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Enzyme-catalysed [6+4] cycloadditions in the biosynthesis of natural products


Pericyclic reactions are powerful transformations for the construction of carbon–carbon and carbon–heteroatom bonds in organic synthesis. Their role in biosynthesis is increasingly apparent, and mechanisms by which pericyclases can catalyse reactions are of major interest1. [4+2] cycloadditions (Diels–Alder reactions) have been widely used in organic synthesis2 for the formation of six-membered rings and are now well-established in biosynthesis3,4,5,6. [6+4] and other ‘higher-order’ cycloadditions were predicted7 in 1965, and are now increasingly common in the laboratory despite challenges arising from the generation of a highly strained ten-membered ring system8,9. However, although enzyme-catalysed [6+4] cycloadditions have been proposed10,11,12, they have not been proven to occur. Here we demonstrate a group of enzymes that catalyse a pericyclic [6+4] cycloaddition, which is a crucial step in the biosynthesis of streptoseomycin-type natural products. This type of pericyclase catalyses [6+4] and [4+2] cycloadditions through a single ambimodal transition state, which is consistent with previous proposals11,12. The [6+4] product is transformed to a less stable [4+2] adduct via a facile Cope rearrangement, and the [4+2] adduct is converted into the natural product enzymatically. Crystal structures of three pericyclases, computational simulations of potential energies and molecular dynamics, and site-directed mutagenesis establish the mechanism of this transformation. This work shows how enzymes are able to catalyse concerted pericyclic reactions involving ambimodal transition states.

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Fig. 1: Cycloadditions in natural product biosynthesis.
Fig. 2: DFT-computed free energies for the [6+4], [4+2] and [3,3]-Cope reactions.
Fig. 3: Crystal structures of three enzymes and catalytic sites for the bispericyclic reaction.

Data availability

Atomic coordinates of StmD, NgnD and 101015D have been deposited in the Protein Data Bank (PDB) with accession codes 6A5G, 6A5F and 6A5H. The crystallographic data of compounds 6 and 8 have been deposited in the Cambridge Crystallographic Data Centre ( with CCDC numbers 1843311 and 1843314. The DNA sequence of the ngn gene cluster has been deposited in GenBank with the accession number MH544245.


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This work was financially supported by Ministry of Science and Technology (2018YFC1706200), the National Natural Science Foundation of China (21572100, 21803030, 81522042, 81773591, 81530089, 81673333, 21861142005, 21761142001 and 21661140001), the National Thousand Young Talents Program, the Jiangsu Specially-Appointed Professor Plan, the Natural Science Foundation of Jiangsu Province (BK20170631) in China, and the US National Institutes of General Medical Sciences, National Institutes of Health (GM 124480). We are grateful to the High Performance Computing Center of Nanjing University for doing the numerical calculations in this paper on its blade cluster system. We thank the staff at beamlines BL17U1 and BL18U1 of Shanghai Synchrotron Radiation Facility and the National Supercomputing Center in Wuxi for their support.

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Nature thanks Fedor Novikov, Satish Nair and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Authors and Affiliations



B.Z., Y.L., R.X.T. and H.M.G. conceived the project. B.Z. and K.B.W. performed all in vivo and in vitro experiments. W.W., H.Y.Q. and R.H.J. performed fermentation, compound isolation and characterization. X.W., F.L., X.Z. and Y.L. conducted the computational studies. J.S. performed LC–MS analysis. L.Y.L., H.H. and K.X. performed NMR experiments. B.Z. and J.Z. carried out the protein crystallographic studies. All authors contributed to the discussion and interpretation of the results. B.Z., K.B.W., K.N.H., Y.L. and H.M.G. prepared the manuscript.

Corresponding authors

Correspondence to Kendall N. Houk, Yong Liang, Ren Xiang Tan or Hui Ming Ge.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Comparative analysis of the stm and ngn gene clusters.

a, Structure of streptoseomycin (1) and structurally related natural products nargenicin (2), coloradocin, nodusmicin and branimycin. b, The stm gene cluster from S. seoulensis A01 and the ngn gene cluster from N. argentinensis ATCC 31306. c, Proposed biosynthetic pathway for 1. d, Proposed biosynthetic pathway for 2.

Extended Data Fig. 2 LC–MS analysis of chemical complementation in the ΔstmA mutant strain.

The experiments were independently repeated twice with similar results.

Extended Data Fig. 3 Time-course 1H NMR spectra for a mixture of 6 and 7.

Spectra were obtained at room temperature. 1H NMR signals highlighted in red are those from 7.

Extended Data Fig. 4 LC–MS analysis of product formation in both in vivo and in vitro enzymatic reactions.

a, b, Time-course analysis of chemical complementation of 6 in the ΔstmA mutant strain. EIC corresponding to 6 and 7 (m/z = 369.0, [M + Na]+) (a); EIC corresponding to 1 (m/z = 622.2, [M + Na]+) (b). c, LC–MS analysis of the production of 68 in mutants and in complementary strains. d, LC–MS analysis of the production for 68 in enzymatic reactions. TE* indicates the StmC/ACP-TE. The experiments were independently repeated twice with similar results.

Extended Data Fig. 5 In vitro biochemical characterization of the efficiency of thioesterase-catalysed macrolactonization and the effects of varying concentrations of StmD in enzymatic cycloaddition.

a, Thioesterase- and cyclase-catalysed tandem reactions. b, LC–MS analysis of different thioesterase-catalysed macrolactonization. c, Effects of varying concentrations of StmD on enzymatic cycloaddition. Each reaction mixture (50 μl 100 mM phosphate buffer at pH 7.0) contained 10 μM StmC/ACP-TE and 200 μM substrate 10, and (i) 0 μM StmD, (ii) 10 μM StmD, (iii) 20 μM StmD, (iv) 40 μM StmD, (v) 80 μM StmD, (vi) 100 μM StmD. (vii) Standard solution of compounds 6, 7 and 8. TE* indicates the StmC/ACP-TE. The experiments were independently repeated three times with similar results.

Extended Data Fig. 6 Sequence alignment of StmD, NgnD and three homologous proteins 101015D, F601D and Root369D.

The secondary structure elements of NgnD are presented at the top. Blue cylinders and green arrows indicate the α-helices and β-strands, respectively. The proteins 101015D (WP_040742972), F601D (WP_057609890) and Root369D (WP_077974259) are from N. tenerifensis NBRC 101015, S. tsukubaensis F601 and Streptomyces sp. Root369, respectively.

Extended Data Fig. 7 Distributions of reactive trajectories initiated from ambimodal transition states TS-1 and TS-3.

a, TS-1. b, TS-3. Fifteen randomly chosen trajectories were plotted in each case. Trajectories leading to a [4+2] adduct are shown in red, and those leading to a [6+4] adduct are shown in blue. The table lists the 100 trajectories that we calculated.

Extended Data Fig. 8 The electrostatic potential analysis of the DFT-optimized transition-state structure TS-1.

The blue and red regions represent electrostatic potential regions of positive and negative potential (repulsive and attractive interactions, respectively) with a positive charge, with darker colour representing a ‘more positive’ or ‘more negative’ potential. Two views of TS-1 are shown from front and back.

Extended Data Fig. 9 Relative activity of NgnD and its site-specific mutants on enzymatic reactions.

Bars represent mean relative activity averaged over three reactions and error bars indicate the standard deviation. The experiments were independently repeated twice with similar results.

Extended Data Table 1 Data collection and refinement statistics (molecular replacement) of NgnD, StmD and 101015D

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

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