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Enzymatic catalysis favours eight-membered over five-membered ring closure in bicyclomycin biosynthesis

Nature Catalysis volume 6, pages 637–648 (2023)Cite this article

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Abstract

The construction of O-heterocycles is an important step in organic synthesis and biosynthesis for producing valuable ring compounds. Although enzyme-catalysed five- or six-membered ring closures in O-heterocycle biosynthesis have been studied extensively, the enzymatic formation of eight-membered O-heterocycles has been proposed only recently. Here we demonstrate a group of enzymes that catalyse an intramolecular attack of alcohol on epoxide for the construction of an eight-membered ring rather than an intrinsically more favourable five-membered tetrahydrofuran. The detailed mechanism is revealed through biochemical experiments, chemical syntheses, crystallographic structural analyses, computational simulations of potential energies and molecular dynamics, and site-directed mutagenesis. This study provides a vivid example of an enzyme that non-covalently protects an intrinsically more active hydroxyl group through a hydrogen-bond network, and reverses the inherent size selectivity in ring-closure reactions, despite the presence of multiple nucleophiles in the substrate.

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Fig. 1: Enzyme-catalysed intramolecular ring-closure reactions.
Fig. 2: Enzyme-catalysed oxa-bridged eight-membered ring formation.
Fig. 3: Crystal structures of SoBcmB.
Fig. 4: DFT-computed transition states for the ring-closure reactions.
Fig. 5: SoBcmB favours eight-membered over five-membered ring closure.

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Data availability

Data that support the findings of this study are available within the paper and its Supplementary Information. The atomic coordinates of SoBcmB·FeII·αKG, Se-Met-SoBcmB·FeII·αKG, SoBcmB·FeII·αKG·2, SoBcmB·FeII·αKG·2f, SoBcmB·FeII·αKG·2a, SoBcmB·FeII·αKG·1, SoBcmB·FeII·αKG·1d, SoBcmB·FeII·αKG·1a and SoBcmBD307A·FeII·αKG·2 have been deposited in the Protein Data Bank (PDB, https://www.rcsb.org/) under accession codes 8HIV, 7V3O, 7V2T, 7V2U, 7V36, 7V2X, 7V34, 7V3E and 7V3N, respectively. The crystallographic data of compound 1d have been deposited in the Cambridge Crystallographic Data Centre, under deposition number CCDC 2105556. Copies of the data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures/. The DNA sequences of genes SsbcmB, SobcmB and SkbcmB were downloaded from GenBank with accession numbers MG018995, NZ_LIQX01000253 and CP023699, and the codon-optimized DNA sequences are listed in Supplementary Methods. All other data are available from the authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Key Research and Development Program of China (2018YFA0901902 to G.-L.T. and J.Z., 2022YFA1503200 to Y.L.), the National Natural Science Foundation of China (22077062 to Y.L., 91856202 to J.Z. and 22207117 to J.-B.H.), the Chinese Academy of Sciences (QYZDJ-SSW-SLH037 to G.-L.T.), the Fundamental Research Funds for the Central Universities (020514380253 to Y.L.), the Natural Science Foundation of Jiangsu Province (BK20200335 to W.W.), and the Jiangsu Innovation & Entrepreneurship Talents Plan. The authors thank the staff of beam lines BL17U1, BL18U1 and BL19U1 of the Shanghai Synchrotron Radiation Facility for access and help with the X-ray data collection. We thank the staff of beam lines BL02U1, BL10U2 and BL19U1 of the Shanghai Synchrotron Radiation Facility for help scanning selenium fluorescence. We thank the High Performance Computing Center (HPCC) of Nanjing University for doing the numerical calculations in this paper on its blade cluster system. We also thank K. N. Houk for polishing the English, and R. Hong, P. Xu and B. Zhang for helpful discussions in chemical synthesis. L.W. thanks Y. Yong and T. Ye for assistance with protein crystallization.

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  1. These authors contributed equally: Jun-Bin He, Lian Wu, Wanqing Wei.

Authors and Affiliations

  1. State Key Laboratory of Bio-organic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences (CAS), CAS, Shanghai, China

    Jun-Bin He, Song Meng, Zheng-Tao Liu, Hai-Xue Pan & Gong-Li Tang

  2. Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, CAS, Shanghai, China

    Lian Wu & Sheng Yang

  3. State Key Laboratory of Coordination Chemistry, Jiangsu Key Laboratory of Advanced Organic Materials, Chemistry and Biomedicine Innovation Center, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, China

    Wanqing Wei, Xuan Wu & Yong Liang

  4. School of Chemistry and Material Sciences, Hangzhou Institute for Advanced Study, University of CAS, Hangzhou, China

    Hai-Xue Pan & Gong-Li Tang

  5. CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, CAS, Shenzhen, China

    Jiahai Zhou

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J.-B.H., Y.L., J.Z. and G.-L.T. conceived the project. J.-B.H. performed all in vitro enzymatic experiments, chemical syntheses, and compound isolation and characterization. Z.-T.L. and S.M. performed protein purification. H.-X.P. and S.M. performed bioinformatic analysis. L.W. performed all crystallographic studies. W.W. conducted all computational studies. All authors analysed and discussed the results. J.-B.H., L.W., W.W., Y.L., J.Z. and G.-L.T. prepared the manuscript.

