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 Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 per month
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
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
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 (http://www.rcsb.org) 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.
Huisgen, R. Cycloadditions—definition, classification, and characterization. Angew. Chem. Int. Ed. Engl. 7, 321–328 (1968).
Woodward, R. B. & Hoffmann, R. The conservation of orbital symmetry. Angew. Chem. Int. Ed. Engl. 8, 781–853 (1969).
Fukui, K., Yonezawa, T. & Shingu, H. A molecular orbital theory of reactivity in aromatic hydrocarbons. J. Chem. Phys. 20, 722–725 (1952).
Alcaide, B., Almendros, P. & Aragoncillo, C. Exploiting [2+2] cycloaddition chemistry: achievements with allenes. Chem. Soc. Rev. 39, 783–816 (2010).
Murakami, M. & Ishida, N. Cleavage of carbon–carbon σ-bonds of four-membered rings. Chem. Rev. 121, 264–299 (2021).
Rattray, J. E. et al. A comparative genomics study of genetic products potentially encoding ladderane lipid biosynthesis. Biol. Direct 4, 8 (2009).
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).
Hong, Y. J. & Tantillo, D. J. How cyclobutanes are assembled in nature—insights from quantum chemistry. Chem. Soc. Rev. 43, 5042–5050 (2014).
Fraga, B. M. Natural sesquiterpenoids. Nat. Prod. Rep. 30, 1226–1264 (2013).
Sancar, A. Structure and function of DNA photolyase. Biochemistry 33, 2–9 (1994).
Jeon, B. S., Wang, S. A., Ruszczycky, M. W. & Liu, H. W. Natural [4 + 2]-cyclases. Chem. Rev. 117, 5367–5388 (2017).
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).
Tang, Z., Wang, H. & Liu, W. in Comprehensive Natural Products III 3rd edn (eds Liu, H.-W. & Begley, T. P.) 187–227 (Elsevier, 2020).
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).
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).
Tian, Z. et al. An enzymatic [4+2] cyclization cascade creates the pentacyclic core of pyrroindomycins. Nat. Chem. Biol. 11, 259–265 (2015).
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).
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).
Ohashi, M. et al. SAM-dependent enzyme-catalysed pericyclic reactions in natural product biosynthesis. Nature 549, 502–506 (2017).
Zhang, B. et al. Enzyme-catalysed [6+4] cycloadditions in the biosynthesis of natural products. Nature 568, 122–126 (2019).
Little, R. et al. Unexpected enzyme-catalysed [4+2] cycloaddition and rearrangement in polyether antibiotic biosynthesis. Nat. Catal. 11, 1045–1054 (2019).
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).
Chen, Q. et al. Enzymatic intermolecular hetero-Diels–Alder reaction in the biosynthesis of tropolonic sesquiterpenes. J. Am. Chem. Soc. 141, 14052–14056 (2019).
Dan, Q. et al. Fungal indole alkaloid biogenesis through evolution of a bifunctional reductase/Diels–Alderase. Nat. Chem. 11, 972–980 (2019).
Gao, L. et al. FAD-dependent enzyme-catalysed intermolecular [4+2] cycloaddition in natural product biosynthesis. Nat. Chem. 12, 620–628 (2020).
Li, Q. et al. Nonspecific heme-binding cyclase, AbmU, catalyzes [4 + 2] cycloaddition during neoabyssomicin biosynthesis. ACS Omega 5, 20548–20557 (2020).
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).
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).
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).
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).
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).
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).
Li, S. et al. Dissecting glycosylation steps in lobophorin biosynthesis implies an iterative glycosyltransferase. Org. Lett. 15, 1374–1377 (2013).
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).
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).
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).
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).
Hoffmann, R. & Woodward, R. B. Selection rules for concerted cycloaddition reactions. J. Am. Chem. Soc. 87, 2046–2048 (2002).
Crimmins, M. T. & Reinhold, T. L. in Organic Reactions (ed. Paquette, L. A.) 297–588 (Wiley, 1993).
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).
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).
Zou, Y. et al. Computational investigation of the mechanism of Diels–Alderase PyrI4. J. Am. Chem. Soc. 142, 20232–20239 (2020).
Green, M. R. & Sambrook, J. Molecular Cloning: A Laboratory Manual 4th edn (Cold Spring Harbor Laboratory Press, 2012).
Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F. & Hopwood, D. A. Practical Streptomyces Genetics (John Innes Foundation, 2000).
Schuck, P. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys. J. 78, 1606–1619 (2000).
Wang, Q.-S. et al. The macromolecular crystallography beamline of SSRF. Nucl. Sci. Technol. 26, 12–17 (2015).
Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).
Vonrhein, C. et al. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. D 67, 293–302 (2011).
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).
Storoni, L. C., McCoy, A. J. & Read, R. J. Likelihood-enhanced fast rotation functions. Acta Crystallogr. D 60, 432–438 (2004).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).
Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002).
Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007).
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).
Frisch, M. J. et al. Gaussian 09, Revision A.02 (Gaussian, 2016).
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).
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).
Case, D. A. et al. Amber 16 (Univ. California San Francisco, 2016).
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).
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).
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).
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.
The authors declare no competing interests.
Peer review information
Nature Chemistry thanks the anonymous reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
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 Figs. 1–24, Tables 1–10, notes, references and uncropped scans of the gels in Supplementary Fig. 1.
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.
About this article
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). https://doi.org/10.1038/s41557-022-01104-x
This article is cited by
Nature Chemistry (2023)