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A two-metalloenzyme cascade constructs the azetidine-containing pharmacophore

Nature Chemistry (2025)Cite this article

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Abstract

Azetidine is a prominent pharmacophore present in dozens of drug-related molecules of both natural and synthetic origins. But how nature builds this moiety has long remained enigmatic. Here we address the full deciphering of a two-metalloenzyme cascade leading to polyoximic acid, an azetidine-containing moiety of the fungicide polyoxin. We demonstrate that the PolE enzyme functions as an Fe2+/pterin-dependent l-isoleucine desaturase. Moreover we illustrate that PolF is a new member of the emerging haem-oxygenase-like diiron oxidases, converting the desaturated l-isoleucine to polyoximic acid via an intramolecular C–N cyclization. Remarkably, we also establish that PolF exhibits dual functionality, orchestrating the sequential desaturation and cyclization with l-isoleucine as the initial substrate. Finally, our combined structural and quantum-mechanics/molecular-mechanics studies show that the PolF enzyme employs an extraordinary mechanism for the construction of the azetidine-containing moiety. These findings expand our knowledge on the catalysis of metalloenzymes and open the way for rational access of more azetidine-related molecules.

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Fig. 1: Structure of POL and in vivo reconstitution of biosynthesis of 1.
Fig. 2: Biochemical characterization of PolE as an Fe2+/pterin-dependent l-isoleucine desaturase.
Fig. 3: In vitro deciphering of PolF as an unusual Fe2+/O2-activating enzyme.
Fig. 4: Biochemical characterization of SzPolF as a new member of the HDO superfamily of enzymes.
Fig. 5: Mechanistic investigations of SzPolF.

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Data availability

The atomic coordinates and structure factors of the reported structures have been deposited in the PDB under accession codes as follows: apo SnPolE, 9J2E; SnPolE–BH4, 9J2M; SnPolE–BH4–l-isoleucine, 9J2P; SnPolEF140A, 9J33; SnPolEY157A, 9J2R; SnPolEY280A, 9J30; apo SmPolE, 9J3G; Fe(II)–SzPolF–l-isoleucine, 9J2C; Fe2(II/II)–SzPolF–l-isoleucine, 9J2B; Fe(II)–SzPolF–2, 9J28; Fe2(II/II)–SzPolF–2, 9J2A; Fe2(II/II)–SzPolF–1, 9J29; Fe2(II/II)–PolF–l-isoleucine, 9J3V; and Fe(II)–SaPolF–l-isoleucine, 9J27. All other data generated and analysed supporting the findings of this study are available within the paper and Supplementary Information. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (32470047 and 32170026 to W.C., 22122305 to B.W., 32371303 to Z.Z., 22403016 to J.L. and 22303043 to X. Zhang), the Scientific Research Innovation Capability Support Project for Young Faculty (ZYGXONJSKYCXNLZCXM-B6 to B.W.), the Hubei Province’s Outstanding Medical Academic Leader Program (Chutian Talent Plan) to W.C. and the China Postdoctoral Science Foundation (2023M742703 and GZC20231989 to R.G.). We are sincerely grateful to the staff members at the BL19U1, BL02U1 and BL10U2 beamlines of the National Facility for Protein Science in Shanghai (NFPS) for providing technical support in the X-ray diffraction data collection and analysis. We also acknowledge the staff members of the Electron Spin Resonance System at the Steady High Magnetic Field Facility for providing technical support and assistance with data collection, and the Core Facility of Wuhan University for LC-HRMS and NMR data collection.

