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The Structure of the Antibiotic Deactivating, N-hydroxylating Rifampicin Monooxygenase.
Journal of Biological Chemistry ( IF 4.0 ) Pub Date : 2016 Aug 24 , DOI: 10.1074/jbc.m116.745315
Li-Kai Liu 1 , Heba Abdelwahab 2, 3 , Julia S Martin Del Campo 2 , Ritcha Mehra-Chaudhary 4 , Pablo Sobrado 5 , John J Tanner 6, 7
Affiliation  

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 pre-catalytic conformation that occurs prior to 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.

中文翻译:


抗生素失活、N-羟基化利福平单加氧酶的结构。



利福平单加氧酶 (RIFMO) 催化天然产物抗生素利福平 (RIF) N-羟基化为 2'-N-羟基-4-氧代-利福平,这是一种抗菌活性低得多的代谢物。 RIFMO 与已充分表征的黄素蛋白单加氧酶具有中等程度的序列相似性,但该蛋白质尚未在分子水平上分离和表征。在此,我们报告了来自诺卡氏菌的 RIFMO 的晶体结构、用小角 X 射线散射测定溶液中寡聚状态以及底物结合的分光光度表征。该结构将 RIFMO 识别为 A 类黄素蛋白单加氧酶,并且在折叠和四级结构上与 MtmOIV 和 OxyS 相似,MtmOIV 和 OxyS 分别是光神霉素和土霉素生物合成途径中的酶。 RIFMO 与其他 A 类黄素蛋白单加氧酶的区别在于其独特的中间结构域,该结构域参与结合 RIF。小角 X 射线散射分析表明,RIFMO 通过 FAD 结合结构域二聚化,在溶液中形成最大尺寸为 110 A 的钟形同二聚体。使用 525 nm 处的吸光度监测 RIF 结合,以确定解离常数13微米。稳态耗氧量测定表明,仅当 RIF 存在时,NADPH 才能有效降低 FAD,这意味着在催化方案中 RIF 在 NADPH 之前结合。 RIFMO 与 RIF 复合的 1.8 A 分辨率结构代表了在三元 E-RIF-NADPH 复合物形成之前发生的预催化构象。 RIF 萘醌阻碍了 FAD N5 原子的进入,这意味着 NADPH 需要发生较大的构象变化才能减少 FAD。提出了这些构象变化的模型。
更新日期:2017-01-31
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