5KOX

Structure of rifampicin monooxygenase complexed with rifampicin

5KOX の概要

• エントリーDOI: 10.2210/pdb5kox/pdb
• 分子名称: Pentachlorophenol 4-monooxygenase, FLAVIN-ADENINE DINUCLEOTIDE, RIFAMPICIN, ... (4 entities in total)
• 機能のキーワード: flavoprotein, monooxygenase, oxidoreductase
• 由来する生物種: Nocardia farcinica
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 53326.07

構造登録者

Tanner, J.J.,Liu, L.-K. (登録日: 2016-07-01, 公開日: 2016-09-07, 最終更新日: 2023-10-04)

主引用文献

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

実験手法

X-RAY DIFFRACTION (1.8 Å)