Elucidation of chalkophomycin biosynthesis reveals -hydroxypyrrole-forming enzymes.

Reactive functional groups, such as -nitrosamines, impart unique bioactivities to the natural products in which they are found. Recent work has illuminated enzymatic -nitrosation reactions in microbial natural product biosynthesis, motivating an interest in discovering additional metabolites constructed using such reactivity. Here, we use a genome mining approach to identify over 400 cryptic biosynthetic gene clusters (BGCs) encoding homologs of the -nitrosating biosynthetic enzyme SznF, including the BGC for chalkophomycin, a Cu-binding metabolite that contains a -type diazeniumdiolate and -hydroxypyrrole. Characterizing chalkophomycin biosynthetic enzymes reveals previously unknown enzymes responsible for -hydroxypyrrole biosynthesis, including the first prolyl--hydroxylase, and a key step in assembly of the diazeniumdiolate-containing amino acid graminine. Discovery of this pathway enriches our understanding of the biosynthetic logic employed in constructing unusual heteroatom-heteroatom bond-containing functional groups, enabling future efforts in natural product discovery and biocatalysis.