Nitration in the biosynthesis of azomycin (2-nitroimidazole) by Streptomyces eurocidicus

Streptomyces eurocidicus bacteria produce the antibiotic azomycin (2-nitroimidazole), which is analogous to the 2,4-dinitroimidazole family of insensitive explosives that are currently synthesized using chemical nitration. A biochemical synthesis of nitroimidazoles would reduce the environmental impact from large-scale acidic nitration reactions. Previous studies identified L-arginine as the precursor for azomycin, but the reaction pathway remains to be confirmed, and no enzymes in this pathway have been identified. A hybrid assembly of S. eurocidicus sequencing reads produced a draft genome sequence with 7.9 Mbp, containing nearly 7000 coding DNA sequences. In order to identify proteins whose abundance correlated to azomycin biosynthesis during late growth stages, the cells were grown in bioreactors using both complex and minimal media, and samples were removed at different times for functional genomic characterization. A semi-quantitative proteomic analysis identified 3,627 proteins, including 293 that were more abundant in late growth-phase cells that produced azomycin compared to early growth phase cells. Bioinformatic analysis identified candidate proteins that may be involved in the separate production of eurocidin and tertiomycin, as well as candidate proteins for azomycin biosynthesis. Inhibitor studies using whole cells indicate that a cytochrome P450 enzyme catalyzes the final reaction:  the oxidation of 2-aminoimidazole to form 2-nitroimidazole. Enzymological studies are investigating the mechanism of 2-aminoimidazole oxidation and the electron transport chain required to activate the putative P450 enzyme. Heterologous expression experiments in other Streptomyces and E. coli host strains are testing the role of candidate genes in producing the targeted oxidase protein.