5KOX

Structure of rifampicin monooxygenase complexed with rifampicin

Summary for 5KOX

• Entry DOI: 10.2210/pdb5kox/pdb
• Descriptor: Pentachlorophenol 4-monooxygenase, FLAVIN-ADENINE DINUCLEOTIDE, RIFAMPICIN, ... (4 entities in total)
• Functional Keywords: flavoprotein, monooxygenase, oxidoreductase
• Biological source: Nocardia farcinica
• Total number of polymer chains: 1
• Total formula weight: 53326.07

Authors

Tanner, J.J.,Liu, L.-K. (deposition date: 2016-07-01, release date: 2016-09-07, Last modification date: 2023-10-04)

Primary citation

Liu, L.K.,Abdelwahab, H.,Martin Del Campo, J.S.,Mehra-Chaudhary, R.,Sobrado, P.,Tanner, J.J.
The Structure of the Antibiotic Deactivating, N-hydroxylating Rifampicin Monooxygenase.
J.Biol.Chem., 291:21553-21562, 2016

PubMed Abstract: 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 Å. RIF binding was monitored using absorbance at 525 nm to determine a dissociation constant of 13 μm 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 Å 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.

PubMed: 27557658
DOI: 10.1074/jbc.M116.745315

Experimental method

X-RAY DIFFRACTION (1.8 Å)