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Mechanistic studies of an unprecedented enzyme-catalysed 1,2-phosphono-migration reaction

Nature volume 496, pages 114–118 (2013)Cite this article

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Abstract

(S)-2-hydroxypropylphosphonate ((S)-2-HPP) epoxidase (HppE) is a mononuclear non-haem-iron-dependent enzyme1,2,3 responsible for the final step in the biosynthesis of the clinically useful antibiotic fosfomycin4. Enzymes of this class typically catalyse oxygenation reactions that proceed via the formation of substrate radical intermediates. By contrast, HppE catalyses an unusual dehydrogenation reaction while converting the secondary alcohol of (S)-2-HPP to the epoxide ring of fosfomycin1,5. Here we show that HppE also catalyses a biologically unprecedented 1,2-phosphono migration with the alternative substrate (R)-1-HPP. This transformation probably involves an intermediary carbocation, based on observations with additional substrate analogues, such as (1R)-1-hydroxyl-2-aminopropylphosphonate, and model reactions for both radical- and carbocation-mediated migration. The ability of HppE to catalyse distinct reactions depending on the regio- and stereochemical properties of the substrate is given a structural basis using X-ray crystallography. These results provide compelling evidence for the formation of a substrate-derived cation intermediate in the catalytic cycle of a mononuclear non-haem-iron-dependent enzyme. The underlying chemistry of this unusual phosphono migration may represent a new paradigm for the in vivo construction of phosphonate-containing natural products that can be exploited for the preparation of new phosphonate derivatives.

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Figure 1: Fosfomycin biosynthetic pathway and HppE-catalysed conversion of various substrate analogues.
Figure 2: 1H NMR time-course for the HppE-catalysed conversion of (S)-7 to 8 (a) and (R)-7 to 9 (b).
Figure 3: Structures of (S)-7 and (R)-7 bound to the iron centre of HppE in a bidentate mode.
Figure 4: Summary of HppE-catalysed reactions and model chemistry examined in this study.
Figure 5: Possible mechanisms for the HppE-catalysed epoxidation of (S)-2-HPP (4) involving C1 cation formation (route a, red arrow) or oxygen-atom rebound (route b, blue arrows).

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Atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB) under accession codes 4J1W and 4J1X.

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Acknowledgements

We thank W. Johnson and S. Sorey for assistance with the NMR experiment setup. This work is supported in part by grants from The National Institutes of Health (GM040541 to H.-w.L.) and the Welch Foundation (F-1511). C.L.D. is a Howard Hughes Medical Institute Investigator. Crystallographic data collection were conducted at the Advanced Light Source, a Department of Energy (DOE) national user facility (Contract DE-AC02-05CH11231), at beamline 8.2.2 operated by the Berkeley Center for Structural Biology, which is supported in part by DOE and National Institutes of Health/National Institute of General Medical Sciences.

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Author notes

  1. Wei-chen Chang, Mishtu Dey, Pinghua Liu, Steven O. Mansoorabadi, Sung-Ju Moon & Zongbao K. Zhao

    Present address: Present addresses: Department of Chemistry, Penn State University, University Park, Pennsylvania 16802, USA (W.-c.C.); Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, USA (M.D.); Department of Chemistry, Boston University, Boston, Massachusetts 02215, USA (P.L.); Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama 36849, USA (S.O.M.); Agensys, Santa Monica, California 90404, USA (S.-J.M.); Department of Biotechnology, Dalian Institute of Chemical Physics, Dalian 116023, China (Z.K.Z.).,

  2. Mishtu Dey, Pinghua Liu and Steven O. Mansoorabadi: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Medicinal Chemistry, and Department of Chemistry and Biochemistry, College of Pharmacy, University of Texas at Austin, Austin, 78712, Texas, USA

    Wei-chen Chang, Pinghua Liu, Steven O. Mansoorabadi, Sung-Ju Moon, Zongbao K. Zhao & Hung-wen Liu

  2. Howard Hughes Medical Institute and the Departments of Chemistry and Biology, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA

    Mishtu Dey & Catherine L. Drennan

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Contributions

H.-w.L. provided the scientific direction and overall experimental design for the studies. W.-c.C. performed many of the experiments described herein. M.D. and C.L.D. collected and interpreted all of the crystallographic data. P.L., S.-J.M. and Z.K.Z. designed and carried out the initial biochemical as well as model system studies. S.O.M. performed the DFT calculations. W.-c.C., M.D., P.L., S.O.M., C.L.D. and H.-w.L. wrote the manuscript.

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Correspondence to Hung-wen Liu.

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The authors declare no competing financial interests.

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This file contains Supplementary Text and Data (see contents for details), Supplementary Figures 1-8, Supplementary Tables 1-2 and Supplementary References. (PDF 2019 kb)

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Chang, Wc., Dey, M., Liu, P. et al. Mechanistic studies of an unprecedented enzyme-catalysed 1,2-phosphono-migration reaction. Nature 496, 114–118 (2013). https://doi.org/10.1038/nature11998

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