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

Abstract

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 biosynthesis^3-6. [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 system^8,9. However, although enzyme-catalysed [6+4] cycloadditions have been proposed^10-12, 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 proposals^11,12. 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.

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 6–8 in mutants and in complementary strains. d, LC–MS analysis of the production for 6–8 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.

Fig. 1 |. Cycloadditions in natural product biosynthesis.

a, Proposed [6+4] and [4+2] cycloadditions involved in the biosynthesis of 1, and crystal structures of 6 and 8. b, HPLC analysis of metabolic extracts from S. seoulensis wild-type, mutant and complementary strains. c, LC–MS analysis of in vitro enzyme-catalysed [6+4]/[4+2] bispericyclic reactions. EIC, extracted ion chromatography. TE* indicates the StmC/ACP-TE. The experiments were independently repeated three times with similar results.

Fig. 2 |. DFT-computed free energies for the [6+4], [4+2] and [3,3]- Cope reactions.

a, Reaction of 3 via TS-1 to give exo-[6+4] and [4+2] products 6 and 7, and the interconversion between 6 and 7 through the Cope rearrangement transition state TS-2. b, Reaction of 3 via TS-3 to give endo-[6+4] and [4+2] products 8 and 9, and the conversion from 9 to 8 through the Cope rearrangement transition state TS-4. The numbers in parentheses show Gibbs free energies in kcal mol⁻¹, computed with CPCM(water)-M06–2X/6–311+G(d,p)//B3LYP-D3/6–31G(d).

Fig. 3 |. Crystal structures of three enzymes and catalytic sites for the bispericyclic reaction.

a, Crystal structures of NgnD, StmD and 101015D. b, The superimposed image of the three homologues. c, Ten residues of NgnD are close to DFT-optimized transition state TS-1. d, Interactions between TS-1 and M69 and Y55 of NgnD. e, Interactions between TS-1 and Y13 and W67 of NgnD.