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.
Recommended Citation
L. K. Liu et al., "The Structure of the Antibiotic Deactivating, N-hydroxylating Rifampicin Monooxygenase," Journal of Biological Chemistry, vol. 291, no. 41, pp. 21553 - 21562, Elsevier; American Society for Biochemistry and Molecular Biology, Oct 2016.
The definitive version is available at https://doi.org/10.1074/jbc.M116.745315
Department(s)
Chemistry
Publication Status
Open Access
International Standard Serial Number (ISSN)
1083-351X; 0021-9258
Document Type
Article - Journal
Document Version
Final Version
File Type
text
Language(s)
English
Rights
© 2024 Elsevier; American Society for Biochemistry and Molecular Biology, All rights reserved.
Creative Commons Licensing
This work is licensed under a Creative Commons Attribution 4.0 License.
Publication Date
07 Oct 2016
PubMed ID
27557658
Comments
National Science Foundation, Grant CHE-1506206