Abstract
Selective activation of aliphatic C–H bonds in polycyclic terpenoids offers a potent strategy for exploring diverse chemical space in drug design, yet challenges persist in achieving site- and diastereoselectivity, especially with increasing structural complexity. Here we report a bioinformatics-driven terpene–P450 discovery strategy to develop bacterial cytochrome P450s for selective activation of aliphatic C–H bonds in structurally intricate pentacyclic triterpenoids (PTs). The identified ApPT demonstrated elegant diastereoselectivity and broad substrate tolerance, facilitating a chemo-enzymatic platform to explore the chemical space of PTs at previously inaccessible sites. Protein crystallization and computational analysis reveal the mechanism of the preliminary C–H bond activation selectivity of ApPT towards various PTs, particularly an example of enzymatic C7-to-C15 relay oxidation mediated by 1,5-hydrogen atom transfer. This work offers ApPT as a valuable biocatalyst to explore the chemical space of PTs via aliphatic C–H bond activation, demonstrating the advantage of our biocatalyst-discovery strategy for the late-stage diversification of polycyclic terpenoids.
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Data availability
The authors declare that all data supporting the findings of this study are available in the main text or Supplementary Information. The X-ray crystal structures of 1b, 3a and 10a are available free from the Cambridge Crystallographic Data Centre under reference numbers: CCDC 2290949 (1b), 2290950 (3a) and 2290948 (10a). The atomic coordinates of ApPT have been deposited in the Protein Data Bank under accession code 8W7G. Source data are provided with this paper.
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Acknowledgements
We appreciate financial support from the National Key Research and Development Program of China (grant no. 2022YFA1503200 to Y.L.), the National Science Foundation of China (grant nos 82473823 to L.-B.D., 22525301 to Y.L. and 22403046 to H.C.), the Project Program of State Key Laboratory of Natural Medicines at China Pharmaceutical University (grant no. SKLNMZZ2024JS44 to L.-B.D.), the Natural Science Foundation of Jiangsu Province (grant nos BK20240097 to L.-B.D., BK20230018 to Y.L. and BK20241231 to H.C.), the Fundamental Research Funds for the Central Universities (grant no. 2632025TD06 to L.-B.D.), the Jiangsu Provincial Major Science and Technology Special Project (grant no. BG2024046 to L.-B.D.) and Postgraduate Research & Practice Innovation Program of Jiangsu Province (grant no. KYCX25_1064 to X.Z.). We thank the staff at beamlines BL02U1, BL10U2 and BL18U1 of Shanghai Synchrotron Radiation Facility for assistance during data collection. We thank the High Performance Computing Center (HPCC) of Nanjing University for doing the numerical calculations in this paper on its blade cluster system. We thank D. Nelson from University of Tennessee for assigning the name CYP161H12 in accordance with the standard Cytochrome P450 Nomenclature. We also express our gratitude to C. Luo from Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Y. Zheng from China Pharmaceutical University, H. Li from Peking University and F. Li from Fudan University for valuable discussions.
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L.-B.D. and Y.L. conceived the project. X.Z., H.C., Y.L. and L.-B.D. designed the experiments. X.Z., Y.W., Z.W., X.L., F.-R.L., X.P. and H.-M.X. performed the experiments. H.C., S.X., J.Q. and Y.L. carried out the computational study. All authors analysed and discussed the results. X.Z., H.C., Y.L. and L.-B.D. wrote the paper with inputs from all co-authors.
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Extended data
Extended Data Fig. 1 Strategies for aliphatic C–H activation of pentacyclic terpenoids (PTs): organic synthesis versus biocatalysis in this work.
The detailed efforts for these strategies are summarized in Supplementary Fig. 1.
Extended Data Fig. 2 Twenty-nine putative terpenoid-producing biosynthetic gene clusters (BGCs) from 11 bacterial strains.
The blue arrows show anchors of putative terpene synthases (TSs) for EFI-GNT. The red arrows show associated CYP450s in the clusters. All terpene synthases in the genome are considered suitable for guiding the discovery of CYP450s, without being restricted to a particular type.
Extended Data Fig. 3 Optimization of the in vitro reaction conditions for ApPT-catalyzed oxidation of 1.
a, Reaction parameters optimization including alternative redox partners (RPs), biocatalyst loads, as well as the pH. Yields were determined by HPLC analysis. Conditions: 2 mM 1 in 5% (v/v) DMSO, 1 mM NADP+, 100 mM Na2HPO3, opt13, and ApPT/RPs in 100 mM kpi buffer at 23 °C, 220 rpm for 20 h. aBiocatalyst loads in entries 1–6 can be effectively determined according to the Supplementary Methods. Due to the biocatalysts in entries 7–10 cannot be purified, the hydroxylated activity was determined using the same amount of crude enzyme lysate with OD600 = 30. b, Different types and expression modes of RPs with ApPT. RP1 and RP2 represent self-sufficient ApPT chimeras that were expressed in the pET28a(+) plasmid. RP3, RP4, and RP5 represent RPs that were individually expressed alongside ApPT in the pRSFDuet-1 plasmid.
Extended Data Fig. 4 In vitro anti-tumor and anti-inflammatory activities of glycyrrhetinic acid derivatives (GADs).
a, Anti-inflammatory activities of the selected GADs in LPS-induced RAW264.7 cells. L-NMMA (NG-monomethyl-L-arginine) was used as a positive control. b, Anti-proliferation activities of the selected GADs against five cancer cell lines. Bardoxolone methyl (CDDO-Me) was used as a positive control. c, Selective inhibitory effects on tumor cell (HeLa) and non-tumor cell (Ect1/E6E7) for representative compounds-25, 26, 27, and CDDO-Me (48 h; n = 3 biologically replicated experiments; mean ± s.d.). HepG2, human liver cancer cell line. SW480, human colorectal adenocarcinoma cell line. A549, human non-small cell lung cancer cell line. PC-3, human prostate cancer cell line. HeLa, human cervical cancer cell line. Ect1/E6E7, human cervical immortalized squamous cell line. N.D., not detected. RAW264.7, mouse mononuclear macrophage cell line. SF (selectivity factor), the ratio of IC50Ect1/E6E7 to IC50HeLa.
Extended Data Fig. 5 DFT calculations and MD simulations for the C7 hydroxylation of 1.
a, Gibbs free energy profiles and DFT-optimized transition states for 1. b, The binding free energies ΔGbind between ApPT and 1-TSC15, 1, and 1-TSC7, respectively. Computed at the PCM (chlorobenzene)-B3LYP-D3/6-311 + G(d,p)+SDD(Fe)//PCM (chlorobenzene)-B3LYP-D3/6-31 G(d)+LanL2DZ(Fe) level of theory. Distances are shown in Å.
Supplementary information
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Supplementary Figs. 1–27, Tables 1–24 and Notes 1–5.
Supplementary Data 1 (download PDF )
DFT-computed Gibbs free energies and Cartesian coordinates.
Supplementary Data 2 (download XLSX )
Numerical data underlying Supplementary Figs. 9, 10 and 17b.
Source data
Source Data Fig. 5 (download XLSX )
Numerical data underlying Fig. 5c.
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Zhang, X., Chen, H., Wang, Y. et al. Exploring bacterial cytochrome P450s for selective activation of aliphatic C–H bonds in pentacyclic triterpenoids. Nat. Chem. (2026). https://doi.org/10.1038/s41557-026-02106-9
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DOI: https://doi.org/10.1038/s41557-026-02106-9
