Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A cyclase that catalyses competing 2 + 2 and 4 + 2 cycloadditions


Cycloaddition reactions are among the most widely used reactions in chemical synthesis. Nature achieves these cyclization reactions with a variety of enzymes, including Diels–Alderases that catalyse concerted 4 + 2 cycloadditions, but biosynthetic enzymes with 2 + 2 cyclase activity have yet to be discovered. Here we report that PloI4, a β-barrel-fold protein homologous to the exo-selective 4 + 2 cyclase that functions in the biosynthesis of pyrroindomycins, catalyses competitive 2 + 2 and 4 + 2 cycloaddition reactions. PloI4 is believed to catalyse an endo-4 + 2 cycloaddition in the biosynthesis of pyrrolosporin A; however, when the substrate precursor of pyrroindomycins was treated with PloI4, an exo-2 + 2 adduct was produced in addition to the exo- and endo-4 + 2 adducts. Biochemical characterizations, computational analyses, (co)crystal structures and mutagenesis outcomes have allowed the catalytic versatility of PloI4 to be rationalized. Mechanistic studies involved the directed engineering of PloI4 to variants that produced the exo-4 + 2, endo-4 + 2 or exo-2 + 2 product preferentially. This work illustrates an enzymatic thermal 2 + 2 cycloaddition and provides evidence of a process through which an enzyme evolves along with its substrate for specialization and activity improvement.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Concerted and stepwise cycloaddition reactions.
Fig. 2: Spiral tetramate and tetronate biosynthesis.
Fig. 3: Computational analysis of the conversion of 7 into adducts 8 and 10 by the exo path.
Fig. 4: Structures of PloI4 and its variants.
Fig. 5: PloI4 engineering, molecular modelling and related biochemical assays.

Data availability

All the data underlying the findings of this study are available in this article and its Supplementary Information. The atomic coordinates of PloI4, PloI4-C16M/D46A/I137V, PloI4-C16M/D46A/I137V complexed with 10 and PloI4-F124L complexed with 9 have been deposited in the Protein Data Bank ( with accession codes 7X7Z, 7X80, 7X81 and 7X86, respectively. The DNA sequence of the plo cluster has been deposited in GenBank with accession number ON045076. Source data are provided with this paper.


  1. Huisgen, R. Cycloadditions—definition, classification, and characterization. Angew. Chem. Int. Ed. Engl. 7, 321–328 (1968).

    Article  CAS  Google Scholar 

  2. Woodward, R. B. & Hoffmann, R. The conservation of orbital symmetry. Angew. Chem. Int. Ed. Engl. 8, 781–853 (1969).

    Article  CAS  Google Scholar 

  3. Fukui, K., Yonezawa, T. & Shingu, H. A molecular orbital theory of reactivity in aromatic hydrocarbons. J. Chem. Phys. 20, 722–725 (1952).

    Article  CAS  Google Scholar 

  4. Alcaide, B., Almendros, P. & Aragoncillo, C. Exploiting [2+2] cycloaddition chemistry: achievements with allenes. Chem. Soc. Rev. 39, 783–816 (2010).

    Article  CAS  Google Scholar 

  5. Murakami, M. & Ishida, N. Cleavage of carbon–carbon σ-bonds of four-membered rings. Chem. Rev. 121, 264–299 (2021).

    Article  CAS  Google Scholar 

  6. Rattray, J. E. et al. A comparative genomics study of genetic products potentially encoding ladderane lipid biosynthesis. Biol. Direct 4, 8 (2009).

    Article  Google Scholar 

  7. Rattray, J. E. et al. Carbon isotope-labelling experiments indicate that ladderane lipids of anammox bacteria are synthesized by a previously undescribed, novel pathway. FEMS Microbiol. Lett. 292, 115–122 (2009).

    Article  CAS  Google Scholar 

  8. Hong, Y. J. & Tantillo, D. J. How cyclobutanes are assembled in nature—insights from quantum chemistry. Chem. Soc. Rev. 43, 5042–5050 (2014).

