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Cryptic chlorination by a non-haem iron enzyme during cyclopropyl amino acid biosynthesis

Nature volume 436, pages 1191–1194 (2005)Cite this article

Abstract

Enzymatic incorporation of chlorine, bromine or iodine atoms occurs during the biosynthesis of more than 4,000 natural products1. Halogenation can have significant consequences for the bioactivity of these products so there is great interest in understanding the biological catalysts that perform these reactions. Enzymes that halogenate unactivated aliphatic groups have not previously been characterized. Here we report the activity of five proteins—CmaA, CmaB, CmaC, CmaD and CmaE—in the construction of coronamic acid (CMA; 1-amino-1-carboxy-2-ethylcyclopropane), a constituent of the phytotoxin coronatine synthesized by the phytopathogenic bacterium Pseudomonas syringae2. CMA derives from l-allo-isoleucine, which is covalently attached to CmaD through the actions of CmaA, a non-ribosomal peptide synthetase module, and CmaE, an unusual acyltransferase. We show that CmaB, a member of the non-haem Fe2+, α-ketoglutarate-dependent enzyme superfamily, is the first of its class to show halogenase activity, chlorinating the γ-position of l-allo-isoleucine. Another previously undescribed enzyme, CmaC, catalyses the formation of the cyclopropyl ring from the γ-Cl-l-allo-isoleucine product of the CmaB reaction. Together, CmaB and CmaC execute γ-halogenation followed by intramolecular γ-elimination, in which biological chlorination is a cryptic strategy for cyclopropyl ring formation.

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Figure 1: Biosynthesis of CMA and coronatine.
Figure 2: The transfer of l -Val from CmaA to CmaD catalysed by CmaE.
Figure 3: Analysis of the reactions catalysed by CmaB and CmaC.
Figure 4: Proposed mechanisms of CmaB and CmaC.

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References

  1. Gribble, G. W. Natural organohalogens: a new frontier for medicinal agents? J. Chem. Educ. 81, 1441–1449 (2004)

    Article  CAS  Google Scholar 

  2. Buell, C. R. et al. The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv. tomato DC3000. Proc. Natl Acad. Sci. USA 100, 10181–10186 (2003)

    Article  ADS  CAS  Google Scholar 

  3. Parry, R. J., Lin, M. T., Walker, A. E. & Mhaskar, S. Biosynthesis of coronatine: investigations of the biosynthesis of coronamic acid. J. Am. Chem. Soc. 113, 1849–1850 (1991)

    Article  CAS  Google Scholar 

  4. Parry, R. J., Mhaskar, S. V., Lin, M. T., Walker, A. E. & Mafoti, R. Investigations of the biosynthesis of the phytotoxin coronatine. Can. J. Chem. 72, 86–99 (1994)

    Article  CAS  Google Scholar 

  5. Young, S. A., Park, S. K., Rodgers, C., Mitchell, R. E. & Bender, C. L. Physical and functional characterization of the gene cluster encoding the polyketide phytotoxin coronatine in Pseudomonas syringae pv. glycinea. J. Bacteriol. 174, 1837–1843 (1992)

    Article  CAS  Google Scholar 

  6. Couch, R., O'Connor, S. E., Seidle, H., Walsh, C. T. & Parry, R. Characterization of CmaA, an adenylation–thiolation didomain enzyme involved in the biosynthesis of coronatine. J. Bacteriol. 186, 35–42 (2004)

    Article  CAS  Google Scholar 

  7. 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  Google Scholar 

  8. Koehntop, K. D., Emerson, J. P. & Que, L. Jr The 2-His-1-carboxylate facial triad: a versatile platform for dioxygen activation by mononuclear non-heme iron(II) enzymes. J. Biol. Inorg. Chem. 10, 87–93 (2005)

    Article  CAS  Google Scholar 

  9. Guenzi, E., Galli, G., Grgurina, I., Gross, D. C. & Grandi, G. Characterization of the syringomycin synthetase gene cluster. A link between prokaryotic and eukaryotic peptide synthetases. J. Biol. Chem. 273, 32857–32863 (1998)

    Article  CAS  Google Scholar 

  10. 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 

  11. Yeh, E., Kohli, R. M., Bruner, S. D. & Walsh, C. T. Type II thioesterase restores activity of a NRPS module stalled with an aminoacyl-S-enzyme that cannot be elongated. Chembiochem 5, 1290–1293 (2004)

    Article  CAS  Google Scholar 

  12. Schofield, C. J. & Zhang, Z. Structural and mechanistic studies on 2-oxoglutarate-dependent oxygenases and related enzymes. Curr. Opin. Struct. Biol. 9, 722–731 (1999)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  14. 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 Fe(IV) complex in taurine/α-ketoglutarate dioxygenase (TauD) from Escherichia coli. Biochemistry 42, 7497–7508 (2003)

    Article  CAS  Google Scholar 

  15. Rohde, J. U. et al. Crystallographic and spectroscopic characterization of a nonheme Fe(IV) = O complex. Science 299, 1037–1039 (2003)

    Article  ADS  CAS  Google Scholar 

  16. Cook, G. K. & Mayer, J. M. C-H bond activation by metal oxo species: oxidation of cyclohexane by chromyl chloride. J. Am. Chem. Soc. 116, 1855–1868 (1994)