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Author notes

  1. These authors contributed equally: Rong Gong, Yao Qu, Jia Liu.

Authors and Affiliations

  1. Department of Anesthesiology, Zhongnan Hospital of Wuhan University, TaiKang Center for Life and Medical Sciences, School of Pharmaceutical Sciences, Wuhan University, Wuhan, China

    Rong Gong, Yao Qu, Liwei Zhou, Zhu Tian, Xinyue Zeng, Boyu Jin, Le Yu, Ruyi Chen, Xuemin Song, You-Sheng Cai, Kun Shen, Zixin Deng, Zhengyu Zhang & Wenqing Chen

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

    Rong Gong, Yao Qu, Liwei Zhou, Zhu Tian, Xinyue Zeng, Boyu Jin, Le Yu, Ruyi Chen, You-Sheng Cai, Kun Shen, Zixin Deng, Zhengyu Zhang & Wenqing Chen

  3. State Key Laboratory Physical Chemistry of Solid Surfaces and Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China

    Jia Liu, Xuan Zhang, Zhen Li, Yang Zhou, Langxing Liao, Liulin Yang & Binju Wang

  4. College of Chemistry and Materials Science, Fujian Normal University, Fuzhou, China

    Jia Liu

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  1. Rong Gong
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Contributions

W.C., B.W. and Z.Z. conceived the project and directed the research. R.G. performed the genetic and biochemical experiments. Y.Q. conducted the structural biology studies. J.L., X. Zhang and Z.L. undertook the QM/MM analysis of the mechanism. L.Z., Z.T. and K.S. carried out the compound synthesis. X. Zeng and B.J. performed the purification of the compounds. L. Yu and R.C. assisted in preliminary genetic experiments. Y.Z. and L.L. carried out the stopped-flow experiments. Y.-S.C. analysed the NMR data. W.C., B.W., Z.Z., R.G., Y.Q. and J.L. wrote the manuscript; Z.D., X.S. and L. Yang made a critical reading of the manuscript.

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Correspondence to Zhengyu Zhang, Binju Wang or Wenqing Chen.

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Extended data

Extended Data Fig. 1 [13C6]l-isoleucine-feeding analysis of E. coli BL21(DE3) recombinants.

a, Schematic of [13C6]l-isoleucine-feeding of the BL21(DE3)/polE + polF recombinant. b, LC-HRMS analysis of the target metabolite 1 produced by the BL21(DE3)/polE + polF recombinant fed with [13C6]l-isoleucine. The expected [M + H]+ ion for 1 and [13C6]-1 is at 128.0706 and 134.0907, respectively, whereas the detected [M + H]+ ion for 1 and [13C6]-1 is individually at 128.0711 and 134.0910. c, Schematic of [13C6]l-isoleucine-feeding of the BL21(DE3)/polF recombinant. d, LC-HRMS analysis of the target metabolite 1 produced by the BL21(DE3)/polF recombinant fed with [13C6]l-isoleucine. The expected [M + H]+ ion for 1 and [13C6]-1 is at 128.0706 and 134.0907, respectively, whereas the detected [M + H]+ ion for 1 and [13C6]-1 is individually at 128.0706 and 134.0907. e, Schematic of [13C6]l-isoleucine-feeding of the BL21(DE3)/polE recombinant. f, LC-HRMS analysis of the target metabolite 2 produced by the BL21(DE3)/polE recombinant fed with [13C6]l-isoleucine. The expected [M + H]+ ion for 2 and [13C6]-2 is at 130.0863 and 136.1064, respectively, whereas the detected [M + H]+ ion for 2 and [13C6]-2 is correspondingly at 130.0866 and 136.1067.

Extended Data Fig. 2 In vitro characterization of PolE as an unusual Fe2+/pterin-dependent desaturase.

