Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Mechanistic considerations of halogenating enzymes

Abstract

In nature, halogenation is a strategy used to increase the biological activity of secondary metabolites, compounds that are often effective as drugs. However, halides are not particularly reactive unless they are activated, typically by oxidation. The pace of discovery of new enzymes for halogenation is increasing, revealing new metalloenzymes, flavoenzymes, S-adenosyl-L-methionine (SAM)-dependent enzymes and others that catalyse halide oxidation using dioxygen, hydrogen peroxide and hydroperoxides, or that promote nucleophilic halide addition reactions.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Halogenated natural products.
Figure 2: Biologically relevant halogenation reactions.
Figure 3: Structure of the active site of haem CPO from C. fumago, with a bound substrate, 1,3-cyclopentanedione25.
Figure 4: Proposed catalytic cycle for haem CPOs24,25,26,27.
Figure 5: Structure of the active site of V-CPO from C. inaequalis35.
Figure 6: Mechanistic considerations of V-HPOs.
Figure 7: Proposed biosynthesis of the napyradiomycins A80915A–A80915D30.
Figure 8: Bromination of 3-oxo-hexanoyl homoserine lactone and subsequent hydrolysis of the dibromo product60.
Figure 9: Structure of the active site of FeNH–αKG in SyrB2 from P. syringae45.
Figure 10: Proposed catalytic cycle of FeNH–αKG halogenases.
Figure 11: Examples of FeNH–αKG halogenase reactions.
Figure 12: Parallel assemblies in the biosynthesis of curacin A and jamaicamide A.

Similar content being viewed by others

References

  1. Faulkner, D. J. Marine natural products. Nat. Prod. Rep. 19, 1–48 (2002).

    CAS  PubMed  Google Scholar 

  2. Gribble, G. W. Naturally occurring organohalogen compounds. Acc. Chem. Res. 31, 141–152 (1998).

    Article  CAS  Google Scholar 

  3. Carpenter, L. J. & Liss, P. S. On temperate sources of bromoform and other reactive bromine gases. J. Geophys. Res. 105, 20539–20547 (2002).

    Article  ADS  Google Scholar 

  4. Hager, L. P., Morris, D. R., Brown, F. S. & Eberwein, H. Chloroperoxidase. II. Utilization of halogen anions. J. Biol. Chem. 241, 1769–1777 (1966).

    CAS  PubMed  Google Scholar 

  5. Fenical, W. Halogenation in the Rhodophyta: a review. J. Phycol. 11, 245–259 (1975).

    CAS  Google Scholar 

  6. Wolinsky, L. E. & Faulkner, D. J. A biomimetic approach to the synthesis of Laurencia metabolites. Synthesis of 10-bromo-α-chamigrene. J. Org. Chem. 41, 597–600 (1976).

    Article  CAS  Google Scholar 

  7. Manthey, J. A. & Hager, L. P. Characterization of the catalytic properties of bromoperoxidase. Biochemistry 28, 3052–3057 (1989).

    Article  CAS  Google Scholar 

  8. Manthey, J. A. & Hager, L. P. Characterization of the oxidized states of bromoperoxidase. J. Biol. Chem. 260, 9654–9659 (1985).

    CAS  PubMed  Google Scholar 

  9. Roach, M. P. et al. Notomastus lobatus chloroperoxidase and Amphitrite ornata dehaloperoxidase both contain histidine as their proximal heme iron ligand. Biochemistry 36, 2197–2202 (1997).

    Article  CAS  Google Scholar 

  10. Vilter, H. Peroxidases from Phaeophyceae. III. Catalysis of halogenation by peroxidases from Ascophyllum nodosum (L.) Le Jol. Bot. Mar. 26, 429–435 (1983).

    CAS  Google Scholar 

  11. Vilter, H. Peroxidases from Phaeophyceae. A vanadium(v)-dependent peroxidase from Ascophyllum nodosum . Phytochemistry 23, 1387–1390 (1984).

    Article  CAS  Google Scholar 

  12. Wever, R., Plat, H. & De Boer, E. Isolation procedure and some properties of the bromoperoxidase from the seaweed Ascophyllum nodosum . Biochim. Biophys. Acta 830, 181–186 (1985).

    Article  CAS  Google Scholar 

  13. Vaillancourt, F. H., Yin, J. & Walsh, C. T. SyrB2 in syringomycin E biosynthesis is a nonheme FeII α-ketoglutarate- and O2-dependent halogenase. Proc. Natl Acad. Sci. USA 102, 10111–10116 (2005).

