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An enzymatic dual-oxa Diels–Alder reaction constructs the oxygen-bridged tricyclic acetal unit of (–)-anthrabenzoxocinone

Nature Chemistry volume 17, pages 1058–1066 (2025)Cite this article

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Abstract

The hetero-Diels–Alder (HDA) reaction is a key method for synthesizing six-membered heterocyclic rings in natural products and bioactive compounds. Despite its importance in synthetic chemistry, naturally occurring enzymatic HDA reactions are rare and limited to a single heteroatom. Here we report Abx(−)F, a bifunctional vicinal oxygen chelate (VOC)-like protein that catalyses dehydration and dual-oxa Diels–Alder reactions to stereoselectively form the oxygen-bridged tricyclic acetal of (–)-anthrabenzoxocinone ((−)-ABX). Isotope assays and density functional theory calculations reveal a dehydration-coordinated, concerted HDA mechanism. The crystal structure of Abx(−)F and NMR complex structures of Abx(−)F with its substrate analogue and (−)-ABX define the reaction’s structural basis. Mutational analysis identifies Asp17 as a general base that mediates dehydration, forming an o-quinone methide intermediate for stereoselective dual-oxa HDA. This work establishes the molecular and structural basis of a polyheteroatomic Diels–Alderase, paving the way for designing polyheteroatomic DA enzymatic tools.

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Fig. 1: An overall schematic view of the oxa DA reaction and natural products containing oxygen-bridged tricyclic acetal.
Fig. 2: (−)-ABX biosynthetic gene cluster and key biosynthetic steps.
Fig. 3: Structure of the methylated derivatives of 2 and functional characterization of Abx(−)E and Abx(−)F.
Fig. 4: The calculated reaction energy profile shows the plausible reactions of 2 towards (±)-ABX.
Fig. 5: Structure and site-directed mutagenesis characterization of Abx(−)F.
Fig. 6: Proposed catalytic mechanism of Abx(−)F.

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Data availability

The crystal structure of Abx(−)F is available in the PDB under accession no. of 9JT3 (revised structure with 2.00 Å resolution and 30.1 Wilson B-factor) and 8STB (original structure with 2.22 Å resolution and 40.45 Wilson B-factor). The NMR structures of Abx(−)F and Abx(−)F in complex with (−)-ABX are available in the PDB under accession nos. 8EO9 and 8EPY, respectively. The complete chemical-shift assignments for Abx(−)F and Abx(−)F in the presence of (−)-ABX are available from BioMagResBank under BMRB ID 31044 and 31046, respectively. Crystallographic data for compound 5 have been deposited at the Cambridge Crystallographic Data Centre, under deposition no. CCDC 2308930. Source data are provided with this paper.

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Acknowledgements

This work was supported in part by the NSFC (32425033 to X.Q., 32300044 to X.Y., 31970041 to Y.-L.Z.), the National Key R&D Program of China (2018YFA0900400 to X.Q., 2020YFA0907700 to Y.-L.Z.), the Program of Shanghai Academic/Technology Research Leader (22XD1421300 to X.Q.), the Shanghai Municipal Science and Technology Major Project (to X.Q.), the Natural Science Foundation of Shanghai (23ZR1432800 to X.Q.), the University of Queensland (UQ Postdoctoral Research Fellowship to X.J., UQ Principal Research Fellowship to M.M.), and the Australian Research Council (ARC Laureate Fellowship FL180100109 to B.K.). We acknowledge the facilities, and the scientific and technical assistance of UQ-ROCX and the Australian Microscopy & Microanalysis Research Facilities at the Centre for Microscopy and Microanalysis, the University of Queensland; as well as the Macromolecular Crystallography (MX) beamlines at the Australian Synchrotron, Melbourne, Victoria, Australia and the Shanghai Synchrotron Radiation Facility BL10U2 beamline. We also thank S. Singh from Merck for kindly gifting the (–)-ABX producing strain S. sp. MA6657, W. Xie for critical discussions, D. Ericsson at Australian Synchrotron for his assistance with screening a potential anomalous signal from metal ions when collecting the diffraction data of Abx(-)F crystals, the Queensland NMR network and the Instrumental Analysis Center of Shanghai Jiao Tong University for access to the 600-, 700- and 900-MHz NMR spectrometer equipped with cryoprobes. We thank X. Bo at the National University of Singapore for his help and discussions on phenix refinement.

