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Volume 31
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10.3390/molecules31111787
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Article

A Substrate-Dependent Bifunctional Dioxygenase from Fraxinus chinensis for O-Demethylation and C8-Hydroxylation of Coumarins

by
Xue-Ping Kong
,
Xue-Qing Zhong
,
Hong-Ling Yan
,
Zhuo-Zheng Xu
,
Jia-Xu Qin
,
Jing Yang
,
Qing-Li He
* and
Qun-Fei Zhao
*
State Key Laboratory of Discovery and Utilization of Functional Components in Traditional Chinese Medicine, Innovation Research Institute of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
*
Authors to whom correspondence should be addressed.
Molecules 2026, 31(11), 1787; https://doi.org/10.3390/molecules31111787
Submission received: 3 April 2026 / Revised: 9 May 2026 / Accepted: 20 May 2026 / Published: 22 May 2026
(This article belongs to the Section Bioorganic Chemistry)
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Abstract

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.
Keywords: Fraxinus chinensis Roxb.; 2-oxoglutarate-dependent dioxygenase; substrate-dependent bifunctionality; coumarins; O-demethylation; hydroxylation Fraxinus chinensis Roxb.; 2-oxoglutarate-dependent dioxygenase; substrate-dependent bifunctionality; coumarins; O-demethylation; hydroxylation

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MDPI and ACS Style

Kong, X.-P.; Zhong, X.-Q.; Yan, H.-L.; Xu, Z.-Z.; Qin, J.-X.; Yang, J.; He, Q.-L.; Zhao, Q.-F. A Substrate-Dependent Bifunctional Dioxygenase from Fraxinus chinensis for O-Demethylation and C8-Hydroxylation of Coumarins. Molecules 2026, 31, 1787. https://doi.org/10.3390/molecules31111787

AMA Style

Kong X-P, Zhong X-Q, Yan H-L, Xu Z-Z, Qin J-X, Yang J, He Q-L, Zhao Q-F. A Substrate-Dependent Bifunctional Dioxygenase from Fraxinus chinensis for O-Demethylation and C8-Hydroxylation of Coumarins. Molecules. 2026; 31(11):1787. https://doi.org/10.3390/molecules31111787

Chicago/Turabian Style

Kong, Xue-Ping, Xue-Qing Zhong, Hong-Ling Yan, Zhuo-Zheng Xu, Jia-Xu Qin, Jing Yang, Qing-Li He, and Qun-Fei Zhao. 2026. "A Substrate-Dependent Bifunctional Dioxygenase from Fraxinus chinensis for O-Demethylation and C8-Hydroxylation of Coumarins" Molecules 31, no. 11: 1787. https://doi.org/10.3390/molecules31111787

APA Style

Kong, X.-P., Zhong, X.-Q., Yan, H.-L., Xu, Z.-Z., Qin, J.-X., Yang, J., He, Q.-L., & Zhao, Q.-F. (2026). A Substrate-Dependent Bifunctional Dioxygenase from Fraxinus chinensis for O-Demethylation and C8-Hydroxylation of Coumarins. Molecules, 31(11), 1787. https://doi.org/10.3390/molecules31111787

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