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An allylic ketyl radical intermediate in clostridial amino-acid fermentation


The human pathogenic bacterium Clostridium difficile thrives by the fermentation of l-leucine to ammonia, CO2, 3-methylbutanoate and 4-methylpentanoate under anaerobic conditions1. The reductive branch to 4-methylpentanoate proceeds by means of the dehydration of (R)-2-hydroxy-4-methylpentanoyl-CoA to 4-methylpent-2-enoyl-CoA, which is chemically the most demanding step. Ketyl radicals have been proposed2 to mediate this reaction catalysed by an iron–sulphur-cluster-containing dehydratase, which requires activation by ATP-dependent electron transfer from a second iron–sulphur protein functionally similar to the iron protein of nitrogenase. Here we identify a kinetically competent product-related allylic ketyl radical bound to the enzyme by electron paramagnetic resonance spectroscopy employing isotope-labelled (R)-2-hydroxy-4-methylpentanoyl-CoA species. We also found that the enzyme generated the stabilized pentadienoyl ketyl radical from the substrate analogue 2-hydroxypent-4-enoyl-CoA, supporting the proposed mechanism. Our results imply that also other 2-hydroxyacyl-CoA dehydratases3 and the related benzoyl-CoA reductases4—present in anaerobically living bacteria—employ ketyl radical intermediates. The absence of radical generators such as coenzyme B12, S-adenosylmethionine or oxygen makes these enzymes unprecedented in biochemistry.

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Figure 1: Proposed mechanism for the enzymatic dehydration of ( R )-2-hydroxy-4-methylpentanoyl-CoA.
Figure 2: EPR spectroscopy of the product-related allylic ketyl radical intermediate in 2-hydroxy-4-methylpentanoyl-CoA dehydratase.
Figure 3: EPR spectrum of the pentadienyl ketyl radical formed from the 2-hydroxypent-4-enoyl-CoA substrate analogue.


  1. Buckel, W. in Biology of the Prokaryotes (eds Lengeler, J. W., Drews, G. & Schlegel, H. G.) 278–326 (Thieme, Stuttgart, 1999)

    Google Scholar 

  2. Buckel, W. & Keese, R. One electron redox reactions of CoASH esters in anaerobic bacteria. A mechanistic proposal. Angew. Chem. Int. Edn Engl. 34, 1502–1506 (1995)

    CAS  Article  Google Scholar 

  3. Buckel, W., Hetzel, M. & Kim, J. ATP-driven electron transfer in enzymatic radical reactions. Curr. Opin. Chem. Biol. 8, 462–467 (2004)

    CAS  Article  Google Scholar 

  4. Boll, M. & Fuchs, G. Unusual reactions involved in anaerobic metabolism of phenolic compounds. Biol. Chem. 386, 989–997 (2005)

    CAS  PubMed  Google Scholar 

  5. Seebach, D. Methods of reactivity Umpolung. Angew. Chem. Int. Edn Engl. 18, 239–258 (1979)

    Article  Google Scholar 

  6. Brückner, R. Reaktionsmechanismen (Spektrum Akademischer, Heidelberg, 2003)

    Google Scholar 

  7. Buckel, W. & Golding, B. T. Radical enzymes in anaerobes. Annu. Rev. Microbiol. 60, 27–49 (2006)

    CAS  Article  Google Scholar 

  8. Smith, D. M., Buckel, W. & Zipse, H. Deprotonation of enoxy radicals: theoretical validation of a 50-year-old mechanistic proposal. Angew. Chem. Int. Edn Engl. 42, 1867–1870 (2003)

    CAS  Article  Google Scholar 

  9. Sebaihia, M. & Thomson, N. R. Colonic irritation. Nature Rev. Microbiol. 4, 882–883 (2006)

    CAS  Article  Google Scholar 

  10. Reineke, J. et al. Autocatalytic cleavage of Clostridium difficile toxin B. Nature 446, 415–419 (2007)

    ADS  CAS  Article  Google Scholar 

  11. Selmer, T. & Andrei, P. I. p-Hydroxyphenylacetate decarboxylase from Clostridium difficile. A novel glycyl radical enzyme catalysing the formation of p-cresol. Eur. J. Biochem. 268, 1363–1372 (2001)

