Fraxinus chinensis Roxb. (Qinpi), a traditional Chinese medicinal plant, accumulates abundant coumarins that contribute to its anti-inflammatory and other bioactivities. However, the enzymatic basis for coumarin structural diversification in this species remains largely unexplored. Here, through transcriptome-wide identification of the 2-oxoglutarate-dependent dioxygenase (2OGD) family in F. chinensis, followed by phylogenetic analysis, heterologous expression, and in vitro enzyme assays, we identified FcDOH2, a member of the DOXC31 subfamily, which exhibits substrate-dependent bifunctionality, catalyzing the C6-O-demethylation of scopoletin (3) to esculetin (2) and the C8-hydroxylation of umbelliferone (1) to daphnetin (6). Using AlphaFold3-based structural modeling, molecular docking, and alanine scanning mutagenesis, we revealed that residues R155 and R221 are essential for both activities through stabilizing hydrogen bonds, whereas residue F312 acts as a functional switch, being critical for demethylation but negatively regulating hydroxylation. These findings uncover a rare bifunctional 2OGD with substrate-dependent catalytic plasticity, providing mechanistic insights into coumarin diversification in medicinal plants and a structural basis for future enzyme engineering.

Coleus strobilifer, the dried rhizome and root of Coleus strobilifer (Roxb.) A.J. Paton, is widely used for dampness-detoxification and detumescence in Chinese folklore. This study marks the first comprehensive investigation into the chemical composition of the whole herb of C. strobilifer, leading to the isolation and identification of two new abietane diterpenes, 10R-carnosuain (1) and 10R-coleon U-3-one (2), along with 34 known compounds (3–36) isolated from C. strobilifer for the first time. Their structures were unambiguously elucidated by analyses of NMR, HRESIMS, IR, CD, and single-crystal X-ray diffraction data, and comparison with the literature. All the isolated compounds were screened for their xanthine oxidase (XO) inhibitory activity. Among them, apigenin (8), luteolin (9), and esculetin (29) showed moderate XO inhibitory activity with IC₅₀ values of 0.034 ± 0.004, 0.067 ± 0.005, and 0.284 ± 0.01 mM, respectively.