Diphthamide biosynthesis requires an organic radical generated by an iron–sulphur enzyme

  • A Corrigendum to this article was published on 26 May 2011

Abstract

Archaeal and eukaryotic translation elongation factor 2 contain a unique post-translationally modified histidine residue called diphthamide, which is the target of diphtheria toxin. The biosynthesis of diphthamide was proposed to involve three steps, with the first being the formation of a C–C bond between the histidine residue and the 3-amino-3-carboxypropyl group of S-adenosyl-l-methionine (SAM). However, further details of the biosynthesis remain unknown. Here we present structural and biochemical evidence showing that the first step of diphthamide biosynthesis in the archaeon Pyrococcus horikoshii uses a novel iron–sulphur-cluster enzyme, Dph2. Dph2 is a homodimer and each of its monomers can bind a [4Fe–4S] cluster. Biochemical data suggest that unlike the enzymes in the radical SAM superfamily, Dph2 does not form the canonical 5′-deoxyadenosyl radical. Instead, it breaks the Cγ,Met–S bond of SAM and generates a 3-amino-3-carboxypropyl radical. Our results suggest that P. horikoshii Dph2 represents a previously unknown, SAM-dependent, [4Fe–4S]-containing enzyme that catalyses unprecedented chemistry.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: The structure of diphthamide and its proposed biosynthesis pathway.
Figure 2: Structure of the PhDPH2 homodimer.
Figure 3: In vitro reconstitution of PhDph2 activity.
Figure 4: Spectroscopic characterization of the [4Fe–4S] cluster in PhDph2.
Figure 5: Identification of SAM-derived small-molecule products in PhDph2-catalysed reactions.
Figure 6: The proposed reaction mechanism for PhDph2.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the crystal structures reported here have been deposited with the Protein Data Bank under accession codes 3LZC for iron-free PhDph2 and 3LZD for reconstituted PhDph2.

References

  1. 1

    Centers for Disease Control and Prevention. Diphtheria. Disease Listing, Diphtheria, Technical Information | CDC Bacterial, Mycotic Diseaseshttp://www.cdc.gov/ncidod/DBMD/diseaseinfo/diptheria_t.htm〉 (2005)

  2. 2

    Collier, R. J. Understanding the mode of action of diphtheria toxin: a perspective on progress during the 20th century. Toxicon 39, 1793–1803 (2001)

    CAS  Article  Google Scholar 

  3. 3

    Liu, S., Milne, G. T., Kuremsky, J. G., Fink, G. R. & Leppla, S. H. Identification of the proteins required for biosynthesis of diphthamide, the target of bacterial ADP-ribosylating toxins on translation elongation factor 2. Mol. Cell. Biol. 24, 9487–9497 (2004)

    CAS  Article  Google Scholar 

  4. 4

    Gomez-Lorenzo, M. G. et al. Three-dimensional cryo-electron microscopy localization of EF2 in the Saccharomyces cerevisiae 80S ribosome at 17.5 Å resolution. EMBO J. 19, 2710–2718 (2000)

    CAS  Article  Google Scholar 

  5. 5

    Ortiz, P. A., Ulloque, R., Kihara, G. K., Zheng, H. & Kinzy, T. G. Translation elongation factor 2 anticodon mimicry domain mutants affect fidelity and diphtheria toxin resistance. J. Biol. Chem. 281, 32639–32648 (2006)

    CAS  Article  Google Scholar 

  6. 6

    Walsh, C. T. Posttranslational Modifications of Proteins: Expanding Nature’s Inventory 321–323 (Roberts, 2006)

    Google Scholar 

  7. 7

    Moehring, J. M., Moehring, T. J. & Danley, D. E. Posttranslational modification of elongation factor 2 in diphtheriatoxin-resistant mutants of CHO-K1 cells. Proc. Natl Acad. Sci. USA 77, 1010–1014 (1980)

    ADS  CAS  Article  Google Scholar 

  8. 8

    Moehring, T. J., Danley, D. E. & Moehring, J. M. In vitro biosynthesis of diphthamide, studied with mutant Chinese hamster ovary cells resistant to diphtheria toxin. Mol. Cell. Biol. 4, 642–650 (1984)

    CAS  Article  Google Scholar 

  9. 9

    Chen, J. Y., Bodley, J. W. & Livingston, D. M. Diphtheria toxin-resistant mutants of Saccharomyces cerevisiae . Mol. Cell. Biol. 5, 3357–3360 (1985)

    CAS  Article  Google Scholar 

  10. 10

    Mattheakis, L. C., Shen, W. H. & Collier, R. J. DPH5, a methyltransferase gene required for diphthamide biosynthesis in Saccharomyces cerevisiae . Mol. Cell. Biol. 12, 4026–4037 (1992)