    CAS  Article  Google Scholar 

  12. Sebaihia, M. et al. The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nature Genet. 38, 779–786 (2006)

    Article  Google Scholar 

  13. Barker, H. A. Amino acid degradation by anaerobic bacteria. Annu. Rev. Biochem. 50, 23–40 (1981)

    CAS  Article  Google Scholar 

  14. Elsden, S. R. & Hilton, M. G. Volatile acid production from threonine, valine, leucine and isoleucine by clostridia. Arch. Microbiol. 117, 165–172 (1978)

    CAS  Article  Google Scholar 

  15. Kim, J., Darley, D., Selmer, T. & Buckel, W. Characterization of (R)-2-hydroxyisocaproate dehydrogenase and a family III coenzyme A transferase involved in reduction of l-leucine to isocaproate by Clostridium difficile. Appl. Environ. Microbiol. 72, 6062–6069 (2006)

    CAS  Article  Google Scholar 

  16. Kim, J., Darley, D. & Buckel, W. 2-Hydroxyisocaproyl-CoA dehydratase and its activator from Clostridium difficile. FEBS J. 272, 550–561 (2005)

    CAS  Article  Google Scholar 

  17. Hans, M., Buckel, W. & Bill, E. The iron–sulfur clusters in 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. Biochemical and spectroscopic investigations. Eur. J. Biochem. 267, 7082–7093 (2000)

    CAS  Article  Google Scholar 

  18. Weil, J. A. & Bolton, J. R. Electron Paramagnetic Resonance: Elementary Theory and Practical Applications 2nd edn (Wiley, Bognor Regis, 2007)

    Google Scholar 

  19. Wu, W. et al. Lysine 2,3-aminomutase and trans-4,5-dehydrolysine: characterization of an allylic analogue of a substrate-based radical in the catalytic mechanism. Biochemistry 39, 9561–9570 (2000)

    CAS  Article  Google Scholar 

  20. Magnusson, O. T., Reed, G. H. & Frey, P. A. Characterization of an allylic analogue of the 5′-deoxyadenosyl radical: an intermediate in the reaction of lysine 2,3-aminomutase. Biochemistry 40, 7773–7782 (2001)

    CAS  Article  Google Scholar 

  21. Layer, G. et al. The substrate radical of Escherichia coli oxygen-independent coproporphyrinogen III oxidase HemN. J. Biol. Chem. 281, 15727–15734 (2006)

    CAS  Article  Google Scholar 

  22. Stubbe, J. & Van der Donk, W. A. Protein radicals in enzyme catalysis. Chem. Rev. 98, 705–762 (1998)

    CAS  Article  Google Scholar 

  23. Buckel, W. The reversible dehydration of (R)-2-hydroxyglutarate to (E)-glutaconate. Eur. J. Biochem. 106, 439–447 (1980)

    CAS  Article  Google Scholar 

  24. Beinert, H. & Albracht, S. P. J. New insights, ideas and unanswered questions concerning iron–sulfur clusters in mitochondria. Biochim. Biophys. Acta 683, 245–277 (1982)

    CAS  Article  Google Scholar 

  25. Tsai, A. L., Berka, V., Kulmacz, R. J., Wu, G. & Palmer, G. An improved sample packing device for rapid freeze-trap electron paramagnetic resonance spectroscopy kinetic measurements. Anal. Biochem. 264, 165–171 (1998)

    CAS  Article  Google Scholar 

  26. Ballinger, M. D., Reed, G. H. & Frey, P. A. An organic radical in the lysine 2,3-aminomutase reaction. Biochemistry 31, 949–953 (1992)

    CAS  Article  Google Scholar 

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We thank R. K. Thauer for the use of his EPR spectrometer, and V. Schünemann and M. Bennati for the use of their freeze-quench instruments.

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Correspondence to Antonio J. Pierik.

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The file contains Supplementary Figures S1-S3 with Legends, Supplementary Methods describing Synthesis and characterization of (labeled) substrates, Supplementary Tables S1-S5, Supplementary Discussion, and additional references. (PDF 980 kb)

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Kim, J., Darley, D., Buckel, W. et al. An allylic ketyl radical intermediate in clostridial amino-acid fermentation. Nature 452, 239–242 (2008).

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