    Article  ADS  CAS  Google Scholar 

  14. Chen, X. & van Pée, K.-H. Catalytic mechanisms, basic roles, and biotechnological and environmental significance of halogenating enzymes. Acta Biochim. Biophys. Sin. (Shanghai) 40, 183–193 (2008).

    Article  CAS  Google Scholar 

  15. Deng, H. & O'Hagan, D. The fluorinase, the chlorinase and the duf-2 enzymes. Curr. Opin. Chem. Biol. 12, 582–592 (2008).

    Article  CAS  Google Scholar 

  16. Wuosmaa, A. M. & Hager, L. P. Methyl chloride transferase: a carbocation route for biosynthesis of halometabolites. Science 249, 160–162 (1990).

    Article  ADS  CAS  Google Scholar 

  17. Blasiak, L. C. & Drennan, C. L. Structural perspective on enzymatic halogenation. Acc. Chem. Res. 42, 147–155 (2009).

    Article  CAS  Google Scholar 

  18. Wagner, C., Omari, E. M. & König, G. M. Biohalogenation: nature's way to synthesize halogenated metabolites. J. Nat. Prod. 72, 540–553 (2009).

    Article  CAS  Google Scholar 

  19. Neumann, C. S., Fujimori, D. G. & Walsh, C. T. Halogenation strategies in natural product biosynthesis. Chem. Biol. 15, 99–109 (2008).

    Article  CAS  Google Scholar 

  20. Fujimori, D. G. & Walsh, C. T. What's new in enzymatic halogenations. Curr. Opin. Chem. Biol. 11, 553–560 (2007).

    Article  CAS  Google Scholar 

  21. Littlechild, J., Rodriguez, E. G. & Isupov, M. Vanadium containing bromoperoxidase — insights into the enzymatic mechanism using X-ray crystallography. J. Inorg. Biochem. 103, 617–621 (2009).

    Article  CAS  Google Scholar 

  22. Vaillancourt, F. H., Yeh, E., Vosburg, D. A., Garneau-Tsodikova, S. & Walsh, C. T. Nature's inventory of halogenation catalysts: oxidative strategies predominate. Chem. Rev. 106, 3364–3378 (2006). This review summarizes the halogenating enzymes with particular insight into the Fe NH –αKG halogenases.

    Article  CAS  Google Scholar 

  23. Butler, A. & Carter-Franklin, J. N. A role for vanadium bromoperoxidase in the biosynthesis of halogenated marine natural products. Nat. Prod. Rep. 21, 180–188 (2004).

    Article  CAS  Google Scholar 

  24. Sundaramoorthy, M., Terner, J. & Poulos, T. L. The crystal structure of chloroperoxidase: a heme peroxidase–cytochrome P450 functional hybrid. Structure 3, 1367–1378 (1995).

    Article  CAS  Google Scholar 

  25. Kuhnel, K., Blankenfeldt, W., Terner, J. & Schlinchting, I. Crystal structures of chloroperoxidase with its bound substrates and complexed with formate, acetate and nitrate. J. Biol. Chem. 281, 23990–23998 (2006).

    Article  Google Scholar 

  26. Wagenknecht, H.-A. & Wolf-Dietrich, W. Identification of intermediates in the catalytic cycle of chloroperoxidase. Chem. Biol. 4, 367–372 (1997).

    Article  CAS  Google Scholar 

  27. Libby, R. D., Beachy, T. M. & Phipps, A. K. Quantitating direct chlorine transfer from enzyme to substrate in chloroperoxidase-catalyzed reactions. J. Biol. Chem. 271, 21820–21827 (1996).

    Article  CAS  Google Scholar 

  28. Reddy, C. M. et al. A chlorine isotope effect for enzyme-catalyzed chlorination. J. Am. Chem. Soc. 124, 14526–14527 (2002). This paper established 35Cl/37Cl isotope fractionation for the first time in a haloperoxidase during turnover.

    Article  CAS  Google Scholar 

  29. Van Schijndel, J. W. P. M., Vollenbroek, E. G. M. & Wever, R. The chloroperoxidase from the fungus Curvularia inaequalis: a novel vanadium enzyme. Biochim. Biophys. Acta 1161, 249–256 (1993).

    Article  CAS  Google Scholar 

  30. Winter, J. M. et al. Molecular basis for chloronium-mediated meroterpene cyclization. Cloning, sequencing, and heterologous expression of the napyradiomycin biosynthetic gene cluster. J. Biol. Chem. 282, 16362–16368 (2007).

    Article  CAS  Google Scholar 

  31. Küpper, F. C. et al. Iodide accumulation provides kelp with an inorganic antioxidant impacting atmospheric chemistry. Proc. Natl Acad. Sci. USA 105, 6954–6958 (2008).

