The anthraquinone-fused enediynes (AFEs) combine an anthraquinone moiety and a ten-membered enediyne core capable of generating a cytotoxic diradical species. AFE cyclization is triggered by opening the F-ring epoxide, which is also the site of the most structural diversity. Previous studies of tiancimycin A, a heavily modified AFE, have revealed a cryptic aldehyde blocking installation of the epoxide, and no unassigned oxidases could be predicted within the tnm biosynthetic gene cluster. Here we identify two consecutively acting cofactorless oxygenases derived from methyltransferase and α/β-hydrolase protein folds, TnmJ and TnmK2, respectively, that are responsible for F-ring tailoring in tiancimycin biosynthesis by comparative genomics. Further biochemical and structural characterizations reveal that the electron-rich AFE anthraquinone moiety assists in catalyzing deformylation, epoxidation and oxidative ring cleavage without exogenous cofactors. These enzymes therefore fill important knowledge gaps for the biosynthesis of this class of molecules and the underappreciated family of cofactorless oxygenases.

蒽醌融合的烯二炔 （AFE） 结合了一个蒽醌部分和一个能够产生细胞毒性二自由基物种的十元烯二炔核心。AFE 环化是通过打开 F 环环氧化物触发的，这也是结构多样性最强的部位。先前对天西霉素 A（一种高度修饰的 AFE）的研究揭示了环氧化物的隐蔽醛阻断装置，并且在 tnm 生物合成基因簇内无法预测未分配的氧化酶。在这里，我们确定了两种连续作用的无辅因子加氧酶，分别来源于甲基转移酶和 α/β-水解酶蛋白折叠，TnmJ 和 TnmK2，它们负责通过比较基因组学在天ci霉素生物合成中进行 F 环定制。进一步的生化和结构表征表明，富含电子的 AFE 蒽醌部分有助于催化变形、环氧化和氧化环裂解，而无需外源性辅因子。因此，这些酶填补了这类分子和被低估的无辅因子加氧酶家族的生物合成的重要知识空白。