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Author notes

  1. These authors contributed equally: Xiaoli Yan, Xinying Jia.

Authors and Affiliations

  1. State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China

    Xiaoli Yan, Shunjia Ji, Meng-Jie Zhang, Kai Jiang, Zhi Lin, Zixin Deng, Xu-Dong Kong, Yi-Lei Zhao & Xudong Qu

  2. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan, China

    Xiaoli Yan, Hui Zhang, Mingjia Yu, Kai Jiang & Xudong Qu

  3. Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, Shanghai, China

    Xiaoli Yan, Meng-Jie Zhang, Kai Jiang, Zhi Lin, Xu-Dong Kong & Xudong Qu

  4. Department of Biochemistry, National University of Singapore, Singapore, Singapore

    Xinying Jia

  5. Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Queensland, Australia

    Xinying Jia & Mehdi Mobli

  6. School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, The University of Queensland, St Lucia, Queensland, Australia

    Zhenyao Luo & Bostjan Kobe

  7. Department of Pharmaceutical Sciences, University of Vienna, Vienna, Austria

    Julien Orts

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  1. Xiaoli Yan
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Contributions

X.Y., X.J., Y.-L.Z., M.M., Z.D. and X.Q. conceived this project. X.Y. performed fermentation, compound isolation, characterization, compound syntheses and in vitro experiments. H.Z. and M.Y. performed in vivo experiments. Z. Luo, M.-J.Z., X.J., X.-D.K. and B.K. solved the crystal structure of Abx(−)F. X.J. prepared labelled protein samples, analysed NMR spectra, performed NMR assignments and determined solution structures. M.M. acquired and processed NMR spectra using non-uniform sampling schemes. S.J. and Y.-L.Z. conducted the computational studies. J.O. performed water refinements of NMR structures. J.O. and M.M. provided advice and help in solving structures by NMR. K.J. and Z. Lin provided advice and help for in vitro experiments. X.Y., X.J., Y.-L.Z. and X.Q. prepared the paper. All authors contributed to paper editing.

Corresponding authors

Correspondence to Yi-Lei Zhao, Mehdi Mobli or Xudong Qu.

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Nature Chemistry thanks Garry Buchko, Marcio Dias and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Cofactor preference of Abx(−)E and characteristics of products of Abx(−)E.

a, Cofactor preference of Abx(−)E. (i) standard (+)-ABX; (ii) standard (−)-ABX; (iii) 100 μM 1 in 80 μM Abx(−)E; (iv) 100 μM 1 in 80 μM Abx(−)E and 1 mM NADPH. (v) 100 μM 1 in 80 μM Abx(−)E and 1 mM NADH; (vi) 100 μM 1 in 80 μM Abx(−)E, 5 μM GDH, 20 mM glucose. There was no addition of NADPH to the reaction system, and the activity detected implies that the endogenous cofactor (*NADP+) was present in Abx(−)E. b, Time-course conversion of 100 μM 1 in the presence of 80 μM Abx(−)E which was analyzed by normal UPLC (λ = 365 nm). The 8 multiplier mean the heights of the tiny peaks corresponding to compound 5 are magnified eight times for a better visibility. c, UV spectra and mass of compound 2, 5 and (±)-ABX.

Extended Data Fig. 2 Kinetic analysis of Abx(−)E and Abx(−)F, and time-course analysis of 2 in the presence of Abx(−)E and Abx(−)F.

a, Michaelis-Menten kinetic analysis of Abx(−)E with 1. Data were presented as mean ± s.d. from triplicate independent experiments (n = 3). b, Michaelis-Menten kinetic analysis of Abx(−)F with 2. Data were presented as mean ± s.d. from triplicate independent experiments (n = 3). c, Time-course analysis of 100 μM 2 in the presence of Abx(−)E which was analyzed by Chiral UPLC (λ = 365 nm). d, Time-course of 100 μM 2 in the presence of Abx(−)F which was analyzed by Chiral UPLC (λ = 365 nm).