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Correspondence to Yong Liang, Jiahai Zhou or Gong-Li Tang.

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The authors declare no competing interests.

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Nature Catalysis thanks Kurt Krause, Frank Hahn 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 Enzymatic and chemical studies of the five-membered THF ring formation.

a, Proposed steps of SoBcmB-catalysed five-membered THF ring formation. b, HPLC chromatograms of SoBcmB-catalysed reactions using compounds 1 and 1a as substrates and chemical transformation of compound 1a with mCPBA. The experiments were repeated independently at least three times with similar results.

Extended Data Fig. 2 Time-course studies of the SoBcmB catalytic reactions using 3 and 4 as substrates.

a and e, Proposed steps of SoBcmB-catalysed reactions using 3 and 4 as substrates, respectively. b and f, HPLC analyses of the SoBcmB catalytic reactions. The enzymatic reaction (50 μl) containing 50 mM Tris–HCl buffer (pH 7.5), 0.6 mM substrate, 2 mM αKG, 2 mM l-ascorbic acid, 50 μM FeSO4·7H2O, and 50 μg purified enzyme (27 μM) was incubated at 37 °C. The reactions were quenched at different times by the addition of 100 μl of precooled methanol and centrifuged at 14,000g for 20 min. The supernatants were analysed by HPLC and LC/MS (Thermo Fisher LTQ Fleet mass spectrometer). Three experiments were repeated independently with similar results. c and g, UV-Vis spectra for 3 and 3a–c and 4, 4a and 4c. d and i, ( + )-ESI-MS spectra of 3, 3a–c, 4, and 4a–4c. h, Extracted ion chromatogram (EIC) corresponding to intermediate 4b in SoBcmB-catalysed reactions. The LC/MS analysis was conducted by Thermo Scientific Q Exactive Plus Orbitrap LC–MS/MS System.

Extended Data Fig. 3 Crystal structures of SoBcmB.

Proteins are shown as sticks in slate and different ligands are shown in different colours. Ligands were modelled into electron density (omit map, grey), contoured to 3.0 σ. The key hydrogen-bond interactions are labelled in black dashed lines. Water molecules that interacted with ligands are shown as a sphere (red), labelled W1, W2, and W3 and the water coordinated with iron is labelled W. a, Overall structure of SoBcmB•FeII•αKG (slate) binary complex (PDB code 8HIV) is shown as a monomer (shown as a cartoon, left). The detail of the active site is shown as the stick and hydrogen bonding interactions are labelled by dash lines in black (right). b, SoBcmB•FeII•αKG•1 (yellow) quaternary complex structure (PDB code 7V2X). c, SoBcmB•FeII•αKG•1a (magenta) quaternary complex structure (PDB code 7V3E). d, SoBcmB•FeII•αKG•1d (orange) quaternary complex structure (PDB code 7V34).

Extended Data Fig. 4 The comparison of SoBcmB complex structures.

a and b, The alignment of SoBcmB•FeII•αKG (orange) and SoBcmB•FeII•αKG•2 (slate), with r.m.s.d. value 0.2 Å. The inconsistent part is highlighted by the green box and the detail is shown in b. The key residue Y311 and D307 were missed in SoBcmB•FeII•αKG and highlighted by magenta dots. c, The alignment of SoBcmB•FeII•αKG•2 (slate) and SoBcmB·FeII·αKG·2a (yellow), with r.m.s.d. value 0.11 Å. d, The alignment of SoBcmB•FeII•αKG•2 (slate) and SoBcmB•FeII•αKG•2f (magenta) with r.m.s.d. value 0.11 Å. e, The alignment of SoBcmB•FeII•αKG•2 (slate) and SoBcmB•FeII•αKG•1 (cyangreen) with r.m.s.d. value 0.12 Å. f, The alignment of SoBcmB•FeII•αKG•1 (cyangreen) and SoBcmB•FeII•αKG•1d (pink), with r.m.s.d. value 0.14 Å. g, The alignment of SoBcmB•FeII•αKG•1 (cyangreen) and SoBcmB•FeII•αKG•1a (bluewhite), with r.m.s.d. value 0.18 Å. Substrates are shown as different coloured sticks. Water molecules are shown as different coloured spheres. The coordination bonds and the hydrogen bonds are labelled by yellow dash line.

