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J Biol Chem. 2016 Oct 7;291(41):21553-21562. Epub 2016 Aug 24.

The Structure of the Antibiotic Deactivating, N-hydroxylating Rifampicin Monooxygenase.

Author information

  • 1From the Departments of Biochemistry and.
  • 2the Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061, and.
  • 3the Department of Chemistry, Faculty of Science, Damietta University, Damietta 34517, Egypt.
  • 4the Structural Biology Core, University of Missouri, Columbia, Missouri 65211.
  • 5the Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061, and psobrado@vt.edu.
  • 6From the Departments of Biochemistry and tannerjj@missouri.edu.
  • 7Chemistry and.

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.

KEYWORDS:

enzyme kinetics; enzyme structure; flavoprotein; small-angle X-ray scattering (SAXS); x-ray crystallography

PMID:
27557658
PMCID:
PMC5076826
[Available on 2017-10-07]
DOI:
10.1074/jbc.M116.745315
[PubMed - in process]
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