Crystal structure of RohS, a heme-oxygenase-like N-oxygenase from azomycin biosynthesis

The nitro group is an important functional group found in the nitroimidazoles, antibiotic therapeutics for anaerobic pathogens. In the biosynthetic pathway to the nitroimidazole antibiotic azomycin, a nitro-forming enzyme RohS-a member of the heme-oxygenase-like dimetal/domain-containing oxidase/oxygenase (HDO) family-catalyzes a six-electron oxidation of 2-aminoimidazole to 2-nitroimidazole. Here, we present the 2.20 Å resolution crystal structure of RohS and identify a potential active site pocket consisting of seven key residues important for metal coordination. By comparing the structures and sequences of two RohS homologs-one functionally active and one inactive-we convert the inactive RohS to its active form, thus revealing a key residue for metal coordination in RohS catalysis. Altogether, our work provides a structural basis for further mechanistic investigation of this six-electron oxidation process and provides insight into the expanding repertoire of the HDO protein family and nitro-formation N-oxygenases.