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

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Nitroimidazoles are vital antibiotics targeting anaerobic pathogens.
• The enzyme RohS, part of the Heme-oxygenase-like dimetal/domain-containing oxidase/oxygenase (HDO) family, synthesizes 2-nitroimidazole from 2-aminoimidazole.
• Understanding RohS's mechanism is key to antibiotic development.

Purpose of the Study:

• To determine the crystal structure of RohS.
• To identify key residues in the active site for metal coordination and catalysis.
• To understand the functional differences between active and inactive RohS homologs.

Main Methods:

• X-ray crystallography to obtain the 2.20 Å resolution structure of RohS.
• Sequence and structural comparison of RohS homologs.
• Site-directed mutagenesis to convert inactive RohS to its active form.

Main Results:

• The crystal structure of RohS revealed a potential active site pocket with seven key residues for metal coordination.
• Comparison of active and inactive homologs identified a critical residue for metal coordination and RohS catalysis.
• The inactive homolog was successfully converted to an active form.

Conclusions:

• The study provides a structural basis for investigating the six-electron oxidation mechanism catalyzed by RohS.
• Findings offer insights into the HDO protein family and nitro-formation N-oxygenases.
• This work facilitates further research into nitroimidazole biosynthesis and antibiotic development.