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Structural basis for stereoselective dehydration and hydrogen-bonding catalysis by the SAM-dependent pericyclase LepI

Nature Chemistry volume 11, pages 812–820 (2019)Cite this article

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Abstract

LepI is an S-adenosylmethionine (SAM)-dependent pericyclase that catalyses the formation of the 2-pyridone natural product leporin C. Biochemical characterization has shown that LepI can catalyse stereoselective dehydration to yield a reactive (E)-quinone methide that can undergo bifurcating intramolecular Diels–Alder (IMDA) and hetero-Diels–Alder (HDA) cyclizations from an ambimodal transition state, as well as a [3,3]-retro-Claisen rearrangement to recycle the IMDA product into leporin C. Here, we solve the X-ray crystal structures of SAM-bound LepI and in complex with a substrate analogue, the product leporin C, and a retro-Claisen reaction transition-state analogue to understand the structural basis for the multitude of reactions. Structural and mutational analysis reveals how nature evolves a classic methyltransferase active site into one that can serve as a dehydratase and a multifunctional pericyclase. Catalysis of both sets of reactions employs H133 and R295, two active-site residues that are not found in canonical methyltransferases. An alternative role of SAM, which is not found to be in direct contact with the substrate, is also proposed.

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Fig. 1: Leporin C biosynthesis pathway highlighting the LepI-catalysed reaction cascade.
Fig. 2: LepI structure and the SAM binding site.
Fig. 3: Crystal structure of the LepI pseudo enzyme–substrate complex and the enzyme–product complex.
Fig. 4: LepI mutant activity.
Fig. 5: Proposed catalytic mechanism of LepI.

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Data availability

The data that support the findings of this study are available within this Article and its Supplementary Information, or are available from the corresponding authors upon reasonable request. The structural factors and coordinates of the LepI–SAM complex, LepI–SAM-1 complex, LepI (C52A)–SAH-1 complex, LepI (C52A)–SAM-8 complex and LepI (C52A)–SAM-10 complex have been deposited in the Protein Data Bank under ID nos. 6IX3, 6IX5, 6IX7, 6IX8 and 6IX9, respectively.

References

  1. Nicolaou, K. C., Snyder, S. A., Montagnon, T. & Vassilikogiannakis, G. The Diels–Alder reaction in total synthesis. Angew. Chem. Int. Ed. 41, 1668–1698 (2002).

    Article  CAS  Google Scholar 

  2. Cooper, S. J., Ohashi, M., Liu, F., Tang, Y. & Houk, K. N. The expanding world of biosynthetic pericyclases: cooperation of experiment and theory for discovery. Nat. Prod. Rep. 36, 698–713 (2018).

    Google Scholar 

  3. Chook, Y. M., Ke, H. & Lipscomb, W. N. Crystal structures of the monofunctional chorismate mutase from Bacillus subtilis and its complex with a transition state analog. Proc. Natl Acad. Sci. USA 90, 8600–8603 (1993).

    Article  CAS  Google Scholar 

  4. Fage, C. D. et al. The structure of SpnF, a standalone enzyme that catalyzes [4+2] cycloaddition. Nat. Chem. Biol. 11, 256–258 (2015).

    Article  CAS  Google Scholar 

  5. Zaitseva, J., Lu, J., Olechoski, K. L. & Lamb, A. L. Two crystal structures of the isochorismate pyruvate lyase from Pseudomonas aeruginosa. J. Biol. Chem. 281, 33441–33449 (2006).

    Article  CAS  Google Scholar 

  6. Shipman, L. W., Li, D., Roessner, C. A., Scott, A. I. & Sacchettini, J. C. Crystal structure of precorrin-8x methyl mutase. Structure 9, 587–596 (2001).

    Article  CAS  Google Scholar 

  7. Zheng, Q. et al. Enzyme-dependent [4+2] cycloaddition depends on lid-like interaction of the N-terminal sequence with the catalytic core in Pyrl4. Cell Chem. Biol. 23, 352–360 (2016).

