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Cofactorless oxygenases guide anthraquinone-fused enediyne biosynthesis

Nature Chemical Biology volume 20, pages 243–250 (2024)Cite this article

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Abstract

The anthraquinone-fused enediynes (AFEs) combine an anthraquinone moiety and a ten-membered enediyne core capable of generating a cytotoxic diradical species. AFE cyclization is triggered by opening the F-ring epoxide, which is also the site of the most structural diversity. Previous studies of tiancimycin A, a heavily modified AFE, have revealed a cryptic aldehyde blocking installation of the epoxide, and no unassigned oxidases could be predicted within the tnm biosynthetic gene cluster. Here we identify two consecutively acting cofactorless oxygenases derived from methyltransferase and α/β-hydrolase protein folds, TnmJ and TnmK2, respectively, that are responsible for F-ring tailoring in tiancimycin biosynthesis by comparative genomics. Further biochemical and structural characterizations reveal that the electron-rich AFE anthraquinone moiety assists in catalyzing deformylation, epoxidation and oxidative ring cleavage without exogenous cofactors. These enzymes therefore fill important knowledge gaps for the biosynthesis of this class of molecules and the underappreciated family of cofactorless oxygenases.

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Fig. 1: Selected AFEs and their BGCs.
Fig. 2: Steps and intermediates in F-ring modification of AFEs.
Fig. 3: TnmJ catalyzes the SAM-independent cofactorless cryptic oxidation of TNM I (8).
Fig. 4: TnmJ deformylates TNM I (8) to yield TNM J (13), H2O and CO2.
Fig. 5: TnmK2 and homologs TnmK1 and DynA4 catalyze different reactions.
Fig. 6: TnmK2 uses a repurposed α/β-hydrolase fold for multiple cofactorless oxidations.

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Data availability

The data that support the findings of this study are available within the main text and its Supplementary Information file. Data are available from the corresponding author upon request. The coordinates of the TnmJ, TnmK2 and TnmK2–9 structures have been deposited to the PDB with the accession codes 8G5S, 8G5T and 8G5U, respectively. Source data are provided with this paper.

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Acknowledgements

This work was supported in part by National Institutes of Health (NIH) grants GM134954 (B.S.) and OD021550 (NMR Core Facility). This work was supported in part by NIH postdoctoral fellowships GM134688 (E.K.) and GM133114 (A.D.S.). We thank X. Kong of the NMR Core Facility at The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, University of Florida, Jupiter, Florida, for assistance with NMR analysis.

Author information

Author notes

  1. These authors contributed equally: Chun Gui, Edward Kalkreuter.

Authors and Affiliations

  1. Department of Chemistry, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, University of Florida, Jupiter, FL, USA

    Chun Gui, Edward Kalkreuter, Yu-Chen Liu, Gengnan Li, Andrew D. Steele, Dong Yang & Ben Shen

  2. Natural Products Discovery Center, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, University of Florida, Jupiter, FL, USA

    Dong Yang & Ben Shen

  3. Structural Biology Center, X-ray Science Division, Argonne National Laboratory, Lemont, IL, USA

    Changsoo Chang

  4. Department of Molecular Medicine, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, University of Florida, Jupiter, FL, USA

    Ben Shen

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Contributions

C.G., E.K., Y.-C.L., G.L., A.D.S., D.Y. and C.C. performed experiments and analysis. C.G., E.K. and B.S. conceived of the project and designed experiments. C.G., E.K. and B.S. wrote the manuscript.

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Correspondence to Ben Shen.

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Extended data

Extended Data Fig. 1 Evaluation of TnmJ for metal, SAM, and reducing agent dependence.

a, No difference was observed upon addition of 10 mM EDTA to a reaction with TnmJ and 8. b, No difference was observed upon addition of 2 mM reducing agents DTT or TCEP. c, No difference was observed upon addition of 200 μM SAM or SAH to a reaction with TnmJ and 8. Likewise, charcoal-treated TnmJ showed no difference. Bars depict the mean; error bars represent standard deviation (n = 3 independent experiments).

Source data

Extended Data Fig. 2 Mutagenesis of TnmJ.

Site-directed mutants of TnmJ were evaluated in vitro with 100 μM 8. Results were used to evaluate possible binding modes from molecular dynamics simulations. Bars depict the mean; error bars represent standard deviation (n = 3 independent experiments).

Source data

Extended Data Fig. 3 Mutagenesis of TnmK2.

TnmK2 mutants were evaluated in vitro with 13 produced in situ by TnmJ from 100 μM 8. N.D. = not detected. Bars depict the mean; error bars represent standard deviation (n = 3 independent experiments).

Source data

Supplementary information

Supplementary Information

Supplementary methods, Figs. 1–44 and Tables 1–10.

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Supplementary Data

Statistical source data for Supplementary Figs. 20, 27, 31, 33 and 43.

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Source Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 2

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Source Data Extended Data Fig. 3

Statistical source data.

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Gui, C., Kalkreuter, E., Liu, YC. et al. Cofactorless oxygenases guide anthraquinone-fused enediyne biosynthesis. Nat Chem Biol 20, 243–250 (2024). https://doi.org/10.1038/s41589-023-01476-2

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