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Iminium catalysis in natural Diels–Alderase

Nature Catalysis volume 8, pages 218–228 (2025)Cite this article

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Abstract

Iminium-catalysed cycloaddition is one of the most prominent examples of organocatalysis, yet a biological counterpart has not been reported, despite the widespread occurrence of iminium adducts in enzymes. Here we present biochemical, structural and computational evidence for iminium catalysis by the natural Diels–Alderase SdnG, which catalyses norbornene formation in sordarin biosynthesis. A Schiff-base adduct between the ε-nitrogen of active site K127 and the aldehyde group of the enal dienophile is revealed by structural analysis and captured under catalytic conditions via borohydride reduction. This Schiff-base adduct positions the substrate into near-attack conformation and decreases the transition-state barrier of Diels–Alder cyclization by 8.3 kcal mol−1 via dienophile activation. A hydrogen-bond network consisting of a catalytic triad is proposed to facilitate the proton transfer required for iminium formation. This work establishes an intriguing mode of catalysis for Diels–Alderases and points the way to the design of iminium-based (bio)catalysts.

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Fig. 1: SdnG is proposed to be a Diels–Alderase that performs iminium catalysis.
Fig. 2: Crystal structures of SdnG with substrate analogue 3 and product 4.
Fig. 3: Biochemical evidence of iminium catalysis in SdnG.
Fig. 4: Proposed mechanism of SdnG.

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Data availability

Data supporting the findings of this study are available within the Article, its Supplementary Information files, and the Source Data files. All unique biological materials, such as plasmids, generated in the study are available from the authors upon request. Crystal structures of Se–Met-SdnG, SdnG–3NC, SdnG–3C, SdnG–4 and SdnG–8 have been deposited in the Protein Data Bank under IDs 8YIA, 8YHG, 8YJ4, 8YI8 and 8YHM, respectively. Data are available from the corresponding authors upon request. Source data are provided with this paper.

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Acknowledgements

This work was supported by NIH (5R01AI141481, to Y.T.), Shenzhen Bay Laboratory Open Program (SZBL2021080601014, to J.Z.) and the NSF (CHE 2153972 and 2409941, to K.N.H.). We thank the staff of beamlines BL17U1, BL18U1 and BL19U1 of Shanghai Synchrotron Radiation Facility for access and help with the X-ray data collection, D. Cascio and M. Sawaya at UCLA-DOE Institute for Genomics and Proteomics for help with discussion of the X-ray data, and Y. Chen at UCLA Molecular Instrumentation Center for help with the peptide MS/MS analysis.

Author information

Author notes

  1. These authors contributed equally: Zuodong Sun, Xin Zang.

Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, CA, USA

    Zuodong Sun, Xin Zang, Masao Ohashi & Yi Tang

  2. Key Laboratory of Quantitative Synthetic Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China

    Xin Zang & Jiahai Zhou

  3. Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA, USA

    Qingyang Zhou, K. N. Houk & Yi Tang

  4. School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, China

    Jiahai Zhou

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  1. Zuodong Sun
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Contributions

Z.S., X.Z., Q.Z., M.O., K.N.H., J.Z. and Y.T. developed the hypothesis and designed the study. Z.S. performed compound isolation, chemical synthesis and all biochemical studies. X.Z. performed crystallization and determined all structures. Q.Z. performed all computational studies. Z.S., X.Z., Q.Z., M.O., K.N.H., J.Z. and Y.T. analysed and discussed the results. Z.S. and Y.T. wrote the manuscript. Z.S., X.Z., Q.Z., M.O., K.N.H., J.Z. and Y.T. read, edited and approved the manuscript.

Corresponding authors

Correspondence to K. N. Houk, Jiahai Zhou or Yi Tang.

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Nature Catalysis thanks Jan Deska, Hui Ming Ge, Shingo Nagano, Masanobu Uchiyama 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 The active sites of SdnG complexed with different ligands are nearly superimposable.

