5KOX: Structure Of Rifampicin Monooxygenase Complexed With Rifampicin

Rifampicin monooxygenase (RIFMO) catalyzes the N-hydroxylation of the natural product antibiotic rifampicin (RIF) to 2'-N-hydroxy-4-oxo-rifampicin, a metabolite with much lower antimicrobial activity. RIFMO shares moderate sequence similarity with well characterized flavoprotein monooxygenases, but the protein has not been isolated and characterized at the molecular level. Herein, we report crystal structures of RIFMO from Nocardia farcinica, the determination of the oligomeric state in solution with small angle x-ray scattering, and the spectrophotometric characterization of substrate binding. The structure identifies RIFMO as a class A flavoprotein monooxygenase and is similar in fold and quaternary structure to MtmOIV and OxyS, which are enzymes in the mithramycin and oxytetracycline biosynthetic pathways, respectively. RIFMO is distinguished from other class A flavoprotein monooxygenases by its unique middle domain, which is involved in binding RIF. Small angle x-ray scattering analysis shows that RIFMO dimerizes via the FAD-binding domain to form a bell-shaped homodimer in solution with a maximal dimension of 110 A. RIF binding was monitored using absorbance at 525 nm to determine a dissociation constant of 13 mum Steady-state oxygen consumption assays show that NADPH efficiently reduces the FAD only when RIF is present, implying that RIF binds before NADPH in the catalytic scheme. The 1.8 A resolution structure of RIFMO complexed with RIF represents the precatalytic conformation that occurs before formation of the ternary E-RIF-NADPH complex. The RIF naphthoquinone blocks access to the FAD N5 atom, implying that large conformational changes are required for NADPH to reduce the FAD. A model for these conformational changes is proposed.
PDB ID: 5KOXDownload
MMDB ID: 142869
PDB Deposition Date: 2016/7/1
Updated in MMDB: 2016/11
Experimental Method:
x-ray diffraction
Resolution: 1.8  Å
Source Organism:
Similar Structures:
Biological Unit for 5KOX: dimeric; determined by author and by software (PISA)
Molecular Components in 5KOX
Label Count Molecule
Proteins (2 molecules)
Pentachlorophenol 4-monooxygenase
Molecule annotation
Chemicals (4 molecules)
* Click molecule labels to explore molecular sequence information.

Citing MMDB