Xanthone-containing natural products display diverse pharmacological properties. The biosynthetic mechanisms of the xanthone formation have not been well documented. Here we show that the flavoprotein monooxygenase FlsO1 in the biosynthesis of fluostatins not only functionally compensates for the monooxygenase FlsO2 in converting prejadomycin to dehydrorabelomycin, but also unexpectedly converts prejadomycin to xanthone-containing products by catalyzing three successive oxidations including hydroxylation, epoxidation and Baeyer-Villiger oxidation. We also provide biochemical evidence to support the physiological role of FlsO1 as the benzo[b]-fluorene C5-hydrolase by using nenestatin C as a substrate mimic. Finally, we resolve the crystal structure of FlsO1 in complex with the cofactor flavin adenine dinucleotide close to the “in” conformation to enable the construction of reactive substrate-docking models to understand the basis of a single enzyme-catalyzed multiple oxidations. This study highlights a mechanistic perspective for the enzymatic xanthone formation in actinomycetes and sets an example for the versatile functions of flavoproteins.

含黄酮的天然产物表现出多种药理特性。黄酮形成的生物合成机制尚未得到充分证明。在这里，我们表明，在氟他汀生物合成中的黄素蛋白单加氧酶 FlsO1 不仅在功能上补偿单加氧酶 FlsO2 将 prejadomycin 转化为脱氢雷贝洛霉素，而且还通过催化羟基化、环氧化和 Baeyer-Villiger 三个连续的氧化反应意外地将 prejadomycin 转化为含黄酮的产物氧化。我们还提供生化证据来支持 FlsO1 作为苯并[ b]-芴 C5-水解酶，使用 nenestatin C 作为底物模拟物。最后，我们解析了 FlsO1 与接近“in”构象的辅因子黄素腺嘌呤二核苷酸复合的晶体结构，从而能够构建反应性底物对接模型以了解单一酶催化的多重氧化的基础。这项研究突出了放线​​菌中酶促黄酮形成的机制观点，并为黄素蛋白的多功能功能树立了榜样。