Camptotheca acuminata is best known as the natural source of camptothecin, the precursor of the anticancer medicines irinotecan and topotecan. The tree also accumulates diverse flavonoids that contribute to plant pigmentation and protection. Although camptothecins and flavonoids belong to different chemical families, both contain aromatic structures that can be refined through O-methylation, a modification that may alter stability, solubility, transport, and biological activity. Earlier studies identified individual enzymes capable of acting on both compound classes, but the broader organization, tissue distribution, substrate range, evolutionary relationship, and structural basis of this shared chemistry remained unclear. Based on these challenges, in-depth research into the enzymes coordinating camptothecin and flavonoid modification is needed.
Researchers from the Laboratory of Medicinal Plant Biotechnology, School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, reported (DOI: 10.1093/hr/uhag145) the study in Horticulture Research on April 21, 2026. Working with one-year-old Camptotheca acuminata plants, the team mapped metabolites and gene activity across seven tissues, then investigated a four-gene Camptotheca acuminata O-methyltransferase (CaOMT) cluster. The study shows that neighboring enzymes can share hydroxycamptothecin activity yet differ sharply in how they methylate flavonoids, linking genomic organization with catalytic specialization and offering a clearer view of how this medicinal tree creates and distributes diverse chemical products.
The researchers first profiled metabolites and sequenced transcriptomes from young and mature leaves, stem xylem, stem phloem, and three root types. Camptothecin and methoxycamptothecin were concentrated in stem phloem at 2.19 and 2.20 milligrams per gram, while methylated quercetin derivatives also showed strong tissue specificity. Integrating these patterns with genome data highlighted a four-gene cluster, CaOMT1–CaOMT4, and connected CaOMT3 expression with rhamnetin accumulation.
The encoded enzymes were tested through transient expression in Nicotiana benthamiana and recombinant production in Escherichia coli, followed by high-performance liquid chromatography with diode-array detection and ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. CaOMT1, CaOMT2, and CaOMT3 methylated both 9- and 10-hydroxycamptothecin. CaOMT1 and CaOMT3 also modified quercetin at the 4′- and 7-hydroxyl positions and produced the dimethylated flavonoid ombuin, whereas CaOMT2 acted only at the 4′ position.
CaOMT3 showed the highest catalytic efficiency toward quercetin. AlphaFold3 modeling, molecular docking, and site-directed mutagenesis identified F166, M313, and L316 as essential catalytic residues: replacing them reduced conversion of all three tested substrates by more than 90%. Additional mutations linked N261, S309, and T312 to CaOMT2’s narrower selectivity, while a 25-amino-acid deletion disrupted the binding pocket and explained CaOMT4’s inactivity.
The authors said the central message is that metabolic pathways do not always operate as isolated production lines. In Camptotheca acuminata, closely positioned genes encode enzymes that can serve both camptothecin and flavonoid chemistry, while small changes in their binding pockets redirect what each enzyme can do. They said this combination of shared function and fine specialization helps explain how plants expand chemical diversity without building every pathway from scratch. The structural evidence also turns the gene cluster from a genomic observation into a practical, testable map for selecting or redesigning enzymes.
The findings could support metabolic engineering of methoxycamptothecins and methylated flavonoids in plants, cultured cells, or microbial production platforms. Because the study identifies both broadly active enzymes and residues that control substrate positioning, researchers may be able to tune product profiles rather than simply increase total pathway output. CaOMT3 is a particularly promising starting point because it combined broad substrate use with strong catalytic efficiency, while CaOMT2 offers a model for position-specific modification. The work may also guide breeding or biotechnology strategies aimed at improving valuable metabolite accumulation in Camptotheca acuminata. However, stable-engineering studies, pathway-flux analysis, scale-up testing, and assessment of product yield, purity, and biological performance will be needed before practical manufacturing applications can be evaluated.
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References
DOI
10.1093/hr/uhag145
Original Source URL
https://doi.org/10.1093/hr/uhag145
Funding information
This work was supported by the National Key Research and Development Program of China (2023YFC3503900), the National Natural Science Foundation of China (82474030), the National “Ten-thousand Talents Program” for Leading Talents of Science and Technology Innovation in China, the National Young Qihuang Scholars Training Program, the Young Talent Support Project of China Association of Chinese Medicine (CACM-2024-QNRC2-B33), the Zhejiang Provincial Natural Science Foundation of China (LZ26H280003), and the Research Project of Zhejiang Chinese Medical University (2025JKZKTS21).
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.