    CAS  Article  Google Scholar 

  11. 11

    Mattheakis, L. C., Sor, F. & Collier, R. J. Diphthamide synthesis in Saccharomyces cerevisiae: structure of the DPH2 gene. Gene 132, 149–154 (1993)

    CAS  Article  Google Scholar 

  12. 12

    Phillips, N. J., Ziegler, M. R. & Deaven, L. L. A cDNA from the ovarian cancer critical region of deletion on chromosome 17p13.3. Cancer Lett. 102, 85–90 (1996)

    CAS  Article  Google Scholar 

  13. 13

    Schultz, D. C., Balasara, B. R., Testa, J. R. & Godwin, A. K. Cloning and localization of a human diphthamide biosynthesis-like protein-2 gene, DPH2L2 . Genomics 52, 186–191 (1998)

    CAS  Article  Google Scholar 

  14. 14

    Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990)

    CAS  Article  Google Scholar 

  15. 15

    Frey, P. A., Hegeman, A. D. & Ruzicka, F. J. The radical SAM superfamily. Crit. Rev. Biochem. Mol. Biol. 43, 63–88 (2008)

    CAS  Article  Google Scholar 

  16. 16

    Sakuraba, H., Tsuge, H., Yoneda, K., Katunuma, N. & Ohshima, T. Crystal structure of the NAD biosynthetic enzyme quinolinate synthase. J. Biol. Chem. 280, 26645–26648 (2005)

    CAS  Article  Google Scholar 

  17. 17

    Sofia, H. J., Chen, G., Hetzler, B. G., Reyes-Spindola, J. F. & Miller, N. E. Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods. Nucleic Acids Res. 29, 1097–1106 (2001)

    CAS  Article  Google Scholar 

  18. 18

    Chatterjee, A. et al. Reconstitution of ThiC in thiamine pyrimidine biosynthesis expands the radical SAM superfamily. Nature Chem. Biol. 4, 758–765 (2008)

    CAS  Article  Google Scholar 

  19. 19

    McGlynn, S. E. et al. Identification and characterization of a novel member of the radical AdoMet enzyme superfamily and implications for the biosynthesis of the Hmd hydrogenase active site cofactor. J. Bacteriol. 192, 595–598 (2010)

    CAS  Article  Google Scholar 

  20. 20

    Makinen, G. B. & Wells, M. W. in ENDOR, EPR and Electron Spin Echo for Probing Coordination Spheres (eds Sigel, H. & Sigel, A.) 129–204 (Dekker, 1987)

    Google Scholar 

  21. 21

    Lieder, K. W. et al. S-adenosylmethionine-dependent reduction of lysine 2,3-aminomutase and observation of the catalytically functional iron–sulfur centers by electron paramagnetic resonance. Biochemistry 37, 2578–2585 (1998)

    CAS  Article  Google Scholar 

  22. 22

    Walsby, C. J. et al. Electron-nuclear double resonance spectroscopic evidence that S-adenosylmethionine binds in contact with the catalytically active [4Fe–4S]+ cluster of pyruvate formate-lyase activating enzyme. J. Am. Chem. Soc. 124, 3143–3151 (2002)

    CAS  Article  Google Scholar 

  23. 23

    Cicchillo, R. M. et al. Escherichia coli quinolinate synthetase does indeed harbor a [4Fe-4S] cluster. J. Am. Chem. Soc. 127, 7310–7311 (2005)

    CAS  Article  Google Scholar 

  24. 24

    Saunders, A. H. et al. Characterization of quinolinate synthases from Escherichia coli, Mycobacterium tuberculosis, and Pyrococcus horikoshii indicates that [4Fe-4S] clusters are common cofactors throughout this class of enzymes. Biochemistry 47, 10999–11012 (2008)

    CAS  Article  Google Scholar 

  25. 25

    Berkovitch, F., Nicolet, Y., Wan, J. T., Jarrett, J. T. & Drennan, C. L. Crystal structure of biotin synthase, an S-adenosylmethionine-dependent radical enzyme. Science 303, 76–79 (2004)

    ADS  CAS  Article  Google Scholar 

  26. 26

    Layer, G., Moser, J., Heinz, D. W., Jahn, D. & Schubert, W.-D. Crystal structure of coproporphyrinogen III oxidase reveals cofactor geometry of radical SAM enzymes. EMBO J. 22, 6214–6224 (2003)

    CAS  Article  Google Scholar 

  27. 27

    Lepore, B. W., Ruzicka, F. J., Frey, P. A. & Ringe, D. The X-ray crystal structure of lysine-2,3-aminomutase from Clostridium subterminale . Proc. Natl Acad. Sci. USA 102, 13819–13824 (2005)

    ADS  CAS  Article  Google Scholar 

  28. 28

    Hänzelmann, P. & Schindelin, H. Crystal structure of the S-adenosylmethionine-dependent enzyme MoaA and its implications for molybdenum cofactor deficiency in humans. Proc. Natl Acad. Sci. USA 101, 12870–12875 (2004)

