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Crystal structure and mechanism of a bacterial fluorinating enzyme

Abstract

Fluorine is the thirteenth most abundant element in the earth's crust, but fluoride concentrations in surface water are low and fluorinated metabolites are extremely rare1,2. The fluoride ion is a potent nucleophile in its desolvated state, but is tightly hydrated in water and effectively inert. Low availability and a lack of chemical reactivity have largely excluded fluoride from biochemistry: in particular, fluorine's high redox potential precludes the haloperoxidase-type mechanism3,4 used in the metabolic incorporation of chloride and bromide ions. But fluorinated chemicals are growing in industrial importance, with applications in pharmaceuticals, agrochemicals and materials products5,6,7. Reactive fluorination reagents requiring specialist process technologies are needed in industry and, although biological catalysts for these processes are highly sought after, only one enzyme that can convert fluoride to organic fluorine has been described8. Streptomyces cattleya can form carbon–fluorine bonds9 and must therefore have evolved an enzyme able to overcome the chemical challenges of using aqueous fluoride. Here we report the sequence and three-dimensional structure of the first native fluorination enzyme, 5′-fluoro-5′-deoxyadenosine synthase, from this organism. Both substrate and products have been observed bound to the enzyme, enabling us to propose a nucleophilic substitution mechanism for this biological fluorination reaction.

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Figure 1: 5′-FDAS from S. cattleya catalyses the formation of 5′-FDA from SAM and an F- ion.
Figure 2: Structure of 5′-FDAS, drawn by PyMOL31.
Figure 3: Fo–Fc electron density maps with phases calculated from models that do not include ligand.
Figure 4: Representation of 5′-FDA and methionine bound to the enzyme, showing hydrogen-bonding to the fluoromethyl group from Ser 158, and the anti relationship between the C–F bond (red) and the disconnected C–S bond (dotted red) of SAM that is indicative of an SN2 reaction course.

References

  1. O'Hagan, D. & Harper, D. B. Fluorine-containing natural products. J. Chem. 100, 127–133 (1999)

    CAS  Google Scholar 

  2. Xu, X.-H. et al. 5-Fluorouracil derivatives from the sponge Phakellia fusca. J. Nat. Prod. 66, 285–288 (2003)

    CAS  Article  Google Scholar 

  3. vanPee, K. H. Biosynthesis of halogenated metabolites by bacteria. Annu. Rev. Microbiol. 50, 375–399 (1996)

    CAS  Article  Google Scholar 

  4. Littlechild, J. Haloperoxidases and their role in biotransformation reactions. Curr. Opin. Chem. Biol. 3, 28–34 (1999)

    CAS  Article  Google Scholar 

  5. Sandford, G. Organofluorine chemistry. Phil. Trans. R. Soc. Lond. A 358, 455–471 (2000)

    ADS  CAS  Article  Google Scholar 

  6. Mann, J. Modern methods for the introduction of fluorine into organic molecules—an approach to compounds with altered chemical and biological activities. Chem. Soc. Rev. 16, 381–436 (1987)

    ADS  CAS  Article  Google Scholar 

  7. Hutchinson, J. & Sandford, G. Elemental fluorine in organic chemistry. Top. Curr. Chem. 193, 1–43 (1997)

    CAS  Article  Google Scholar 

  8. Zechel, D. L. et al. Enzymatic synthesis of carbon–fluorine bonds. J. Am. Chem. Soc. 123, 4350–4351 (2001)

    CAS  Article  Google Scholar 

  9. Sanada, M. et al. Biosynthesis of fluorothreonine and fluoroacetic acid by the thienamycin producer, Streptomyces cattleya. J. Antibiotics 141, 259–265 (1986)

    Article  Google Scholar 

  10. O'Hagan, D., Schaffrath, C., Cobb, S. L., Hamilton, J. T. G. & Murphy, C. D. Enzyme catalysed organofluorine synthesis. Nature 416, 279 (2002)

    ADS  CAS  Article  Google Scholar 

  11. Schaffrath, C., Deng, H. & O'Hagan, D. Isolation and characterisation of 5′-fluorodeoxyadenosine synthetase, a fluorination enzyme from Streptomyces cattleya. FEBS Lett. 547, 111–114 (2003)

    CAS  Article  Google Scholar 

  12. Boutselakis, H. et al. E-MSD: the European Bioinformatics Institute Macromolecular Structure Database. Nucleic Acids Res. 31, 458–462 (2003)