    Article  CAS  Google Scholar 

  8. Byrne, M. J. et al. The catalytic mechanism of a natural Diels–Alderase revealed in molecular detail. J. Am. Chem. Soc. 138, 6095–6098 (2016).

    Article  CAS  Google Scholar 

  9. Cogan, D. P. et al. Structural insights into enzymatic [4+2] aza-cycloaddition in thiopeptide antibiotic biosynthesis. Proc. Natl Acad. Sci. USA 114, 12928–12933 (2017).

    Article  CAS  Google Scholar 

  10. Zheng, Q. et al. Structural insights into a flavin-dependent [4+2] cyclase that catalyzes trans-decalin formation in pyrroindomycin biosynthesis. Cell Chem. Biol. 25, 718–727 (2018).

    Article  CAS  Google Scholar 

  11. Newmister, S. A. et al. Structural basis of the Cope rearrangement and cyclization in hapalindole biogenesis. Nat. Chem. Biol. 14, 345–351 (2018).

    Article  CAS  Google Scholar 

  12. Ohashi, M. et al. SAM-dependent enzyme-catalysed pericyclic reactions in natural product biosynthesis. Nature 549, 502–506 (2017).

    Article  Google Scholar 

  13. Liscombe, D. K., Louie, G. V. & Noel, J. P. Architectures, mechanisms and molecular evolution of natural product methyltransferases. Nat. Prod. Rep. 29, 1238–1250 (2012).

    Article  CAS  Google Scholar 

  14. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    Article  CAS  Google Scholar 

  15. Newmister, S. A. et al. Unveiling sequential late-stage methyltransferase reactions in the meleagrin/oxaline biosynthetic pathway. Org. Biomol. Chem. 16, 6450–6459 (2018).

    Article  CAS  Google Scholar 

  16. Holm, L. & Rosenström, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, w545–w549 (2010).

    Article  CAS  Google Scholar 

  17. Horowitz, S. et al. Conservation and functional importance of carbon–oxygen hydrogen bonding in AdoMet-dependent methyltransferases. J. Am. Chem. Soc. 135, 15536–15548 (2013).

    Article  CAS  Google Scholar 

  18. Fick, R. J. et al. Water-mediated carbon–oxygen hydrogen bonding facilitates S-adenosylmethionine recognition in the reactivation domain of cobalamin-dependent methionine synthase. Biochemistry 57, 3733–3740 (2018).

    Article  CAS  Google Scholar 

  19. Jeon, B.-S., Wang, S.-A., Ruszczycky, M. W. & Liu, H.-W. Natural [4+2]-cyclases. Chem. Rev. 117, 5367–5388 (2017).

    Article  CAS  Google Scholar 

  20. Preiswerk, N. et al. Impact of scaffold rigidity on the design and evolution of an artificial Diels–Alderase. Proc. Natl Acad. Sci. USA 111, 8013–8018 (2014).

    Article  CAS  Google Scholar 

  21. Minami, A. & Oikawa, H. Recent advances of Diels–Alderases involved in natural product biosynthesis. J. Antibiot. 69, 500–506 (2016).

    Article  CAS  Google Scholar 

  22. Breslow, R. Hydrophobic effects on simple organic reactions in water. Acc. Chem. Res. 24, 159–164 (1991).

    Article  CAS  Google Scholar 

  23. Blokzijl, W., Blandamer, M. J. & Engberts, J. B. F. N. Diels–Alder reactions in aqueous solutions. Enforced hydrophobic interactions between diene and dienophile. J. Am. Chem. Soc. 113, 4241–4246 (1991).

    Article  CAS  Google Scholar 

  24. Lee, A. Y., Stewart, J. D., Clardy, J. & Ganem, B. New insight into the catalytic mechanism of chorismite mutase from structural studies. Chem. Biol. 2, 195–203 (1995).

    Article  CAS  Google Scholar 

  25. Burschowsky, D. et al. Electrostatic transition state stabilization rather than reactant destabilization provides the chemical basis for efficient chorismate mutase catalysis. Proc. Natl Acad. Sci. USA 111, 17516–17521 (2014).

