Multienzyme-Mediated Atypical Mode of Dehydration in Anthracycline Biosynthesis

Anthracyclines featuring a tetracyclic (A–D rings) scaffold, such as clinically approved doxorubicin and aclarubicin, are essential natural products. The D-ring presents an intriguing structural puzzle: while predominantly nonaromatic and typically decorated with one or two α-oriented oxygenated substituents, its aromatization constitutes a critical biosynthetic prerequisite for the conversion of resomycin C into chartreusin, a promising antitumor drug candidate. Given the intriguing and biologically significant nature of the “D-ring state”, we elucidate here the mechanism of D-ring aromatization during resomycin C biosynthesis. This process features an unprecedented dehydration pattern mediated by the collaborative catalysis of two enzymes, ChaU and ChaX. Combining gene inactivation, biochemical assays, isotope labeling, protein crystallography, and site-directed mutagenesis, we demonstrated that ChaU facilitates the stereoselective C2–C19 cyclization and plays a critical role in prompting the attack of water on C17 from the pro-R face, followed by the 17-dehydroxylation mediated by ChaX, thereby triggering the aromatization of the D-ring. Furthermore, molecular dynamics simulations and density functional theory calculations elucidated the stepwise catalytic mechanism of the ChaU/ChaX-mediated cascade reactions that drive D-ring aromatization, uncovering a previously unrecognized dehydration pathway. Collectively, this work reveals sophisticated enzymatic strategies for anthracycline biosynthesis, enabling a more rational biotechnological production of valuable anthracyclines.