Extended Data Fig. 5 Computational analyses for the SoBcmB-catalysed desaturation reaction.

a, Representative MD snapshot of the reactant FeIV-oxo species of wild-type (WT) SoBcmB in complex with 2. Key residues (carbon in purple blue) and substrate 2 (carbon in green) are in stick modes. b, r.m.s.d. of backbone heavy atoms relative to the first snapshot during 10 ns classical MD simulation on enzyme-substrate 2 complex. c, Distances (d1 to d3) between the oxygen of the FeIV-oxo species and the hydrogen atoms from C1 or C1’ of 2. d, Free energy profiles (kcal mol-1) relative to complex reactants for substrate desaturation based on a theozyme model. All data refer to Gibbs free energies obtained in the quintet state at the B3LYP-D3/6-311 + G(2d,p)[SDD(Fe)] level with ZPE corrections at the B3LYP-D3/6-31 G(d,p)[LanL2DZ(Fe)] level of theory. Solvation by chlorobenzene was considered by using the CPCM model for all the above calculations.

Extended Data Fig. 6 Computational analyses for the SoBcmB-catalysed epoxidation reaction.

a, Representative MD snapshot of the reactant FeIV-oxo species of WT SoBcmB in complex with 2a. Key residues (carbon in purple blue) and intermediate 2a (carbon in cyan) are in stick modes. b, r.m.s.d. of backbone heavy atoms relative to the first snapshot during 10 ns classical MD simulation on enzyme-intermediate 2a complex. c, Distances (d1 to d2) between the oxygen of the FeIV-oxo species and C1 or C1’ of 2a. d, Free energy profiles (kcal mol-1) relative to complex reactants for epoxidation of the desaturated intermediate 2a based on a theozyme model. All data refer to Gibbs free energies obtained in the quintet state at the B3LYP-D3/6-311 + G(2d,p)[SDD(Fe)] level with ZPE corrections at the B3LYP-D3/6-31 G(d,p)[LanL2DZ(Fe)] level of theory. Solvation by chlorobenzene was considered by using the CPCM model for all the above calculations.

Extended Data Fig. 7 Computational analyses for the ring-closure reaction.

a, Representative MD snapshot in SoBcmB complexed with the epoxide-containing intermediate 2b. Key residues (carbon in purple blue) and the epoxide intermediate 2b (carbon in light pink) are in stick modes. b, r.m.s.d. of backbone heavy atoms relative to the first snapshot during 10 ns classical MD simulation on enzyme-intermediate 2b complex. c, Distances (d1 and d2) between the amino group nitrogen (NE2) of Q120 and the phenol hydroxyl group oxygen (OH) of Y311 with 2’-OH (O4) and 3’-OH (O3) in 2b. d, Numbers of snapshots missing one hydrogen-bond with the OH in Y311 or the NH2 in Q120.

Extended Data Fig. 8 In vitro assays of the SoBcmB mutants using compound 2 as the substrate.

a, Enzymatic activities of SoBcmB and its mutants. Reactions were performed at 37 °C for 1 h using compound 2 as the substrate in 50 μl 50 mM Tris–HCl (pH 7.5) buffer containing 50 μg SoBcmB or one of its mutants. Different colour columns represent different products. Bars represent mean conversion yield averaged over three reactions and error bars indicate standard deviation of three independent replicates. Asterisks represent enzyme activity not detected. b, Structures of products 2a, 2d, and 2f–2i. See Extended Data Fig. 9a and Supplementary Fig. 17 for HPLC chromatograms.

Source data

Extended Data Fig. 9 HPLC analysis of in vitro reaction of BcmB and its mutants.

a, HPLC chromatograms of the SoBcmB-catalysed reaction mixtures using 2 as the substrate. b, HPLC chromatograms of the SoBcmB-catalysed reaction mixtures using 2a as the substrate. c, HPLC chromatograms of the SsBcmB-catalysed reaction mixtures using 2 as the substrate. d, HPLC chromatograms of the SkBcmB-catalysed reaction mixtures using 2 as the substrate. Reaction conditions are detailed in Methods unless otherwise specified. The data show one representative experiment from at least three independent replicates.

Extended Data Table 1 Data collection and refinement statistics for SoBcmB crystal structures
Full size table

Supplementary information

Supplementary Information

Supplementary Methods, Tables 1–15, Figs. 1–148 and References.

Reporting Summary

Supplementary Data 1

The crystallographic data of both protein and compound 1d reported in this article.

Supplementary Data 2

The atomic coordinates of the optimized computational models studied in this article.

Supplementary Data 3

The initial and final configurations investigated by molecular dynamics simulations.

Source data

Source Data Fig. 5

Statistical source data for Fig. 5i.

Source Data Extended Data Fig./Table 8

Statistical source data for Extended Data Fig. 8a.

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He, JB., Wu, L., Wei, W. et al. Enzymatic catalysis favours eight-membered over five-membered ring closure in bicyclomycin biosynthesis. Nat Catal 6, 637–648 (2023). https://doi.org/10.1038/s41929-023-00987-4

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