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Structural insight into antibiotic fosfomycin biosynthesis by a mononuclear iron enzyme

Nature volume 437, pages 838–844 (2005)Cite this article

Abstract

The biosynthetic pathway of the clinically important antibiotic fosfomycin uses enzymes that catalyse reactions without precedent in biology. Among these is hydroxypropylphosphonic acid epoxidase, which represents a new subfamily of non-haem mononuclear iron enzymes. Here we present six X-ray structures of this enzyme: the apoenzyme at 2.0 Å resolution; a native Fe(ii)-bound form at 2.4 Å resolution; a tris(hydroxymethyl)aminomethane–Co(ii)-enzyme complex structure at 1.8 Å resolution; a substrate–Co(ii)-enzyme complex structure at 2.5 Å resolution; and two substrate–Fe(ii)-enzyme complexes at 2.1 and 2.3 Å resolution. These structural data lead us to suggest how this enzyme is able to recognize and respond to its substrate with a conformational change that protects the radical-based intermediates formed during catalysis. Comparisons with other family members suggest why substrate binding is able to prime iron for dioxygen binding in the absence of α-ketoglutarate (a co-substrate required by many mononuclear iron enzymes), and how the unique epoxidation reaction of hydroxypropylphosphonic acid epoxidase may occur.

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Figure 1: Fosfomycin biosynthesis.
Figure 2: Overall structure of Fe( ii )-HppE.
Figure 3: HppE active sites displayed with 2 Fo - Fc maps contoured from 1.0–1.5σ.
Figure 4: Structural insights into catalysis.
Figure 5: Schematic of interactions of bidentate-bound S -HPP with protein, water and Fe( ii).
Figure 6: Possible mechanisms for HppE.

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References

  1. Liu, P. et al. Protein purification and functional assignment of the epoxidase catalyzing the formation of fosfomycin. J. Am. Chem. Soc. 123, 4619–4620 (2001)

    Article  CAS  PubMed  Google Scholar 

  2. Lobel, B. Short term therapy for uncomplicated urinary tract infection today. Clinical outcome upholds the theories. Int. J. Antimicrob. Agents 22, 85–87 (2003)

    Article  PubMed  Google Scholar 

  3. Nakazawa, H., Kikuchi, Y., Honda, T., Isago, T. & Nozaki, M. Enhancement of antimicrobial effects of various antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) by combination with fosfomycin. J. Infect. Chemother. 9, 304–309 (2003)

    Article  CAS  PubMed  Google Scholar 

  4. Cassone, M., Campanile, F., Pantosti, A., Venditti, M. & Stefani, S. Identification of a variant “Rome clone” of methicillin-resistant Staphylococcus aureus with decreased susceptibility to vancomycin, responsible for an outbreak in an intensive care unit. Microb. Drug Resist. 10, 43–49 (2004)

    Article  CAS  PubMed  Google Scholar 

  5. Hidaka, T. et al. Cloning and nucleotide sequence of fosfomycin biosynthetic genes of Streptomyces wedmorensis. Mol. Gen. Genet. 249, 274–280 (1995)

    Article  CAS  PubMed  Google Scholar 

  6. Hammerschmidt, F. Biosynthesis of natural-products with a P-C bond. 8. On the origin of the oxirane oxygen atom of fosfomycin in Streptomyces fradiae. J. Chem. Soc. Perkin Trans. 1 8, 1993–1996 (1991)

    Article  Google Scholar 

  7. Liu, P. et al. Biochemical and spectroscopic studies on (S)-2-hydroxypropylphosphonic acid epoxidase: a novel mononuclear non-heme iron enzyme. Biochemistry 42, 11577–11586 (2003)

    Article  CAS  PubMed  Google Scholar 

  8. Guengerich, F. P. Cytochrome P450 oxidations in the generation of reactive electrophiles: epoxidation and related reactions. Arch. Biochem. Biophys. 409, 59–71 (2003)