a, Schematic of the reaction catalyzed by PolE. b, PolE reactions with individual addition of BH4, 6-MePH4, and THF. Std, the authentic l-isoleucine and 2 standards; +BH4, the reaction with Fe2+, BH4, and FolA added; +6-MePH4, the reaction with Fe2+, 6-MePH4, and FolA added; +THF, the reaction with Fe2+, THF, and FolA added; NC, the reaction without PolE added as negative control. c, Evaluation of the metal dependence of PolE. PolE was firstly chelated with EDTA, and then its reactions were individually supplemented with various divalent metal ions. d, Schematic of the hypothetic catalytic-process by PolE. e, LC-HRMS analysis of the PolE reactions using 3 or 4 as substrate. Std, the authentic 2, 3 and 4 standards; +(3+PolE), the PolE reaction using 3 as substrate; +3, the reaction without PolE added as negative control; +(4+PolE), the PolE reaction using 4 as substrate; +4, the reaction without PolE added as negative control. f, LC-HRMS analysis of the PolE reactions using 5 or 6 as substrate. Std, the authentic 2, 5 and 6 standards; +(5+PolE), the PolE reaction using 5 as substrate; +5, the reaction without PolE added as negative control; +(6+PolE), the PolE reaction using 6 as substrate; +6, the reaction without PolE added as negative control.

Extended Data Fig. 3 Structural characterization of SnPolE and functional analysis of its substrate-coordination sites.

a, Surface representation of SnPolE with Fe (orange sphere), BH4 (purple stick), and l-isoleucine (blue stick). b, The structure alignment of apo SnPolE (pink cartoon), SnPolE-BH4 (yellow cartoon), and SnPolE-BH4-l-isoleucine (green cartoon). c, Electron map of apo SnPolE. The 2Fo − Fc map is shown for Fe-coordinating residues and water (grey mesh, contoured at 1.0σ). Fe-coordinating residues are shown in lines and sticks, Fe is illustrated as orange sphere, and water is indicated as red sphere. d, Electron map of ligands bond in SnPolE-BH4 binary complex. The 2Fo − Fc map is shown for Fe-coordinating residues, water, and BH4 (grey mesh, contoured at 1.0σ). BH4 is shown in purple stick. e, Electron map of ligands bound in the SnPolE-BH4-l-isoleucine ternary complex structure. The Fo − Fc omit maps of l-isoleucine (cyan stick), Fe ions (orange spheres), the Fe-coordinating water molecule (red sphere) and Fe-coordinating residues (sticks) are contoured at 2.5σ (green mesh). f, Comparison of the binding pockets of apo SnPolE, SnPolE-BH4 binary complex, and SnPolE-BH4-l-isoleucine ternary complex. SnPolE, SnPolE-BH4, SnPolE-BH4-l-isoleucine is correspondingly shown in pink, yellow, and green. g, Crystal structure of SnPolEY157A variant with l-isoleucine mistakenly coordinated to iron. The selected amino acid residues and l-isoleucine are shown in the stick and ball, and Fe is illustrated as sphere. h, Comparison of active sites of SnPolEY157A (yellow) and SnPolE (white). i, Comparison of active sites of SnPolEF140A (deep teal) and SnPolE (white). j, Comparison of active sites of SnPolEY280A (pink) and SnPolE (white).

Extended Data Fig. 4 Mechanistic investigations of SnPolE.

a, The potential energy surface (in kcal mol−1) of SnPolE-catalyzed desaturation reaction obtained from QM/MM calculations. b, The structures of key intermediates involved in the SnPolE-catalyzed desaturation reaction.

Extended Data Fig. 5 In vitro characterization and monitoring of the catalytic process of PolF.