(a) Overlay of active sites of SdnG-3NC (ivory) and SdnG-3C (gray). Noncovalently bound 3 and covalently bound 3 are shown in cyan and green respectively. Water molecules interacting with 3 and Y41 are omitted for clarity. (b) Overlay of active sites of SdnG-3NC (ivory) and SdnG-4NC (gray). 3 and 4 are shown in cyan and green respectively. (c) Overlay of active sites of SdnG-3C (ivory) and SdnG-7 (gray). Covalently bound 3 and adduct 7 are shown in cyan and green respectively.

Extended Data Fig. 2 K127 adducts corresponding to the rDA and Cope rearrangement products of intermediate 7 were observed in the crystal structure of SdnG with 4.

(a) Active site of SdnG-7-5 (the rDA product of 7, chain C of SdnG-4, PDB 8YI8). Adducts 7 and 5 are shown in cyan and green respectively. Residues around 4 Å of the ligands are shown in ivory. The Polder omit map of 7-5 (contoured at 2.0 σ) is shown in blue mesh. Maps contoured at higher levels (2.5 σ and 3.0 σ) are shown in Supplementary Fig. 12. (b) Distance between diene and dienophile (highlighted in purple) of 5 in SdnG-7-5. Active site residues and 5 are shown in ivory and green respectively. Adduct 7 is omitted from the view for clarity. (c) Active site of SdnG-8 (the Cope rearrangement product of 7, PDB 8YHM). Adduct 8 is shown in cyan. The Polder omit map of 8 (blue mash) is contoured at 3.0 σ. Distances between atoms are shown in dashed lines. Bonds corresponding to the diene and the dienophile in 1 are highlighted in purple. Other coloring schemes are the same as in (a). (d) DFT-calculated transition states of rDA and Cope rearrangement of 7. All energies are relative to the ground state energy of 5 (iminium form). Bonds corresponding to the diene and the dienophile in 1 are highlighted in blue in 5 and green in 8. The transition state structure of rDA (TS-5) is the same as that of the forward reaction (TS-4). But the barriers of the forward and reverse reactions differ substantially due to the large energy gap between 5 and 7.

Extended Data Fig. 3 Catalytic activity of SdnG and K127X mutants.

All reactions were carried out for 1 min with 100 µM 1. Values and error bars are obtained from the average and standard deviation of three independent measurements (black circles) respectively (n = 3). (a) Absolute rates of non-enzymatic and enzymatic DA cyclization of 1. Asterisks indicate no measurable substrate consumption during the course of the reaction. (b) Relative activity of SdnG and K127X variants normalized by enzyme concentration. All activities are shown relative to the rate acceleration of the DA reaction exhibited by the wild-type enzyme (WT, 100%). A 0% value (asterisks) indicates no rate acceleration compared to uncatalyzed DA reaction.

Source data

Extended Data Fig. 4 Effect of the concentration of 4 on formation of iminium 7 in SdnG and the H72A variant.

SdnG or its mutational variants (5 µM) were mixed with varied amounts of 4 and the mixture was immediately treated with 20 mM NaBH4. The reaction was subsequently analyzed by UHPLC-HRMS and the deconvoluted ESI-MS spectra were shown in the figure. Increased concentration of 4 promotes imine formation of SdnG but not the H72A variant. The result suggests that ligand binding is rate-determining for iminium formation in SdnG but not in the H72A variant. Therefore, diminished iminium formation in H72A is not a result of compromised ligand binding.

Supplementary information

Supplementary Information

Supplementary Tables 1–4, Figs. 1–20, cartesian coordinates of calculated structures and source data.

Reporting Summary

Supplementary Data 1

Statistical source data for Supplementary Fig. 16.

Supplementary Data 2

Cartesian coordinates of calculated structures.

Source data

Source Data Fig. 3d

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

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Sun, Z., Zang, X., Zhou, Q. et al. Iminium catalysis in natural Diels–Alderase. Nat Catal 8, 218–228 (2025). https://doi.org/10.1038/s41929-025-01294-w

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