Source data

Extended Data Fig. 3 Characteristics of the enantiomers (S)-5 and (R)-5.

a, Analysis (λ = 400 nm) of the enantiomers (S)-5 and (R)-5 using HPLC equipped with a chiral column (OD-H, 5 μm, 4.6 × 250 mm). (i) The mixture of (S)-5 and (R)-5 isolated from the fermentation extract of the ∆abx(−)F mutant strain; (ii) The mixture of (S)-5 and (R)-5 from the crystal. b, The measured CD and calculated ECD spectra of (S)-5 and (R)-5. c, Structures of (S)-5 and (R)-5.

Extended Data Fig. 4 Proposed dehydrative cyclization model of Abx(−)F.

a, Proposed reaction diagram of dehydrative cyclization. b, MS spectra of 18O labeled or unlabeled substrate and products in Abx(−)E and Abx(−)F assay. (i) MS spectra of 18O-1 in boiled Abx(−)E and boiled Abx(−)F assay; (ii) MS spectra of 1 in boiled Abx(−)E and boiled Abx(−)F assay; (iii) MS spectra of products 18O-2, 18O-5, 18O-(+)-ABX and 18O-(−)-ABX in Abx(−)E and boiled Abx(−)F assay; (iv) MS spectra of products 2, (+)-ABX and (−)-ABX in Abx(−)E and boiled Abx(−)F assay; (v) MS spectra of product 18O-(−)-ABX in Abx(−)E and Abx(−)F assay; (vi) MS spectra of product (−)-ABX in Abx(−)E and Abx(−)F assay.

Extended Data Fig. 5 Proposed SN1 and SN2 model of Abx(−)F and the DFT calculation.

a, Proposed reaction diagram of SN1 and SN2. b, The high strain in the 8-membered ring prevents the C24 hydroxyl group from approaching the C8 atom. (i) The ground state of the 8-membered ring intermediate upon hemiketalization, in which the C24 hydroxyl group points outward; (ii) The local minimal with the shortest C8–O(C24) distance, the conformation of which is higher by 8.3 kcal mol−1 at the B3LYP(D3)/def2tzvp (SMD) level of theory. c, The energy will further increase to as high as 83.1 kcal mol−1 when the distance decreases to 1.8 Å. Thus, an SN2 mechanism is unlikely to take place. Moreover, there is no specific reason to form high-energy carbon cation species through an SN1 mechanism as well.

Extended Data Fig. 6 Proposed annulation of phenol and the dual-oxa DA reaction model of Abx(−)F.

a, Proposed reaction diagram of annulation of phenol and the dual-oxa DA reaction. Intermediate 3 either undergo a disrotatory oxa-6π electrocyclization or a 1,4-Michael addition to form compound 5. b, MS spectra of 2 and (−)-ABX in Abx(−)F assay which buffer configured with D2O and H2O. (i) MS spectra of 2 in boiled Abx(−)F and D2O buffer; (ii) MS spectra of (−)-ABX in Abx(−)F and D2O buffer; (iii) MS spectra of 2 in boiled Abx(−)F and H2O buffer; (iv) MS spectra of (−)-ABX in Abx(−)F and H2O buffer.

Extended Data Fig. 7 The quantum chemical calculations support and explain the innate stereospecific dual-oxa Diels-Alder cyclization of Abx(−)F.

a, Transition states for the formation of (Z, Ra)-3 in both the non-enzymatic (TS1 and TS2) and enzymatic pathways (TS0). In TS0, the assumed base is displayed as faint sticks behind the solid TS0. b, Comparison of energy profiles between the non-enzymatic and enzymatic pathway. The enzymatic pathway was calculated using the theozyme model. c, Four plausible orientations of the oxa Diels-Alder reactions between the o-QM and the ketone group. d, Transition states and e, energy profiles for the oxa-DA reaction of both E- and Z- o-QM, along with enthalpies and Gibbs free energies (in parentheses).