    Article  ADS  Google Scholar 

  32. Ortiz-Bermudez, P. et al. Chlorination of lignin by ubiquitous fungi has a likely role in global organochlorine production. Proc. Natl Acad. Sci. USA 104, 3895–3900 (2007).

    Article  ADS  CAS  Google Scholar 

  33. Weyand, M. et al. X-ray structure determination of a vanadium-dependent haloperoxidase from Ascophyllum nodosum at 2.0 Å resolution. J. Mol. Biol. 293, 595–611 (1999).

    Article  CAS  Google Scholar 

  34. Isupov, M. N. et al. Crystal structure of dodecameric vanadium-dependent bromoperoxidase from the red algae Corallina officinalis . J. Mol. Biol. 299, 1035–1049 (2000).

    Article  CAS  Google Scholar 

  35. Messerschmidt, A. & Wever, R. X-ray structure of a vanadium containing enzyme: chloroperoxidase from the fungus Curvularia inaequalis . Proc. Natl Acad. Sci. USA 93, 392–396 (1996).

    Article  ADS  CAS  Google Scholar 

  36. Colpas, G. J., Hamstra, B. J., Kampf, J. W. & Pecoraro, V. L. Functional models for vanadium haloperoxidases: reactivity and mechanism of halide oxidation. J. Am. Chem. Soc. 118, 3469–3478 (1996).

    Article  CAS  Google Scholar 

  37. Hemrika, W., Rokus, R., Macedo-Ribeiro, S., Messerschmidt, A. & Wever, R. Heterologous expression of the vanadium-containing chloroperoxidases from Curvularia inaequalis in Saccharomyces cerevisiae and site-directed mutagenesis of the active site residues His496, Lys353, Arg360, and Arg490 . J. Biol. Chem. 274, 23820–23827 (1999).

    Article  CAS  Google Scholar 

  38. Everett, R. R., Kanofsky, J. R. & Butler, A. Mechanism of dioxygen formation catalyzed by vanadium bromoperoxidase. Steady state kinetic analysis and comparison to the mechanism of bromination. J. Biol. Chem. 265, 15671–15679 (1990).

    CAS  PubMed  Google Scholar 

  39. Tschirret-Guth, R. A. & Butler, A. Evidence for organic substrate binding to vanadium bromoperoxidase. J. Am. Chem. Soc. 116, 411–412 (1994).

    Article  CAS  Google Scholar 

  40. Carter-Franklin, J. N., Parrish, J. D., Tschirret-Guth, R. A., Little, R. D. & Butler, A. Vanadium haloperoxidase-catalyzed bromination and cyclization of terpenes. J. Am. Chem. Soc. 125, 3688–3689 (2003).

    Article  CAS  Google Scholar 

  41. Carter-Franklin, J. N. & Butler, A. Vanadium bromoperoxidase-catalyzed biosynthesis of halogenated marine natural products. J. Am. Chem. Soc. 126, 15060–15066 (2004). This paper shows diasteroselectivity of a V-BPO-catalysed reaction for the first time in the bromination and cyclization of the terpene ( E )-(+)-nerolidol.

    Article  CAS  Google Scholar 

  42. Wang, Y. J., Huang, J. J. & Leadbetter, J. R. Acyl-HSL signal decay: intrinsic to bacterial cell-cell communications. Adv. Appl. Microbiol. 61, 27–58 (2007).

    Article  CAS  Google Scholar 

  43. Steinberg, P. D., de Nys, R. & Kjelleberg, S. in Marine Chemical Ecology (eds McClintock, J. B. & Baker, B. J.) 355–387 (CRC, 2001).

    Google Scholar 

  44. Borchardt, S. A. et al. Reaction of acylated homoserine lactone bacterial signaling molecules with oxidized halogen antimicrobials. Appl. Environ. Microbiol. 67, 3174–3179 (2001).

    Article  CAS  Google Scholar 

  45. Blasiak, L. C., Vaillancourt, F. H., Walsh, C. T. & Drennan, C. L. Crystal structure of the non-haem iron halogenase SyrB2 in syringomycin biosynthesis. Nature 440, 368–371 (2006).

    Article  ADS  CAS  Google Scholar 

  46. Hanauske-Abel, H. M. & Popowicz, A. M. The HAG mechanism: a molecular rationale for the therapeutic application of iron chelators in human diseases involving the 2-oxoacid utilizing dioxygenases. Curr. Med. Chem. 10, 1005–1019 (2003).

