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Structural and mechanistic insights into iminium-catalysed macrocyclization by nuclear transport factor 2-like enzymes

Nature Synthesis (2026)Cite this article

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Abstract

Macrocyclization is crucial in natural product biosynthesis for enhancing molecular rigidity and stability. Although thioesterase-mediated macrolactonization or macrolactamization is the predominant mechanism in type I polyketide synthases, here we report an alternative macrocyclization mechanism in which nuclear transport factor 2 (NTF2)-like enzymes catalyse a tandem stereoselective Michael addition and Knoevenagel condensation to construct a tetrahydrofuran-fused macrocyclic carbocycle. Genome mining identified a family of NTF2-like proteins that share this tandem cyclization capability. X-ray crystal structures complexed with substrate mimics and structure-based mutagenesis reveal that a lysine residue forms an iminium intermediate with the terminal aldehyde to enable cyclization, while an aspartic acid acts as a general base to mediate proton transfers. Structures capturing distinct states, from linear precursor to precyclization, provide direct insight into the ring-closure process. This work elucidates an iminium-catalysed tandem cyclization mechanism, expanding the known catalytic repertoire of NTF2-like enzymes and highlighting the potential of iminium-based biocatalysis in natural product biosynthesis.

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Fig. 1: Biosynthesis of AKA (1).
Fig. 2: The macrocyclization process for AKA and MAN.
Fig. 3: Crystal structures of CatM–6b and CatM–W86A–6a.
Fig. 4: Iminium catalysis involved in the macrocyclization reactions.
Fig. 5: Captured conformation of ligands and proposed reaction mechanism for CatM.

Data availability

Data supporting the findings of this work are available within the paper and its Supplementary Information files. Crystal structures of AkaM, SacM, CatM, MicM, SacM–3, SacM–6b, CatM–6b and CatM–W86A–6a have been deposited in the Protein Data Bank under accession numbers 9IQ3, 9IQ7, 9IQ5, 9IQD, 9IQK, 9IQL, 9IQM and 9IQN, respectively.

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Acknowledgements

H.M.G. acknowledges funding support from the NSFC (82525108, 22193071, 22437003, W2412037 and 22377051), the Natural Science Foundation of Jiangsu Province (BK20253010 and BF2025075), Hainan Province Science and Technology Special Fund (ZDYF2023SHFZ107), Fundamental and Interdisciplinary Disciplines Breakthrough Plan of the Ministry of Education of China (JYB2025XDXM507) and the Fundamental Research Fund for the Central Universities (14380205). B.Z. acknowledges funding support from the NSFC (22277051) and the Natural Science Foundation of Jiangsu Province (BK20220123). We thank beamlines BL02U1, BL10U2 and BL19U1 of the Shanghai Synchrotron Radiation Facility for assistance with the X-ray data collection.

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Authors and Affiliations

  1. State Key Laboratory of Pharmaceutical Biotechnology, Institute of Functional Biomolecules, Department of Neurology, Nanjing Drum Tower Hospital, School of Life Sciences, Nanjing University, Nanjing, China

    Cheng Li Liu, Bo Zhang, Cheng Yuan Yuan, Zi Jie Wang, Ao Zhu, Si Yuan Wang, Zhang Yuan Yan, Ren Xiang Tan & Hui Ming Ge

  2. UKRI–STFC, Rutherford Appleton Laboratory, Research Complex at Harwell, Didcot, UK

    Andrey Lebedev

  3. Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, Toyama, Japan

    Yasuhiro Igarashi

  4. Chemistry and Biomedicine Innovation Center (ChemBIC), ChemBioMed Interdisciplinary Research Center at Nanjing University, Nanjing, China

    Hui Ming Ge

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Contributions

C.L.L., B.Z. and H.M.G. conceived the idea for the study and designed the experiments. C.L.L., C.Y.Y. and Z.J.W. performed fermentation, compound isolation, chemical synthesis and all biochemical studies. B.Z., A.Z. and A.L. performed crystallization and solved all the structures. S.Y.W. and Z.Y.Y. assisted in NMR and MS data measurement and analysis. Y.I. and R.X.T. contributed materials and equipment. C.L.L, B.Z. and H.M.G. wrote the manuscript. B.Z. and H.M.G. supervised the work. All authors discussed the results and analysed the data.

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Correspondence to Bo Zhang or Hui Ming Ge.

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Nature Synthesis thanks Robin Teufel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Thomas West, in collaboration with the Nature Synthesis team.