    Article  CAS  Google Scholar 

  17. Mayer, J. M. Hydrogen atom abstraction by metal-oxo complexes: understanding the analogy with organic radical reactions. Acc. Chem. Res. 31, 441–450 (1998)

    Article  CAS  Google Scholar 

  18. Kojima, T., Leising, R. A., Yan, S. & Que, L. Jr Alkane functionalization at nonheme iron center. Stoichiometric transfer of metal-bound ligands to alkane. J. Am. Chem. Soc. 115, 11328–11335 (1993)

    Article  CAS  Google Scholar 

  19. McCarthy, A. A., Baker, H. M., Shewry, S. C., Patchett, M. L. & Baker, E. N. Crystal structure of methylmalonyl-coenzyme A epimerase from P. shermanii: a novel enzymatic function on an ancient metal binding scaffold. Structure 9, 637–646 (2001)

    Article  CAS  Google Scholar 

  20. Armstrong, R. N. Mechanistic diversity in a metalloenzyme superfamily. Biochemistry 39, 13625–13632 (2000)

    Article  CAS  Google Scholar 

  21. Gerlt, J. A. & Babbitt, P. C. Divergent evolution of enzymatic function: mechanistically diverse superfamilies and functionally distinct suprafamilies. Annu. Rev. Biochem. 70, 209–246 (2001)

    Article  CAS  Google Scholar 

  22. 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 

  23. Massey, V. Activation of molecular oxygen by flavins and flavoproteins. J. Biol. Chem. 269, 22459–22462 (1994)

    CAS  PubMed  Google Scholar 

  24. Bugg, T. D. Oxygenases: mechanisms and structural motifs for O2 activation. Curr. Opin. Chem. Biol. 5, 550–555 (2001)

    Article  CAS  Google Scholar 

  25. Ryle, M. J. & Hausinger, R. P. Non-heme iron oxygenases. Curr. Opin. Chem. Biol. 6, 193–201 (2002)

    Article  CAS  Google Scholar 

  26. Entsch, B., Ballou, D. P. & Massey, V. Flavin–oxygen derivatives involved in hydroxylation by ρ-hydroxybenzoate hydroxylase. J. Biol. Chem. 251, 2550–2563 (1976)

    CAS  PubMed  Google Scholar 

  27. Yeh, E., Garneau, S. & Walsh, C. T. Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis. Proc. Natl Acad. Sci. USA 102, 3960–3965 (2005)

    Article  ADS  CAS  Google Scholar 

  28. Vaillancourt, F. H., Han, S., Fortin, P. D., Bolin, J. T. & Eltis, L. D. Molecular basis for the stabilization and inhibition of 2, 3-dihydroxybiphenyl 1,2-dioxygenase by t-butanol. J. Biol. Chem. 273, 34887–34895 (1998)

    Article  CAS  Google Scholar 

  29. Haigler, B. E. & Gibson, D. T. Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816. J. Bacteriol. 172, 457–464 (1990)

    Article  CAS  Google Scholar 

  30. Molnar-Perl, I. Derivatization and chromatographic behaviour of the o-phthaldialdehyde amino acid derivatives obtained with various SH-group-containing additives. J. Chromatogr. A 913, 283–302 (2001)

    Article  CAS  Google Scholar 

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Acknowledgements

We thank M. R. Rondon for providing Pseudomonas syringae pv. tomato DC3000, and M. G. Thomas for discussion. This work was supported in part by an NIH grant (C.T.W.), a Merck-sponsored Fellowship of the Helen Hay Whitney Foundation (F.H.V.), an NSERC Postdoctoral Fellowship (F.H.V.), an NIH Medical Scientist Training Program Fellowship (E.Y.), a Jane Coffin Childs Memorial Fund for Medical Research Fellowship (D.A.V.), and an Irving S. Sigal Postdoctoral Fellowship (S.E.O.).

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Author notes

  1. Sarah E. O'Connor

    Present address: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139, USA

  2. David A. Vosburg

    Present address: Department of Chemistry, Harvey Mudd College, Claremont, California, 91711, USA

  3. Frédéric H. Vaillancourt and Ellen Yeh: *These authors contributed equally to this work

Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Massachusetts, 02115, Boston, USA

    Frédéric H. Vaillancourt, Ellen Yeh, David A. Vosburg, Sarah E. O'Connor & Christopher T. Walsh

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Correspondence to Christopher T. Walsh.

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Supplementary information

Supplementary Methods

This file describes the detailed synthesis protocol of γ-Cl-L-valine and γ-Cl-L-allo-isoleucine, the cloning of the cma genes, the overproduction and purification of the Cma proteins, and the biochemical assays that were performed. (PDF 168 kb)

Supplementary Table S1

dThis table contains the sequences of the oligonucleotides used to amplify the cmaA/B/C/D/E genes from Pseudomonas syringae pv. tomato DC3000 genomic DNA. (PDF 80 kb)

Supplementary Table S2

This table contains the kinetic parameters obtained using the ATP-[32P]PPi exchange assay with highly pure CmaA. (PDF 79 kb)

Supplementary Figure S1

This figure describes the chemical steps performed in the syntheses of γ-Cl-L-valine and γ-Cl-L-allo-isoleucine. (PDF 195 kb)

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Vaillancourt, F., Yeh, E., Vosburg, D. et al. Cryptic chlorination by a non-haem iron enzyme during cyclopropyl amino acid biosynthesis. Nature 436, 1191–1194 (2005). https://doi.org/10.1038/nature03797

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