    Article  CAS  Google Scholar 

  9. Fraga, B. M. Natural sesquiterpenoids. Nat. Prod. Rep. 30, 1226–1264 (2013).

    Article  CAS  Google Scholar 

  10. Sancar, A. Structure and function of DNA photolyase. Biochemistry 33, 2–9 (1994).

    Article  CAS  Google Scholar 

  11. Jeon, B. S., Wang, S. A., Ruszczycky, M. W. & Liu, H. W. Natural [4 + 2]-cyclases. Chem. Rev. 117, 5367–5388 (2017).

    Article  CAS  Google Scholar 

  12. Jamieson, C. S., Ohashi, M., Liu, F., Tang, Y. & Houk, K. N. The expanding world of biosynthetic pericyclases: cooperation of experiment and theory for discovery. Nat. Prod. Rep. 36, 698–713 (2019).

    Article  CAS  Google Scholar 

  13. Tang, Z., Wang, H. & Liu, W. in Comprehensive Natural Products III 3rd edn (eds Liu, H.-W. & Begley, T. P.) 187–227 (Elsevier, 2020).

  14. Zheng, Q., Tian, Z. & Liu, W. Recent advances in understanding the enzymatic reactions of [4+2] cycloaddition and spiroketalization. Curr. Opin. Chem. Biol. 31, 95–102 (2016).

    Article  CAS  Google Scholar 

  15. Kim, H. J., Ruszczycky, M. W., Choi, S. H., Liu, Y. N. & Liu, H. W. Enzyme-catalysed [4+2] cycloaddition is a key step in the biosynthesis of spinosyn A. Nature 473, 109–112 (2011).

    Article  CAS  Google Scholar 

  16. Tian, Z. et al. An enzymatic [4+2] cyclization cascade creates the pentacyclic core of pyrroindomycins. Nat. Chem. Biol. 11, 259–265 (2015).

    Article  CAS  Google Scholar 

  17. Hashimoto, T. et al. Biosynthesis of versipelostatin: identification of an enzyme-catalyzed [4+2]-cycloaddition required for macrocyclization of spirotetronate-containing polyketides. J. Am. Chem. Soc. 137, 572–575 (2015).

    Article  CAS  Google Scholar 

  18. Byrne, M. J. et al. The catalytic mechanism of a natural Diels–Alderase revealed in molecular detail. J. Am. Chem. Soc. 138, 6095–6098 (2016).

    Article  CAS  Google Scholar 

  19. Ohashi, M. et al. SAM-dependent enzyme-catalysed pericyclic reactions in natural product biosynthesis. Nature 549, 502–506 (2017).

    Article  Google Scholar 

  20. Zhang, B. et al. Enzyme-catalysed [6+4] cycloadditions in the biosynthesis of natural products. Nature 568, 122–126 (2019).

    Article  CAS  Google Scholar 

  21. Little, R. et al. Unexpected enzyme-catalysed [4+2] cycloaddition and rearrangement in polyether antibiotic biosynthesis. Nat. Catal. 11, 1045–1054 (2019).

    Article  Google Scholar 

  22. Cai, Y. et al. Structural basis for stereoselective dehydration and hydrogen-bonding catalysis by the SAM-dependent pericyclase LepI. Nat. Chem. 11, 812–820 (2019).

    Article  CAS  Google Scholar 

  23. Chen, Q. et al. Enzymatic intermolecular hetero-Diels–Alder reaction in the biosynthesis of tropolonic sesquiterpenes. J. Am. Chem. Soc. 141, 14052–14056 (2019).

    Article  CAS  Google Scholar 

  24. Dan, Q. et al. Fungal indole alkaloid biogenesis through evolution of a bifunctional reductase/Diels–Alderase. Nat. Chem. 11, 972–980 (2019).

    Article  CAS  Google Scholar 

  25. Gao, L. et al. FAD-dependent enzyme-catalysed intermolecular [4+2] cycloaddition in natural product biosynthesis. Nat. Chem. 12, 620–628 (2020).

    Article  CAS  Google Scholar 

  26. Li, Q. et al. Nonspecific heme-binding cyclase, AbmU, catalyzes [4 + 2] cycloaddition during neoabyssomicin biosynthesis. ACS Omega 5, 20548–20557 (2020).

    Article  CAS  Google Scholar 

  27. Sato, M. et al. Catalytic mechanism and endo-to-exo selectivity reversion of an octalin-forming natural Diels–Alderase. Nat. Catal. 4, 223–232 (2021).

    Article  CAS  Google Scholar 

  28. Lam, K. S. et al. Pyrrolosporin A, a new antitumor antibiotic from Micromonospora sp. C39217-R109-7. I. Taxonomy of producing organism, fermentation and biological activity. J. Antibiot. 49, 860–864 (1996).

