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The structure of SpnF, a standalone enzyme that catalyzes [4 + 2] cycloaddition

Abstract

In the biosynthetic pathway of the spinosyn insecticides, the tailoring enzyme SpnF performs a [4 + 2] cycloaddition on a 22-membered macrolactone to forge an embedded cyclohexene ring. To learn more about this reaction, which could potentially proceed through a Diels-Alder mechanism, we determined the 1.50-Å-resolution crystal structure of SpnF bound to S-adenosylhomocysteine. This sets the stage for advanced experimental and computational studies to determine the precise mechanism of SpnF-mediated cyclization.

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Figure 1: Reactions mediated by SpnF and accompanying tailoring enzymes.
Figure 2: SpnF structure and consensus docking of its substrate and product.
Figure 3: Cyclization assays of SpnF mutants.

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Acknowledgements

We thank M. Ruszczycky for helpful discussions. We thank K. Diederichs for assistance in processing diffraction data. We thank the National Institutes of Health (GM106112 to A.T.K.-C. and GM035906 and GM040541 to H.L.) and the Welch Foundation (F-1712 to A.T.K.-C. and F-1511 to H.L.) for financial support. Instrumentation and technical assistance for this work were provided by the Macromolecular Crystallography Facility, with financial support from the College of Natural Sciences, the Office of the Executive Vice President and Provost and the Institute for Cellular and Molecular Biology at the University of Texas at Austin. Use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy Office of Science by Argonne National Laboratory, was supported by the US DOE under contract no. DE-AC02-06CH11357.

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Contributions

C.D.F. and E.A.I. performed the crystallography and generated mutants for activity assays. C.D.F. managed the consensus docking studies. D.T.W. assessed the stability of SpnF mutants. Y.L. conducted the cyclization assays. All authors analyzed and discussed the results. C.D.F., E.A.I., H.L. and A.T.K.-C. prepared the manuscript.

Corresponding authors

Correspondence to Hung-wen Liu or Adrian T Keatinge-Clay.

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The authors declare no competing financial interests.

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Supplementary Results, Supplementary Figures 1–7 and Supplementary Tables 1 and 2. (PDF 20983 kb)

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Fage, C., Isiorho, E., Liu, Y. et al. The structure of SpnF, a standalone enzyme that catalyzes [4 + 2] cycloaddition. Nat Chem Biol 11, 256–258 (2015). https://doi.org/10.1038/nchembio.1768

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