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

Nature Chemistry volume 15, pages 177–184 (2023)Cite this article

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Abstract

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.

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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.

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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 (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.

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Acknowledgements

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

Author notes

  1. These authors contributed equally: Hongbo Wang, Yike Zou, Miao Li, Zhijun Tang.

Authors and Affiliations

  1. State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Shanghai, China

    Hongbo Wang, Miao Li, Zhijun Tang, Jiabao Wang, Zhenhua Tian, Qian Yang, Qingfei Zheng, Yujiao Guo, Wen Liu & Lifeng Pan

  2. Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA

    Yike Zou, Nina Strassner & K. N. Houk

  3. School of Chemistry and Materials Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China

    Miao Li & Lifeng Pan

  4. Department of Chemistry, Shanghai Normal University, Shanghai, China

    Jiabao Wang & Wen Liu

  5. Abiochem Biotechnology Co., Ltd, Shanghai, China

    Zhenhua Tian

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Contributions

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.

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

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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.

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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

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