    CAS  Article  Google Scholar 

  13. Holm, L. & Sander, C. Protein folds and families: sequence and structure alignments. Nucleic Acids Res. 27, 244–247 (1999)

    CAS  Article  Google Scholar 

  14. Bateman, A. et al. The Pfam Protein Families Database. Nucleic Acids Res. 30, 276–280 (2002)

    CAS  Article  Google Scholar 

  15. Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997)

    CAS  Article  Google Scholar 

  16. Allen, F. H. The Cambridge Structural Database: a quarter of a million crystal structures and rising. Acta Crystallogr. B 58, 380–388 (2002)

    Article  Google Scholar 

  17. Berman, H. M. et al. The Protein Data Bank. Acta Crystallogr. D 58, 899–907 (2002)

    Article  Google Scholar 

  18. Kollman, P. A. et al. Calculating structures and free energies of complex molecules: combining molecular mechanics and continuum models. Acc. Chem. Res. 33, 889–897 (2000)

    CAS  Article  Google Scholar 

  19. O'Hagan, D. et. al. An assay for the enantiomeric assay of [2H1]-fluoroacetic acid: Insight into the stereochemical course of fluorination during fluorometabolite biosynthesis in Streptomyces cattleya. J. Am. Chem. Soc. 125, 379–387 (2003)

    CAS  Article  Google Scholar 

  20. Dunitz, J. & Taylor, R. Organic fluorine hardly ever accepts hydrogen bonds. Chem. Eur. J. 3, 89–98 (1997)

    CAS  Article  Google Scholar 

  21. Howard, J. A. K., Hoy, J. V., O'Hagan, D. & Smith, G. T. How good is fluorine as a hydrogen bond acceptor? Tetrahedron 38, 12613–12622 (1996)

    Article  Google Scholar 

  22. Smart, O. S., Neduvelil, J. G., Wang, X., Wallace, B. A. & Sansom, M. S. P. HOLE: A program for the analysis of the pore dimensions of ion channel structural models. J. Mol. Graph. Model 14, 354–360 (1996)

    CAS  Article  Google Scholar 

  23. Dong, C. et al. Crystallization and X-ray diffraction of the 5′-fluoro-5′-deoxyadenosine synthase, a fluorination enzyme from Streptomyces cattleya. Acta Crystallogr. D 60, 760–763 (2003)

    Google Scholar 

  24. Doublie, S. Preparation of selenomethionyl proteins for phase determination. Methods Enzymol. 276, 523–530 (1997)

    CAS  Article  Google Scholar 

  25. Bailey, S. The CCP4 Suite—programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Article  Google Scholar 

  26. Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D 55, 849–861 (1999)

    CAS  Article  Google Scholar 

  27. Morris, R. J., Perrakis, A. & Lamzin, V. S. ARP/wARP's model-building algorithms. I. The main chain. Acta Crystallogr. D 58, 968–975 (2002)

    Article  Google Scholar 

  28. Murshudov, G. N., Vagin, A. A., Lebedev, A., Wilson, K. S. & Dodson, E. J. Efficient anisotropic refinement of macromolecular structures using FFT. Acta Crystallogr. D 55, 247–255 (1999)

    CAS  Article  Google Scholar 

  29. van Aalten, D. M. F. et al. PRODRG, a program for generating molecular topologies and unique molecular descriptors from coordinates of small molecules. J. Comput. Aided Mol. Des. 10, 255–262 (1996)

    ADS  CAS  Article  Google Scholar 

  30. Schaffrath, C., Cobb, S. L. & O'Hagan, D. Cell-free biosynthesis of fluoroacetate and 4-fluorothreonine in Streptomyces cattleya. Angew. Chem. Int. Edn Engl. 41, 3913–3915 (2002)

    CAS  Article  Google Scholar 

  31. DeLano, W. L., The PyMOL Molecular Graphics Systemhttp://www.pymol.org/〉 (2003).

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Acknowledgements

We thank M. Dorward for technical assistance and G. Leonard for help with data collection. J.H.N. is a Biotechnology and Biological Sciences Research Council (BBSRC) Career Development Fellow; H.D. was supported by the BBSRC; D.O.H. thanks the BBSRC for financial support; J.H.N. thanks the Wellcome Trust.

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Correspondence to James H. Naismith.

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Dong, C., Huang, F., Deng, H. et al. Crystal structure and mechanism of a bacterial fluorinating enzyme. Nature 427, 561–565 (2004). https://doi.org/10.1038/nature02280

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