    Article  PubMed  Google Scholar 

  9. Costas, M., Mehn, M. P., Jensen, M. P. & Que, L. Jr Dioxygen activation at mononuclear nonheme iron active sites: enzymes, models, and intermediates. Chem. Rev. 104, 939–986 (2004)

    Article  CAS  PubMed  Google Scholar 

  10. Dunwell, J. M. Cupins: a new superfamily of functionally diverse proteins that include germins and plant storage proteins. Biotechnol. Genet. Eng. Rev. 15, 1–32 (1998)

    Article  CAS  PubMed  Google Scholar 

  11. Zhao, Z. et al. Mechanistic studies of HPP epoxidase: configuration of the substrate governs its enzymatic fate. Angew. Chem. Int. Edn Engl. 41, 4529–4532 (2002)

    Article  CAS  Google Scholar 

  12. Roach, P. L. et al. Structure of isopenicillin N synthase complexed with substrate and the mechanism of penicillin formation. Nature 387, 827–830 (1997)

    Article  ADS  CAS  PubMed  Google Scholar 

  13. Dunwell, J. M., Culham, A., Carter, C. E., Sosa-Aguirre, C. R. & Goodenough, P. W. Evolution of functional diversity in the cupin superfamily. Trends Biochem. Sci. 26, 740–746 (2001)

    Article  CAS  PubMed  Google Scholar 

  14. Dunwell, J. M., Purvis, A. & Khuri, S. Cupins: the most functionally diverse protein superfamily? Phytochemistry 65, 7–17 (2004)

    Article  CAS  PubMed  Google Scholar 

  15. Hausinger, R. P. Fe(II)/α-ketoglutarate-dependent hydroxylases and related enzymes. Crit. Rev. Biochem. Mol. Biol. 39, 21–68 (2004)

    Article  CAS  PubMed  Google Scholar 

  16. Liu, P. et al. Oxygenase activity in the self-hydroxylation of (S)-2-hydroxypropylphosphonic acid epoxidase involved in fosfomycin biosynthesis. J. Am. Chem. Soc. 126, 10306–10312 (2004)

    Article  CAS  PubMed  Google Scholar 

  17. Ryle, M. J. et al. O2- and α-ketoglutarate-dependent tyrosyl radical formation in TauD, an α-keto acid-dependent non-heme iron dioxygenase. Biochemistry 42, 1854–1862 (2003)

    Article  CAS  PubMed  Google Scholar 

  18. Roach, P. L., Schofield, C. J., Baldwin, J. E., Clifton, I. J. & Hajdu, J. Crystallization and preliminary X-ray diffraction studies on recombinant isopenicillin N synthase from Aspergillus nidulans. Protein Sci. 4, 1007–1009 (1995)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Valegård, K. et al. The structural basis of cephalosporin formation in a mononuclear ferrous enzyme. Nature Struct. Mol. Biol. 11, 95–101 (2004)

    Article  Google Scholar 

  20. Price, J. C., Barr, E. W., Tirupati, B., Bollinger, J. M. Jr & Krebs, C. The first direct characterization of a high-valent iron intermediate in the reaction of an α-ketoglutarate-dependent dioxygenase: a high-spin FeIV complex in taurine/α-ketoglutarate dioxygenase (TauD) from Escherichia coli. Biochemistry 42, 7497–7508 (2003)

    Article  CAS  PubMed  Google Scholar 

  21. Zhou, J. et al. Spectroscopic studies of substrate interactions with clavaminate synthase 2, a multifunctional α-KG-dependent non-heme iron enzyme: correlation with mechanisms and reactivities. J. Am. Chem. Soc. 123, 7388–7398 (2001)

    Article  CAS  PubMed  Google Scholar 

  22. Solomon, E. I. et al. Geometric and electronic structure/function correlations in non-heme iron enzymes. Chem. Rev. 100, 235–350 (2000)

    Article  CAS  PubMed  Google Scholar 

  23. Roach, P. L. et al. Anaerobic crystallisation of an isopenicillin N synthase.Fe(II).substrate complex demonstrated by X-ray studies. Eur. J. Biochem. 242, 736–740 (1996)

