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：

---

抗生素失活的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）的N-羟基化为2'-N-羟基-4-氧代-利福平，这是一种具有较低抗菌活性的代谢物。RIFMO与特征明确的黄素单加氧酶具有适度的序列相似性，但该蛋白尚未在分子水平上分离和鉴定。在这里，我们报告了诺卡氏菌的RIFMO的晶体结构，用小角X射线散射测定溶液中的低聚状态，以及底物结合的分光光度法表征。该结构将RIFMO识别为A类黄素蛋白单加氧酶，其折叠和四级结构类似于MtmOIV和OxyS，后者分别是光神霉素和土霉素的生物合成途径中的酶。RIFMO与其他A类黄素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结合结构域二聚形成最大尺寸为110 A的溶液中的钟形均二聚体。使用525 nm处的吸光度监控RIF结合，确定解离常数为13毫米 稳态耗氧量分析表明，只有当存在RIF时，NADPH才能有效地降低FAD，这意味着在催化方案中，RIF会先于NADPH结合。与RIF络合的RIFMO的1.8 A拆分结构表示在三元E-RIF-NADPH络合物形成之前发生的催化前构象。RIF萘醌可阻止进入FAD N5原子，暗示NADPH需要大量构象变化以减少FAD。提出了这些构象变化的模型。