a, Schematic of the proposed catalytic process by PolF. b, LC-HRMS analysis of PolF activity in the presence of Fe2+ or other divalent metal ions. PolF was firstly chelated with EDTA, and then its reactions were individually supplemented with various divalent metal ions. c, LC-HRMS analysis of PolF reactions with l-isoleucine as substrate. Activity assays of PolF were performed under aerobic atmosphere. Std, the authentic standards of l-isoleucine and 1; Full reaction, the PolF reaction with Fe2+ and VC added; -O2, the full reaction incubated under anaerobic atmosphere; -Fe2+, the PolF reaction without Fe2+ added; -Fe2+(+EDTA), the PolF reaction with EDTA added; -VC, reaction without VC added; -PolF, the reaction without PolF added; -l-Ile, the reaction without l-isoleucine added. d, LC-HRMS analysis of the PolF reactions using 3 and 4 as substrate. Std, the authentic standards of 3, 4, and 1; +(3+PolF), the PolF reaction using 3 as substrate; +3, the reaction without PolF added as negative control; +(4+PolF), the PolF reaction using 4 as substrate; +4, the reaction without PolF added as negative control. e, LC-HRMS analysis of the PolF reactions using 5 and 6 as substrate. Std, the authentic standards of 5, 6, and 1; +(5+PolF), the PolF reaction using 5 as substrate; +5, the reaction without PolF added as negative control; +(6+PolF), the PolF reaction using 6 as substrate; +6, the reaction without PolF added as negative control.

Extended Data Fig. 6 Structure comparison of PolF and its homologues.

a, Overall structure of Fe2(II/II)-PolF (yellow). b, Overall structure of Fe(II)-SaPolF (blue). c, Overall structure of Fe2(II/II)-SzPolF (green). d, Structure alignment of PolF, SaPolF, and SzPolF with highlight of the core helix α3.

Extended Data Fig. 7 Comparison of the active pocket sites of Fe(II)-SzPolF and Fe2(II/II)-SzPolF.

a, The three core α helices (core α1, α2, and α3) harbor metal-binding ligands. b, Fe coordination sites of Fe2(II/II)-SzPolF. Six-coordinate Fe1 has an overall octahedral geometry, whereas the five-coordinate Fe2 attains roughly square pyramidal symmetry. c, Comparison of the binding pockets of Fe(II)-SzPolF and Fe2(II/II)-SzPolF. Fe(II)-SzPolF is shown in purple sticks and Fe2(II/II)-SzPolF is shown in yellow sticks. d, Slice-surface view of Fe(II)-SzPolF (left) and Fe2(II/II)-SzPolF (right). Electrostatic surface potential is colored by red (-79.400 kT/e), blue (+79.400 kT/e), and white (neutral). e, Electron map of ligand bond in Fe(II)-SzPolF-2 complex. Structure of Fe(II)-SzPolF reveals that Fe1 (orange sphere) recruits 2 (green sticks) as a bidentate ligand. Selected amino acids are shown in green stick format. The 2Fo − Fc map is shown for residues, Fe1 and substrate (grey mesh, contoured at 1.0σ). f, Hydrogen-bonding and hydrophobic-interaction with selected amino acids and 2.

Extended Data Fig. 8 Stopped-flow absorption spectroscopy demonstrates the accumulation of intermediate in the SzPolF reaction.

Absorption spectra acquired after rapid mixing an O2-free solution of SzPolF (0.3 mM) and Fe2+ (0.6 mM, 2 molar equiv.) in the absence (a) or presence of (b) 2 mM 2 (without VC), (c) 2 mM 2 (with VC), (d) 2 mM l-isoleucine (with VC), with an equal volume of O2-saturated buffer.

Extended Data Fig. 9 Mechanistic investigations of SzPolF catalyzed desaturation in the presence of reductants.

a, The potential energy surface (in kcal mol−1) of SzPolF-catalyzed desaturation reaction obtained from QM/MM calculations. b, The structures of key intermediates involved in the SzPolF-catalyzed desaturation reaction.

Extended Data Fig. 10 Distribution of selected PolF homologues in microbial genomes.

a, Phylogenetic analysis of PolF and its homologues with diverse microbial origins. The branches corresponding to PolF, SaPolF, SrPolF, and SzPolF are highlighted. b, Selected biosynthetic gene clusters that encode the homologues of PolF.

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Gong, R., Qu, Y., Liu, J. et al. A two-metalloenzyme cascade constructs the azetidine-containing pharmacophore. Nat. Chem. (2025). https://doi.org/10.1038/s41557-025-01949-y

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