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

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.

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.

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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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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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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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