    ADS  Article  Google Scholar 

  29. 29

    Vey, J. L. et al. Structural basis for glycyl radical formation by pyruvate formate-lyase activating enzyme. Proc. Natl Acad. Sci. USA 105, 16137–16141 (2008)

    ADS  CAS  Article  Google Scholar 

  30. 30

    Marinoni, I. et al. Characterization of l-aspartate oxidase and quinolinate synthase from Bacillus subtilis . FEBS J. 275, 5090–5107 (2008)

    CAS  Article  Google Scholar 

  31. 31

    Rekittke, I. et al. Structure of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase, the terminal enzyme of the non-mevalonate pathway. J. Am. Chem. Soc. 130, 17206–17207 (2008)

    CAS  Article  Google Scholar 

  32. 32

    Walsby, C. J., Ortillo, D., Broderick, W. E., Broderick, J. B. & Hoffman, B. M. An anchoring role for FeS clusters: chelation of the amino acid moiety of S-adenosylmethionine to the unique iron site of the [4Fe–4S] cluster of pyruvate formate-lyase activating enzyme. J. Am. Chem. Soc. 124, 11270–11271 (2002)

    CAS  Article  Google Scholar 

  33. 33

    Krogan, N. J. et al. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae . Nature 440, 637–643 (2006)

    ADS  CAS  Article  Google Scholar 

  34. 34

    Gavin, A.-C. et al. Proteome survey reveals modularity of the yeast cell machinery. Nature 440, 631–636 (2006)

    ADS  CAS  Article  Google Scholar 

  35. 35

    Collins, S. R. et al. Toward a comprehensive atlas of the physical interactome of Saccharomyces cerevisiae . Mol. Cell. Proteomics 6, 439–450 (2007)

    CAS  Article  Google Scholar 

  36. 36

    Proudfoot, M. et al. Biochemical and structural characterization of a novel family of cystathionine β-synthase domain proteins fused to a Zn ribbon-like domain. J. Mol. Biol. 375, 301–315 (2008)

    CAS  Article  Google Scholar 

  37. 37

    Johnson, D. C., Dean, D. R., Smith, A. D. & Johnson, M. K. Structure, function, and formation of biological iron-sulfur clusters. Annu. Rev. Biochem. 74, 247–281 (2005)

    CAS  Article  Google Scholar 

  38. 38

    Lill, R. & Mühlenhoff, U. Maturation of iron-sulfur proteins in eukaryotes: mechanisms, connected processes, and diseases. Annu. Rev. Biochem. 77, 669–700 (2008)

    CAS  Article  Google Scholar 

  39. 39

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    CAS  Article  Google Scholar 

  40. 40

    Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008)

    ADS  CAS  Article  Google Scholar 

  41. 41

    Collaborative. Computation Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  42. 42

    Terwilliger, T. C. Reciprocal-space solvent flattening. Acta Crystallogr. D 55, 1863–1871 (1999)

    CAS  Article  Google Scholar 

  43. 43

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

    CAS  Article  Google Scholar 

  44. 44

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  45. 45

    Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    CAS  Article  Google Scholar 

  46. 46

    Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    Article  Google Scholar 

  47. 47

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

    Article  Google Scholar 

Download references

Acknowledgements

We thank L. Kinsland for assistance with manuscript preparation, the Dreyfus Foundation for a New Faculty Award to H.L., NIH/NIGMS R01GM088276 to H.L. and S.E.E., and NIH/NCRR P41-RR016292 for the ACERT Center Grant to J.F. This work is based upon research conducted at the Advanced Photon Source (APS), Argonne National Laboratory, on the Northeastern Collaborative Access Team beamlines, which are supported by award RR-15301 from the US National Center for Research Resources at the US National Institutes of Health. Use of the APS is supported by the US Department of Energy, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357.

Author information

Affiliations

Authors

Contributions

Y.Z. determined the crystal structure of iron-free PhDph2, X.Z. performed the biochemical studies and prepared protein samples for spectroscopic and structural studies, A.T.T. determined the crystal structure of anaerobically purified PhDph2, M.L. and C.K. performed the Mössbauer spectroscopy, B.D. and J.F. performed the EPR spectroscopy, R.M.K. prepared the initial PhDph2 crystals, E.W. prepared the PhEF2 mutant proteins, S.E.E. supervised the crystallographic studies, H.L. supervised the biochemical studies and H.L., S.E.E. and C.K. prepared the manuscript.

Corresponding authors

Correspondence to Steven E. Ealick or Hening Lin.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Table 1 and Supplementary Figures 1-9 with legends. (PDF 9956 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Zhang, Y., Zhu, X., Torelli, A. et al. Diphthamide biosynthesis requires an organic radical generated by an iron–sulphur enzyme. Nature 465, 891–896 (2010). https://doi.org/10.1038/nature09138

Download citation

Further reading

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.