    Article  CAS  Google Scholar 

  29. Schroeder, D. R. et al. Pyrrolosporin A, a new antitumor antibiotic from Micromonospora sp. C39217-R109-7. II. Isolation, physicochemical properties, spectroscopic study and X-ray analysis. J. Antibiot. 49, 865–872 (1996).

  30. Jia, X. Y. et al. Genetic characterization of the chlorothricin gene cluster as a model for spirotetronate antibiotic biosynthesis. Chem. Biol. 13, 575–585 (2006).

    Article  CAS  Google Scholar 

  31. Zhang, H. et al. Elucidation of the kijanimicin gene cluster: insights into the biosynthesis of spirotetronate antibiotics and nitrosugars. J. Am. Chem. Soc. 129, 14670–14683 (2007).

    Article  CAS  Google Scholar 

  32. Fang, J. et al. Cloning and characterization of the tetrocarcin A gene cluster from Micromonospora chalcea NRRL 11289 reveals a highly conserved strategy for tetronate biosynthesis in spirotetronate antibiotics. J. Bacteriol. 190, 6014–6025 (2008).

    Article  CAS  Google Scholar 

  33. Li, S. et al. Dissecting glycosylation steps in lobophorin biosynthesis implies an iterative glycosyltransferase. Org. Lett. 15, 1374–1377 (2013).

    Article  CAS  Google Scholar 

  34. Zheng, Q. et al. Structural insights into a flavin-dependent [4+2] cyclase that catalyzes trans-decalin formation in pyrroindomycin biosynthesis. Cell Chem. Biol. 25, 718–727 (2018).

  35. Li, B. et al. Mechanism of the stereoselective catalysis of Diels–Alderase PyrE3 involved in pyrroindomycin biosynthesis. J. Am. Chem. Soc. 11, 5099–5107 (2022).

    Article  Google Scholar 

  36. Wu, Q., Wu, Z., Qu, X. & Liu, W. Insights into pyrroindomycin biosynthesis reveal a uniform paradigm for tetramate/tetronate formation. J. Am. Chem. Soc. 134, 17342–17345 (2012).

    Article  CAS  Google Scholar 

  37. Zheng, Q. et al. A linear hydroxymethyl tetramate undergoes an acetylation-elimination process for exocyclic methylene formation in the biosynthetic pathway of pyrroindomycins. Org. Biomol. Chem. 15, 88–91 (2016).

    Article  Google Scholar 

  38. Hoffmann, R. & Woodward, R. B. Selection rules for concerted cycloaddition reactions. J. Am. Chem. Soc. 87, 2046–2048 (2002).

    Article  Google Scholar 

  39. Crimmins, M. T. & Reinhold, T. L. in Organic Reactions (ed. Paquette, L. A.) 297–588 (Wiley, 1993).

  40. Svatunek, D., Pemberton, R. P., Mackey, J. L., Liu, P. & Houk, K. N. Concerted [4+2] and stepwise (2+2) cycloadditions of tetrafluoroethylene with butadiene: DFT and DLPNO-UCCSD(T) explorations. J. Org. Chem. 85, 3858–3864 (2020).

  41. Zheng, Q. et al. Enzyme-dependent [4+2] cycloaddition depends on lid-like interaction of the N-terminal sequence with the catalytic core in PyrI4. Cell Chem. Biol. 23, 352–360 (2016).

  42. Zou, Y. et al. Computational investigation of the mechanism of Diels–Alderase PyrI4. J. Am. Chem. Soc. 142, 20232–20239 (2020).

    Article  CAS  Google Scholar 

  43. Green, M. R. & Sambrook, J. Molecular Cloning: A Laboratory Manual 4th edn (Cold Spring Harbor Laboratory Press, 2012).

  44. Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F. & Hopwood, D. A. Practical Streptomyces Genetics (John Innes Foundation, 2000).

  45. Schuck, P. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys. J. 78, 1606–1619 (2000).

    Article  CAS  Google Scholar 

  46. Wang, Q.-S. et al. The macromolecular crystallography beamline of SSRF. Nucl. Sci. Technol. 26, 12–17 (2015).

    Google Scholar 

  47. Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).

