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.

Structure of the molybdopterin-bound Cnx1G domain links molybdenum and copper metabolism

Abstract

The molybdenum cofactor is part of the active site of all molybdenum-dependent enzymes1, except nitrogenase. The molybdenum cofactor consists of molybdopterin, a phosphorylated pyranopterin2, with an ene-dithiolate coordinating molybdenum. The same pyranopterin-based cofactor is involved in metal coordination of the homologous tungsten-containing enzymes found in archea3. The molybdenum cofactor is synthesized by a highly conserved biosynthetic pathway4. In plants, the multidomain protein Cnx1 catalyses the insertion of molybdenum into molybdopterin. The Cnx1 G domain (Cnx1G), whose crystal structure has been determined in its apo form, binds molybdopterin with high affinity and participates in the catalysis of molybdenum insertion. Here we present two high-resolution crystal structures of Cnx1G in complex with molybdopterin and with adenylated molybdopterin (molybdopterin–AMP), a mechanistically important intermediate. Molybdopterin–AMP is the reaction product of Cnx1G and is subsequently processed in a magnesium-dependent reaction by the amino-terminal E domain of Cnx1 to yield active molybdenum cofactor. The unexpected identification of copper bound to the molybdopterin dithiolate sulphurs in both structures, coupled with the observed copper inhibition of Cnx1G activity, provides a molecular link between molybdenum and copper metabolism.

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: Structure of the Cnx1G–MPT and Ser583Ala–MPT–AMP complexes.
Figure 2: Mg2+- and Cnx1E-dependent Moco synthesis by Cnx1G–MPT complexes.
Figure 3: Cu–MPT dithiolate complex.
Figure 4: Hypothetical mechanism of the molybdenum insertion reaction.

References

  1. Hille, R. The mononuclear molybdenum enzymes. Chem. Rev. 96, 2757–2816 (1996)

    Article  CAS  Google Scholar 

  2. Romao, M. J. et al. Crystal structure of the xanthine oxidase-related aldehyde oxido- reductase from D. gigas. Science 270, 1170–1176 (1995)

    Article  ADS  CAS  Google Scholar 

  3. Chan, M. K., Mukund, S., Kletzin, A., Adams, M. W. W. & Rees, D. C. Structure of a hyperthermophilic tungstopterin, aldehyde ferredoxin oxidoreductase. Science 267, 1463–1469 (1995)

    Article  ADS  CAS  Google Scholar 

  4. Mendel, R. R. & Schwarz, G. Biosynthesis and molecular biology of the molybdenum cofactor (Moco). Met. Ions Biol. Syst. 39, 317–368 (2002)

    CAS  PubMed  Google Scholar 

  5. Stiefel, E. I. Molybdenum bolsters the bioinorganic brigade. Science 272, 1599–1600 (1996)

    Article  ADS  CAS  Google Scholar 

  6. Enemark, J. H. & Cosper, M. M. Molybdenum enzymes and sulfur metabolism. Met. Ions Biol. Syst. 39, 621–654 (2002)

    CAS  PubMed  Google Scholar 

  7. Mendel, R. R. & Schwarz, G. Molybdoenzymes and molybdenum cofactor in plants. Crit. Rev. Plant. Sci. 18, 33–69 (1999)

    Article  CAS  Google Scholar 

  8. Johnson, J. L. et al. Inborn errors of molybdenum metabolism: combined deficiencies of sulfite oxidase and xanthine dehydrogenase in a patient lacking the molybdenum cofactor. Proc. Natl Acad. Sci. USA 77, 3715–3719 (1980)

    Article  ADS  CAS  Google Scholar 

  9. Shih, V. E. et al. Sulfite oxidase deficiency. Biochemical and clinical investigations of a hereditary metabolic disorder in sulfur metabolism. N. Engl. J. Med. 297, 1022–1028 (1977)

    Article  CAS  Google Scholar 

  10. Kuper, J., Winking, J., Hecht, H. J., Mendel, R. R. & Schwarz, G. The active site of the molybdenum cofactor biosynthetic protein domain Cnx1G. Arch. Biochem. Biophys. 411, 36–46 (2003)