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Extended data

Extended Data Table 1 LC-MS/MS data of the trypsin-digested peptide fragment of CatM-5a adduct
Full size table
Extended Data Table 2 Data collection, refinement and validation statistics
Full size table

Extended Data Fig. 1 HPLC analysis of DMP oxidation reactions of 6a and 6b.

6a, 6b accumulated in ΔakaM mutant strain were probably derived from the reduction of 5a and 5b, by an endogenous reductase. DMP oxidation of 6a, 6b were carried out to obtain corresponding aldehyde products 5a and 5b. DMP: Dess–Martin periodinane.

Source data

Extended Data Fig. 2 Biochemical evidences of Michael addition catalysed by AkaM or its homologues.

Standard of 5b obtained from DMP oxidation of 6b (i); standard of 5a obtained from DMP oxidation of 6a (ii); standard of 8 (iii); HPLC analysis of non-enzymatic products 5 generated in AkaP1-catalysed reaction, which is conducted in an optimized condition (iv); high-revolution HPLC analysis of one-pot enzymatic reactions of AkaP1 and AkaM or its homologues (v-x).

Source data

Extended Data Fig. 3 In vitro kinetic studies of AkaM with different substrates.

a, In vitro enzymatic activity assays of AkaM using 5a as substrate. b, In vitro enzymatic activity assays of AkaM using 5b as substrate. n = 3 replicates and data are presented as mean ± s.e.m.

Source data

Extended Data Fig. 4 Crystal structures of NTF2-like proteins and characterized enzymatic reactions.

a, Crystal structures of AkaM, SacM, CatM and MicM. b, The superimposed image of the four homologues. c, Representative crystal structures of NTF2-like proteins, including NgnD, SdnG, SnoaL, and tKSI. d, Diverse enzymatic reactions catalysed by NTF2-like proteins.

Extended Data Fig. 5 Highly conserved residues in these NTF2-like proteins.

a, Residues surround the K113 and distance between K113, W31, and D101. b, Residues around K113 are highly conserved across these NTF2-like proteins. c, Residues located at the side of active tunnel in AkaM. d, Residues located at the side of active tunnel in CatM. e-f, The two different geometries of active side tunnel in AkaM and CatM. The AkaM, SacM, CatM, and MicM are shown in green, wheat, lime, and light blue, respectively.

Extended Data Fig. 6 Crystal structures of SacM in complex with substrate analogue 3 and 6b.

a, Close-up active site view of SacM-3. Compound 3 and the surrounding residues within 4 Å are shown in cyan and wheat, respectively. The polder (omit) electron density map of 3 (contoured at 3.0 σ) is displayed in grey mesh. b, Close-up view of the active site in SacM-6b. Compound 6b and the surrounding residues within 4 Å are shown in light pink and wheat, respectively. The polder (omit) electron density map of 6b (contoured at 3.0 σ) is shown in grey mesh. Compounds 3 and 6b are coloured as cyan and light pink, respectively, while SacM is shown in wheat.

Extended Data Fig. 7 Comparation of CatM-6b, CatM-W86A-6a and SacM-3 crystal structures.

a, Overlay of CatM-6b (lime) and CatM-W86A-6a (light blue). The two flexible loop regions are highlighted by red circles. b, the overlay of CatM-6b and CatM-W86A-6a shows a steric clash between W86 and ligand 6a. c, The crystal structure of CatM-W86A-6a. No water molecule was built within 4 Å of ligand 6a, by Phenix. d, The crystal structure of SacM-3. One water molecule was built in the active cavity by Phenix, which may function as general base for the deprotonation of key lysine. Similar water molecules were also observed in crystal structures of AkaM, SacM, CatM, MicM and SacM-6b.

Supplementary information

Supplementary Information

Experimental methods, Supplementary Tables 1–15 and Figs. 1–111.

Reporting Summary

Supplementary Data

Source data for Supplementary Fig. 23.

Source data

Source Data Fig. 1

Source data for HPLC analysis.

Source Data Fig. 2

Source data for HPLC analysis.

Source Data Fig. 3

Source data for enzymatic reaction analysis.

Source Data Extended Data Fig. 1

Source data for HPLC analysis.

Source Data Extended Data Fig. 2

Source data for HPLC analysis.

Source Data Extended Data Fig. 3

Source data for kinetic analysis.

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Liu, C.L., Zhang, B., Yuan, C.Y. et al. Structural and mechanistic insights into iminium-catalysed macrocyclization by nuclear transport factor 2-like enzymes. Nat. Synth (2026). https://doi.org/10.1038/s44160-025-00989-z

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