Extended Data Fig. 8 The calculated reaction energy profile shows the plausible reactions of 2 toward 5.

The Gibbs free energies, given in kcal mol−1, were used to quantify the thermodynamics of these processes at the B3LYP(D3)/def2-TZVP(SMD) level of theory. The energy profile of the two potential dehydration pathways involved in the conversion of 2 to enantiomeric intermediates 3. Following this, besides undergoing oxo-Diels-Alder cyclization to yield the products (−)-ABX and (+)-ABX, the compound 3 can undergo disrotatory oxa-6π electrocyclization to form less stable compounds 5 after hydrogen abstraction from the acetyl methylene substituent. Alternatively, compound 3 form an enolate ion under basic condition and go 1,4-Michael addition forming the anionic form of compound 5 (blue dotted box).

Extended Data Fig. 9 The critical steps of forming (±)-ABX, (R/S)-5 and 6.

‘spon.’ represent spontaneous reaction whose reaction pathways are indicated by grey dotted arrows; reactions catalyzed by Abx(−)E and Abx(−)F are indicated by blank and red arrows; chiral axle is highlighted by orange.

Extended Data Fig. 10 Abx(−)F structure and molecular dynamics simulations.

a, Crystal structure of Abx(−)F. the dashed line represents the amino acids whose electron densities are missing. b, Superimposed structures of crystal Abx(−)F (chain A with purple, chain B with lightblue) and solution NMR Abx(−)F (pink). For Abx(−)F_chain A and solution NMR Abx(−)F, RMSD is 1.370 Å; For Abx(−)F_chain B and solution NMR Abx(−)F, RMSD is 1.398 Å; For Abx(−)F_chain A and Abx(−)F_chain B, the RMSD is 0.522 Å. The missing loop regions in the crystal Abx(−)F structure are highlight in green in NMR structure. c, Superimposed structures of crystal Abx(−)F (purple), MtRv0577 (green, PDB: 3OXH) and the BphC enzyme (light pink, PDB: 1DHY). The backbone RMSD values were 1.168 Å between Abx(−)F and MtRv0577 and 10.261 Å between Abx(−)F and BphC. In BphC enzyme, Fe ion is coordinated by residues H145, H209 and E260. However, no residues in MtRv0577 or Abx(−)F were appropriate for binding a metal ion. d, Ribbon representation of Abx(−)F without (−)-ABX (pink) and Abx(−)F (purple) with (−)-ABX (cyan), including the active residues (W15, D17, L19, Y48, S60, N67, Y78, I105 and W125, pink represent residues of Abx(−)F without (−)-ABX; red represent residues of Abx(−)F with (−)-ABX) displayed as sticks. Regions that undergo apparent conformational changes are highlighted as a non-transparent cartoon against a transparent background. The blue spheres identify the backbone amides with significant chemical-shift perturbations in the presence of (−)-ABX (Supplementary Fig. 19). The conformational changes are manifested by three labelled distances: 11.8 Å between the Cα atoms of Gly44, 8.5 Å between the Cα atoms of Asn67, and 4.6 Å between the Cα atoms of Ile105. e, Molecular dynamics simulations of hydrogen bond between D17 and 6-OH group of 1.

Supplementary information

Supplementary Information

Supplementary Methods 1.1–1.4, Tables 1–11, Figs. 1–20, HADDOCK NOE restraints, computational analysis and references.

Reporting Summary

Supplementary Data 1

Source data for graphs in Supplementary Figs. 14b and 18.

Supplementary Data 2

Crystallographic data of compound 5.

Source data

Source Data Fig. 5

Source data for graph in Fig. 5d.

Source Data Extended Data Fig./Table 2

Source data for graph in Extended Data Fig. 2a and 2b

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Yan, X., Jia, X., Luo, Z. et al. An enzymatic dual-oxa Diels–Alder reaction constructs the oxygen-bridged tricyclic acetal unit of (–)-anthrabenzoxocinone. Nat. Chem. 17, 1058–1066 (2025). https://doi.org/10.1038/s41557-025-01804-0

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