Biosynthesis of Methylated Ditryptophan 2,5-Diketopeprazine Involves Cryptic <i>N</i> -nitrosation

Modifications to cyclodipeptides endow this class of natural products with structural diversity and bioactive significance. Herein, we identified a group of natural products, named ditrypzocin A–G, featuring a cyclo-di-l-tryptophan scaffold and N-nitroso modification on the indole moiety through intensive spectroscopic analysis and isotope labeling. In vivo and in vitro investigations revealed that these compounds are biosynthesized by a three-gene cluster, encoding a tRNA-dependent cyclodipeptide synthase, a SAM-dependent methyltransferase, and an acyl-CoA-synthetase-like N-nitrosylase. The N-nitrosation of indole side chain is catalyzed by the N-nitrosylase Mr3558 in an ATP and nitrite-dependent manner, which is a prerequisite to the methylation of the diketopiperazine ring but was neglected in previous study. Molecular docking and site-directed mutagenesis evidenced that a noncanonical substrate binding mode probably accounts for the dedicated catalysis of Mr3558. These findings enlarge the structural diversity of cyclodipeptides and supply a beneficial tool for the discovery and engineering of N-nitroso-containing compounds.