    Article  CAS  PubMed  Google Scholar 

  24. Hegg, E. L. & Que, L. Jr The 2-His-1-carboxylate facial triad—an emerging structural motif in mononuclear non-heme iron(II) enzymes. Eur. J. Biochem. 250, 625–629 (1997)

    Article  CAS  PubMed  Google Scholar 

  25. Brünger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  PubMed  Google Scholar 

  26. Terwilliger, T. C. Automated main-chain model building by template matching and iterative fragment extension. Acta Crystallogr. D 59, 38–44 (2003)

    Article  PubMed  Google Scholar 

  27. Terwilliger, T. C. Maximum-likelihood density modification. Acta Crystallogr. D 56, 965–972 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Navaza, J. AMoRe: an Automated Package for Molecular Replacement. Acta Crystallogr. A50, 164–182 (1994)

    Google Scholar 

  29. Kissinger, C. R., Gehlhaar, D. K. & Fogel, D. B. Rapid automated molecular replacement by evolutionary search. Acta Crystallogr. D 55, 484–491 (1999)

    Article  CAS  PubMed  Google Scholar 

  30. Laskowski, R., MacArthur, M., Moss, D. & Thornton, J. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993)

    Article  CAS  Google Scholar 

  31. DeLano, W. L. The PyMOL Molecular Graphics System (DeLano Scientific, San Carlos, California, 2002)

    Google Scholar 

Download references

Acknowledgements

This research is supported in part by the National Institutes of Health (C.L.D. and H-W.L.), the National Institute of Environmental Health Sciences (L.J.H.), the Searle Scholars Program (C.L.D.), Alfred P. Sloan Foundation (C.L.D.), and a Lester Wolfe Predoctoral Fellowship (L.J.H.). Synchrotron facilities are funded by the NIH National Center of Research Resources (Advanced Photon Source NE-CAT 8BM) and the US Department of Energy, Division of Materials Sciences and Division of Chemical Sciences (National Synchrotron Light Source, Brookhaven National Laboratory).

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Authors and Affiliations

  1. Department of Chemistry, Massachusetts Institute of Technology, Massachusetts, 02139, Cambridge, USA

    Luke J. Higgins, Pinghua Liu & Catherine L. Drennan

  2. Department of Chemistry and Biochemistry, University of Texas at Austin, Texas, 78712, Austin, USA

    Feng Yan, Pinghua Liu & Hung-wen Liu

Authors
  1. Luke J. Higgins
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Corresponding author

Correspondence to Catherine L. Drennan.

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Competing interests

Atomic coordinates for the following structures have been deposited in the Protein Data Bank: apo-HppE (1ZZ6), Fe(ii)-HppE (1ZZ9), form-1 S-HPP–Fe(ii)-HppE (1ZZ7), form-2 S-HPP–Fe(ii)-HppE (1ZZ8), Tris–Co(ii)-HppE (1ZZC) and S-HPP–Co(ii)-HppE (1ZZB). Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Methods

Site-directed mutagenesis, protein purification, assays, crystallization and data collection.

Supplementary Figure S1

Stereoviews of HppE active sites displayed with 2Fo-Fc maps contoured from 1.0-1.5ω .

Supplementary Figure S2

Structural insights into catalysis (stereoviews).

Supplementary Figure S3

Stereoviews of S-HPP-Co(II)-HppE active sites displayed with 2Fo-Fc maps contoured from 1.0-1.5ω.

Supplementary Tables

Supplementary Table S1 details data and refinement statistics for Co and Apo HppE structures. Supplementary Table S2 details data and refinement statistics for Fe HppE structures

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Higgins, L., Yan, F., Liu, P. et al. Structural insight into antibiotic fosfomycin biosynthesis by a mononuclear iron enzyme. Nature 437, 838–844 (2005). https://doi.org/10.1038/nature03924

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