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Unexpected enzyme-catalysed [4+2] cycloaddition and rearrangement in polyether antibiotic biosynthesis


Enzymes that catalyse remarkable Diels–Alder-like [4+2] cyclizations have been previously implicated in the biosynthesis of spirotetronate and spirotetramate antibiotics. Biosynthesis of the polyether antibiotic tetronasin is not expected to require such steps, yet the tetronasin gene cluster encodes enzymes Tsn11 and Tsn15, which are homologous to authentic [4+2] cyclases. Here, we show that deletion of Tsn11 led to accumulation of a late-stage intermediate, in which the two central rings of tetronasin and four of its twelve asymmetric centres remain unformed. In vitro reconstitution showed that Tsn11 catalyses an apparent inverse-electron-demand hetero-Diels–Alder-like [4+2] cyclization of this species to form an unexpected oxadecalin compound that is then rearranged by Tsn15 to form tetronasin. To gain structural and mechanistic insight into the activity of Tsn15, the crystal structure of a Tsn15-substrate complex has been solved at 1.7 Å resolution.

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Fig. 1: [4+2] cyclases in polyether tetronate biosynthesis.
Fig. 2: Functional characterization of the Diels-Alderase homologues Tsn11 and Tsn15 in tetronasin biosynthesis.
Fig. 3: The crystal structure of Tsn15.
Fig. 4: Structural homologues of Tsn15 and their respective reactions.
Fig. 5: Structure of Tsn15 and a Tsn15-substrate complex.
Fig. 6: The proposed mechanism for formation of the cyclohexane and tetrahydropyran rings of tetronasin.

Data availability

The tetronasin biosynthetic gene cluster sequence is available on GenBank (accession code: FJ462704). The crystal structure data is available on the PDB (accession codes: 6NOI (Tsn15) and 6NNW (Tsn15-substrate complex)). All other data that supports the findings of this study are available from the corresponding author on reasonable request.


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R.L was supported by the Woolf Fisher Trust and the Cambridge Commonwealth European and International Trust. M.V.B.D was supported by the São Paulo Research Foundation under grant nos 2015/09188-8, 2017/50140-4 and 2018/00351-1. F.C.R.P was supported by a CNPq (National Council for Scientific and Technological Development) fellowship (141090/2016-2). M.T and R.J gratefully acknowledge EPSRC (DTA PhD studentship to R.J.); BBSRC (project grant no. BB/J007250/1 to M.T.) and the FAPESP–Warwick Joint Fund (for a SPRINT award to M.V.B.D. and M.T.).

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R.L., P.F.L., F.J.L., M.T. and M.V.B.D. developed the hypothesis and designed the study. Y.D., Y.S. and M.S. cloned, sequenced and analysed the gene clusters. R.L., Y.S. and H.H. performed gene deletions, whereas M.T. and R.J. performed and analysed the experiments with chain-terminating probes. R.L. performed protein expressions and purifications, in vitro experiments and compound isolations. R.L. and F.J.L. performed compound characterizations. F.C.R.P., R.L. and M.V.B.D. solved the crystal structures. All authors analysed and discussed the results. P.F.L., R.L. and F.C.R.P. prepared the manuscript.

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Correspondence to Peter F. Leadlay.

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

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