    Article  CAS  Google Scholar 

  26. Warshel, A. et al. Electrostatic basis for enzyme catalysis. Chem. Rev. 106, 3210–3235 (2006).

    Article  CAS  Google Scholar 

  27. Smith, P. J. & Wilcox, C. S. The chemistry of functional group arrays. Electrostatic catalysis and the ‘intramolecular salt effect’. Tetrahedron 47, 2617–2628 (1991).

    Article  CAS  Google Scholar 

  28. Fried, S. D. & Boxer, S. G. Electric fields and enzyme catalysis. Annu. Rev. Biochem. 86, 387–415 (2017).

    Article  CAS  Google Scholar 

  29. Shaik, S., Mandal, D. & Ramanan, R. Oriented electric fields as future smart reagents in chemistry. Nat. Chem. 8, 1091–1098 (2016).

    Article  CAS  Google Scholar 

  30. Aragonès, A. C. et al. Electrostatic catalysis of a Diels–Alder reaction. Nature 531, 88–91 (2016).

    Article  Google Scholar 

  31. Welborn, V. V., Ruiz, P. L. & Head-Gordon, T. Computational optimization of electric fields for better catalysis design. Nat. Catal. 1, 649–655 (2018).

    Article  Google Scholar 

  32. Yu, J., Zhou, Y., Tanaka, I. & Yao, M. Roll: a new algorithm for the detection of protein pockets and cavities with a rolling probe sphere. Bioinformatics 26, 46–52 (2010).

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the NSFC (91856202), CAS (XDB20000000) and SMSTC (18430721500, 19XD1404800) (J.Z.), the NIH (1R01AI141481) and NSF (CHE-1806581) (Y.T. and K.N.H.). Chemical characterization studies were supported by shared instrumentation grants from the NSF (CHE-1048804) and NIH NCRR (S10RR025631). The authors thank the staff of beamlines BL17U1, BL18U1 and BL19U1 of Shanghai Synchrotron Radiation Facility for access and help with the X-ray data collection. The authors also thank J. Gan for help with structure refinement and J. Long for help with ultracentrifugation sedimentation measurements. The computational resources from the UCLA Institute of Digital Research and Education (IDRE) are acknowledged. M.O. is supported by an overseas postdoctoral fellowship from The Uehara Memorial Foundation, Japan. Y.H. is a Life Sciences Research Foundation fellow sponsored by the Mark Foundation for Cancer Research. The authors thank E.B. Go for careful proofreading of the manuscript.

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Author notes

  1. These authors contributed equally: Yujuan Cai, Yang Hai, Masao Ohashi.

Authors and Affiliations

  1. State Key Laboratory of Bio-organic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Shanghai, China

    Yujuan Cai & Jiahai Zhou

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

    Yang Hai, Masao Ohashi, Cooper S. Jamieson, Marc Garcia-Borras, K. N. Houk & Yi Tang

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Contributions

Y.C., Y.H., M.O., K.N.H., J.Z. and Y.T. developed the hypothesis and designed the study. Y.C. and Y.H. purified and crystallized protein samples and solved the X-ray structures. M.O. and Y.H. performed in vivo and in vitro experiments. M.O. performed compound isolation and characterization. C.S.J. and M.G.-B. performed the computational analysis. All authors analysed and discussed the results. Y.C., Y.H., K.N.H., J.Z. and Y.T. prepared the manuscript. Y.C., Y.H. and M.O. contributed equally to this work.

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Correspondence to K. N. Houk, Jiahai Zhou or Yi Tang.

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The Supplementary Information file includes methods, Supplementary tables and Supplementary figures

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Cai, Y., Hai, Y., Ohashi, M. et al. Structural basis for stereoselective dehydration and hydrogen-bonding catalysis by the SAM-dependent pericyclase LepI. Nat. Chem. 11, 812–820 (2019). https://doi.org/10.1038/s41557-019-0294-x

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