Tang, HaoyuHaoyuTangWu, Min-HaoMin-HaoWuLin, Hsiao-YuHsiao-YuLinHan, Meng-RuMeng-RuHanTu, Yueh-HuaYueh-HuaTuYang, Zhi-JieZhi-JieYangChien, Tun-ChengTun-ChengChienNEI-LI CHANChang, Wei-ChenWei-ChenChang2022-02-102022-02-102022-01-0400278424https://scholars.lib.ntu.edu.tw/handle/123456789/593986Deoxypodophyllotoxin contains a core of four fused rings (A to D) with three consecutive chiral centers, the last being created by the attachment of a peripheral trimethoxyphenyl ring (E) to ring C. Previous studies have suggested that the iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, deoxypodophyllotoxin synthase (DPS), catalyzes the oxidative coupling of ring B and ring E to form ring C and complete the tetracyclic core. Despite recent efforts to deploy DPS in the preparation of deoxypodophyllotoxin analogs, the mechanism underlying the regio- and stereoselectivity of this cyclization event has not been elucidated. Herein, we report 1) two structures of DPS in complex with 2OG and (±)-yatein, 2) in vitro analysis of enzymatic reactivity with substrate analogs, and 3) model reactions addressing DPS's catalytic mechanism. The results disfavor a prior proposal of on-pathway benzylic hydroxylation. Rather, the DPS-catalyzed cyclization likely proceeds by hydrogen atom abstraction from C7', oxidation of the benzylic radical to a carbocation, Friedel-Crafts-like ring closure, and rearomatization of ring B by C6 deprotonation. This mechanism adds to the known pathways for transformation of the carbon-centered radical in Fe/2OG enzymes and suggests what types of substrate modification are likely tolerable in DPS-catalyzed production of deoxypodophyllotoxin analogs.enC−C coupling; cyclization; natural product; oxygenase; reaction mechanism[SDGs]SDG3[SDGs]SDG6journal article10.1073/pnas.2113770119349698442-s2.0-85122700364https://api.elsevier.com/content/abstract/scopus_id/85122700364