    Article  CAS  Google Scholar 

  48. Vonrhein, C. et al. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. D 67, 293–302 (2011).

    Article  CAS  Google Scholar 

  49. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  50. Storoni, L. C., McCoy, A. J. & Read, R. J. Likelihood-enhanced fast rotation functions. Acta Crystallogr. D 60, 432–438 (2004).

    Article  Google Scholar 

  51. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  Google Scholar 

  52. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002).

    Article  Google Scholar 

  53. Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007).

    Article  Google Scholar 

  54. Grimme, S. Exploration of chemical compound, conformer, and reaction space with meta-dynamics simulations based on tight-binding quantum chemical calculations. J. Chem. Theory Comput. 15, 2847–2862 (2019).

    Article  CAS  Google Scholar 

  55. Frisch, M. J. et al. Gaussian 09, Revision A.02 (Gaussian, 2016).

  56. Luchini, G., Alegre-Requena, J. V., Funes-Ardoiz, I. & Paton, R. S. GoodVibes: automated thermochemistry for heterogeneous computational chemistry data [version 1; peer review: 2 approved with reservations]. F1000Res. 9, 291 (2020).

    Article  Google Scholar 

  57. Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31, 455–461 (2010).

    CAS  Google Scholar 

  58. Case, D. A. et al. Amber 16 (Univ. California San Francisco, 2016).

  59. Søndergaard, C. R., Olsson, M. H. M., Rostkowski, M. & Jensen, J. H. Improved treatment of ligands and coupling effects in empirical calculation and rationalization of pKa values. J. Chem. Theory Comput. 7, 2284–2295 (2011).

    Article  Google Scholar 

  60. Olsson, M. H. M., Søndergaard, C. R., Rostkowski, M. & Jensen, J. H. PROPKA3: consistent treatment of internal and surface residues in empirical pKa predictions. J. Chem. Theory Comput. 7, 525–537 (2011).

    Article  CAS  Google Scholar 

  61. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983).

    Article  CAS  Google Scholar 

Download references


We thank R. Wang (Fudan University) for help with the TDDFT-ECD theoretical calculations, the Nuclear Magnetic Resonance Facility at SIOC for NMR analysis and the SSRF BL19U1, BL17B1 and BL10U2 beam lines for X-ray beam time. This work was supported in part by grants from the National Natural Science Foundation of China (22193070, 32030002 and 21621002 for W.L. and 21822705 for L.P.), the National Key R&D Program of China (2022YFC2303100 and 2019YFA0905400 for W.L.), STCSM (20XD1425200 for L.P.) and the National Institutes of Health (GM 124480, R01GM116961 and CHE100024P for K.N.H.). The funders had no input into the study design, data collection and analysis, the decision to publish or the preparation of the manuscript.

Author information

Authors and Affiliations



H.W., Z. Tang, J.W., Z. Tian and Q.Z. performed the biochemical experiments. Y.Z. and N.S. performed the computational experiments. M.L. and Y.G. performed the structural biology experiments. H.W., Z. Tang and J.W. engineered the proteins. Z. Tang and Z. Tian performed the sequence analysis. H.W. and Q.Y. isolated and characterized the compounds. All authors analysed and discussed the results. H.W., Y.Z., Z. Tang, W.L., L.P. and K.N.H. prepared the manuscript. W.L., L.P. and K.N.H. developed the hypothesis. W.L. designed and organized the research.

Corresponding authors

Correspondence to Wen Liu, Lifeng Pan or K. N. Houk.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Chemistry thanks the anonymous reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Chemical structures of PLO-A and structurally related, naturally occurring tetramates/tetronates.

The dialkyldecalin and spiro-conjugated portions in each structure are indicated in blue and red, respectively.

Extended Data Fig. 2 Biogenesis for PLO-A and PYRs.

a, gene clusters of PLO-A (top, from S. rugosporus) and PYRs (bottom, from M. sp. C39217-R109-7). For the detailed assignments of gene functions, which are annotated here by colored rectangular blocks, see Supplementary Table 10. b, Proposed biosynthetic pathways of PLO-A (top) and PYRs (bottom). M, module. For domain functions, KS, ketosynthase; AT, acyltransferase; ACP, acyl carrier protein; DH, dehydrotase; ER, enoylreductase; KR, ketoreductase; A, adenylation; PCP, peptidyl carrier protein; and C, condensation.

Extended Data Fig. 3 Computational analyses of the transition states and energetics in the endo-path for converting 7 into endo-4 + 2 adduct 9 and endo-2 + 2 adduct 13.