    Article  CAS  Google Scholar 

  47. Matthews, M. L. et al. Substrate-triggered formation and remarkable stability of the C–H bond-cleaving chloroferryl intermediate in the aliphatic halogenase, SyrB2. Biochemistry 48, 4331–4343 (2009). This paper provides significant mechanistic insight into the reactions catalysed by the Fe NH –αKG halogenases in comparison with those catalysed by the Fe NH –αKG oxygenases.

    Article  CAS  Google Scholar 

  48. Galonic, D. P., Vaillancourt, F. H. & Walsh, C. T. Halogenation of unactivated carbon centers in natural product biosynthesis: trichlorination of leucine during barbamide biosynthesis. J. Am. Chem. Soc. 128, 3900 (2006).

    Article  CAS  Google Scholar 

  49. Chang, Z. et al. The barbamide biosynthetic gene cluster: a novel marine cyanobacterial system of mixed polyketide synthase (PKS)-non ribosomal peptide synthetase (NRPS) origin involving an unusual trichloroleucyl starter unit. Gene 296, 235–247 (2002).

    Article  CAS  Google Scholar 

  50. Ueki, M. et al. Enzymatic generation of the antimetabolite γ, γ-dichloroaminobutyrate by NRPS and mononuclear iron halogenase action in a streptomycete. Chem. Biol. 13, 1183–1191 (2006).

    Article  CAS  Google Scholar 

  51. Vaillancourt, F. H., Yeh, E., Vosburg, D. A., O'Connor, S. E. & Walsh, C. T. Cryptic chlorination by a non-haem iron enzyme during cyclopropyl amino acid biosynthesis. Nature 436, 1191–1194 (2005). This paper is the first report of cyclopropyl formation via a chlorinated precursor.

    Article  ADS  CAS  Google Scholar 

  52. Chang, Z. et al. Biosynthetic pathway and gene cluster analysis of curacin A, an antitubulin natural product from the tropical marine cyanobacterium Lyngbya majuscula . J. Nat. Prod. 67, 1356–1367 (2004).

    Article  CAS  Google Scholar 

  53. Edwards, D. J. et al. Structure and biosynthesis of the jamaicamides, new mixed polyketide-peptide neurotoxins from the marine cyanobacterium Lyngbya majuscula . Chem. Biol. 11, 817–833 (2004).

    Article  MathSciNet  CAS  Google Scholar 

  54. Gu, L. et al. Metamorphic enzyme assembly in polyketide diversification. Nature 459, 731–735 (2009). This paper demonstrates coevolution of enzymes for metabolic diversification in the biosynthetic pathways leading to β -branched cyclopropane in curacin A and a vinyl chloride in jamaicamide A.

    Article  ADS  CAS  Google Scholar 

  55. Ni, X. & Hager, L. P. cDNA cloning of Batis maritima methyl chloride transferase and purification of the enzyme. Proc. Natl Acad. Sci. USA 95, 12866–12871 (1998).

    Article  ADS  CAS  Google Scholar 

  56. Eustaquio, A. S., Pojer, F., Noe, J. P. & Moore, B. S. Discovery and characterization of a marine bacterial SAM-dependent chlorinase. Nature Chem. Biol. 4, 69–74 (2008).

    Article  CAS  Google Scholar 

  57. Dong, C. et al. Crystal structure and mechanism of a bacterial fluorinating enzyme. Nature 427, 561–565 (2004).

    Article  ADS  CAS  Google Scholar 

  58. Deng, H. et al. The fluorinase from Streptomyces cattleya is also a chlorinase. Angew. Chem. Int. Ed. 45, 759–762 (2006).

    Article  CAS  Google Scholar 

  59. Cadicamo, C. D., Courtieu, J., Deng, H., Meddour, A. & O'Hagan, D. Enzymatic fluorination in Streptomyces cattleya takes place with an inversion of configuration consistent with an SN2 reaction mechanism. ChemBioChem 5, 685–690 (2004).

    Article  CAS  Google Scholar 

  60. Michels, J. J., Allain, E. J., Borchardt, S. A., Hu, P. & McCoy, W. F. Degradation pathway of homoserine lactone bacterial signal molecules by halogen antimicrobials identified by liquid chromatography with photodiode array and mass spectrometric detection. J. Chromatogr. A 898, 153–165 (2000).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

A.B. greatly acknowledges US National Science Foundation Division of Chemistry award number 0719553 for support of her research.

Author information

Authors and Affiliations

Authors

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Reprints and permissions information is available at http://www.nature.com/reprints.

Correspondence should be addressed to A.B. (butler@chem.ucsb.edu).

Rights and permissions

Reprints and permissions

About this article

Cite this article

Butler, A., Sandy, M. Mechanistic considerations of halogenating enzymes. Nature 460, 848–854 (2009). https://doi.org/10.1038/nature08303

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08303

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing