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.

蒽醌稠合烯二炔 (AFE) 结合了蒽醌部分和十元烯二炔核心，能够产生细胞毒性双自由基。 AFE 环化是通过打开 F 环环氧化物触发的，这也是最具结构多样性的位点。先前对天西霉素 A（一种经过大量修饰的 AFE）的研究揭示了环氧化物的神秘醛阻断装置，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，我们通过比较基因组学鉴定了两种连续作用的无辅因子加氧酶，它们分别源自甲基转移酶和α/β-水解酶蛋白折叠，TnmJ和TnmK2，它们负责天西霉素生物合成中的F环剪裁。进一步的生化和结构表征表明，富电子的 AFE 蒽醌部分有助于催化去甲酰化、环氧化和氧化环裂解，无需外源辅因子。因此，这些酶填补了此类分子和未被充分重视的无辅因子加氧酶家族生物合成的重要知识空白。