Elucidation of chalkophomycin biosynthesis revealsN-hydroxypyrrole-forming enzymes

Reactive functional groups, such asN-nitrosamines, impart unique bioactivities to the natural products in which they are found. Recent work has illuminated enzymaticN-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 theN-nitrosating biosynthetic enzyme SznF, including the BGC for chalkophomycin, a CuII-binding metabolite that contains aC-type diazeniumdiolate andN-hydroxypyrrole. Characterizing chalkophomycin biosynthetic enzymes reveals previously unknown enzymes responsible forN-hydroxypyrrole biosynthesis and an unexpected role for a heme-dependent enzyme inN-nitrosation. 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.