Gibbs free energies in kcal/mol are shown. * indicates open-shell energies.

Extended Data Fig. 4 Structural alignment for comparison.

a, dimers of PloI4 (green and yellow) and PyrI4 (blue and purple) in apo-form. b, monomers of apo-form PloI4 (green) and PyrI4 (purple) in complex with exo-4 + 2 product 8. The central cavity in each monomer is indicated. c, comparison of PloI4 (green) with PyrI4 (purple) in the central cavity.

Extended Data Fig. 5 Computational docking of various transition states at the active site of wild-type PloI4.

a, binding interface of PloI4 with the TS2-A of substrate 3 (purple) that leads to endo-4 + 2 adduct 4. b, binding interface of PloI4 with the TS2a of substrate 7 (grey) that leads to exo-4 + 2 adduct 8. c, binding interface of PloI4 with the TS2b of substrate 7 (light green) that leads to endo-4 + 2 adduct 9. d, binding interface of PloI4 with the TS3a of substrate 7 (yellow) that leads to exo-2 + 2 adduct 10.

Extended Data Fig. 6 Determination of the product profiles of PloI4 and its engineered variants by HPLC.

Reactions, compounds and structures related to exo-4 + 2 (green), endo-4 + 2 (blue) and exo-2 + 2 (yellow) cycloadditions are indicated by color. a, PloI4-catalyzed conversion of substrate 7. b, engineering toward the production of exo-4 + 2 adduct 8. With the enzyme-free mixture as a control (i), reactions were conducted in the presence of wild-type PloI4 (ii), PloI4-I137Y (iii), and PloI4-I137F (iv), respectively. c, engineering toward the production of endo-4 + 2 adduct 9. With the enzyme-free mixture as a control (i), reactions were conducted in the presence of wild-type PloI4 (ii), PloI4-F124V (iii), PloI4-F124L (iv), PloI4-F124I (v), and PloI4-F124A (vi), respectively. d, engineering toward the production of exo-2 + 2 adduct 10. With the enzyme-free mixture as a control (i), reactions were conducted in the presence of wild-type PloI4 (ii), PloI4-C16M (iii), PloI4-D46A (iv), PloI4-I137V (v), PloI4-C16M/I137V (vi), PloI4-C16M/D46A (vii), PloI4-D46A/I137V (viii), and PloI4-C16M/D46A/I137V (ix), respectively.

Extended Data Fig. 7 Determination of the overall activities of PloI4 and its engineered variants.

The overall activity of each enzyme was examined by quantitative analysis of the production of all three products, that is, 8, 9 and 10. For comparison, the average activity of wild-type PloI4 (100%) was used as the control. Each central value means the average of three independent replicates with error bars representing the standard deviation.

Source data

Extended Data Fig. 8 Computational modeling of various transition states of substrate 7 at the active sites of PloI4 variants.

a, overlaid exo-2 + 2-selective TS3a (yellow) and exo-4 + 2-selective TS2a (green) in PloI4-I137F. b, overlaid TS3a (green) and exo-4 + 2-selective TS2a (yellow) in PloI4-C16M/D46A/I137V. c, exo-selective TS1a in PloI4-I137F. d, exo-selective TS1b in PloI4-F124A. For c and d, substructures indicated by green are overlaid endo (for TS1a) and exo (for TS1b) rotamers of the diene group.

Extended Data Fig. 9 Product alignment in the central cavities of PloI4 and its variants by combined surface and stick representation.

In each cavity (shown in wireframe mode), products 8 (left), 9 (middle), and 10 (right) are indicated by grey, orange and yellow, respectively. a, wild-type PloI4. The side chains of related residues C16, D46, F124, and I137 at the active side are shown. b, variant PloI4-F124L. The side chain of related residues M97, L124 and I137 are shown. c, variant PloI4-C16M/D46A/I137V. The side chains of related residues M16, A46, Y95 and V137 residues are shown.

Supplementary information

Supplementary Information

Supplementary Figs. 1–24, Tables 1–10, notes, references and uncropped scans of the gels in Supplementary Fig. 1.

Reporting Summary

Supplementary Data

xyz coordinates.

Source data

Source Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 7

Statistical source data.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wang, H., Zou, Y., Li, M. et al. A cyclase that catalyses competing 2 + 2 and 4 + 2 cycloadditions. Nat. Chem. 15, 177–184 (2023).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing