrogen and primary alcohol as nucleophiles, it retained the ability to employ the amine nucleophile, showing a clear correlation between the catalytic residues and the nucleophile scope. A model of the acyl-enzyme complex revealed how the substrate is recognized, including interactions involving a unique second lid-like structural motif in BulbE TE. This study provides an enzymatic basis for indole -acylation and offers important insights into the nucleophile specificity in TE-mediated macrocyclization.