    Article  CAS  Google Scholar 

  11. Schwarz, G. et al. The molybdenum cofactor biosynthetic protein Cnx1 complements molybdate-repairable mutants, transfers molybdenum to the metal binding pterin, and is associated with the cytoskeleton. Plant Cell 12, 2455–2472 (2000)

    Article  CAS  Google Scholar 

  12. Kuper, J. et al. In vivo detection of molybdate-binding proteins using a competition assay with ModE in Escherichia coli. FEMS Microbiol. Lett. 218, 187–193 (2003)

    Article  CAS  Google Scholar 

  13. Kisker, C. et al. Molecular basis of sulfite oxidase deficiency from the structure of sulfite oxidase. Cell 91, 973–983 (1997)

    Article  CAS  Google Scholar 

  14. Kuper, J., Palmer, T., Mendel, R. R. & Schwarz, G. Mutations in the molybdenum cofactor biosynthetic protein Cnx1G from Arabidopsis thaliana define functions for molybdopterin bind, Mo-insertion and molybdenum cofactor stabilization. Proc. Natl Acad. Sci. USA 97, 6475–6480 (2000)

    Article  ADS  CAS  Google Scholar 

  15. Lake, M. W., Wuebbens, M. M., Rajagopalan, K. V. & Schindelin, H. Mechanism of ubiquitin activation revealed by the structure of a bacterial MoeB–MoaD complex. Nature 414, 325–329 (2001)

    Article  ADS  CAS  Google Scholar 

  16. Schindelin, H., Kisker, C., Hilton, J., Rajagopalan, K. V. & Rees, D. C. Crystal structure of DMSO reductase: redox-linked changes in molybdopterin coordination. Science 272, 1615–1621 (1996)

    Article  ADS  CAS  Google Scholar 

  17. Schrader, N. et al. The crystal structure of plant sulfite oxidase provides insights into sulfite oxidation in plants and animals. Structure 11, 1251–1263 (2003)

    Article  CAS  Google Scholar 

  18. Santamaria-Araujo, J. A. et al. The tetrahydropyranopterin structure of the sulfur-free and metal-free molybdenum cofactor precursor. J. Biol. Chem. 279, 15994–15999 (2004)

    Article  CAS  Google Scholar 

  19. Bertero, M. G. et al. Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A. Nature Struct. Biol. 10, 681–687 (2003)

    Article  CAS  Google Scholar 

  20. Nason, A. et al. In vitro formation of assimilatory reduced nicotinamide adenine dinucleotide phosphate: nitrate reductase from a Neurospora mutant and a component of molybdenum-enzymes. Proc. Natl Acad. Sci. USA 68, 3242–3246 (1971)

    Article  ADS  CAS  Google Scholar 

  21. Xiang, S., Nichols, J., Rajagopalan, K. V. & Schindelin, H. The crystal structure of Escherichia coli MoeA and its relationship to the multifunctional protein Gephyrin. Structure 9, 299–310 (2001)

    Article  CAS  Google Scholar 

  22. Schrag, J. D. et al. The crystal structure of Escherichia coli MoeA, a protein from the molybdopterin synthesis pathway. J. Mol. Biol. 310, 419–431 (2001)

    Article  CAS  Google Scholar 

  23. Moorhead, G. B. et al. Purification of a plant nucleotide pyrophosphatase as a protein that interferes with nitrate reductase and glutamine synthetase assays. Eur. J. Biochem. 270, 1356–1362 (2003)

    Article  CAS  Google Scholar 

  24. Mercer, J. F. The molecular basis of copper-transport diseases. Trends Mol. Med. 7, 64–69 (2001)

    Article  CAS  Google Scholar 

  25. Mason, J. Thiomolybdates: mediators of molybdenum toxicity and enzyme inhibitors. Toxicology 42, 99–109 (1986)

    Article  CAS  Google Scholar 

  26. Navaza, J. AMORE—an automated package for molecular replacement. Acta Crystallogr. A 50, 157–163 (1994)

    Article  Google Scholar 

  27. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  29. Guse, A. et al. Biochemical and structural analysis of the molybdenum cofactor biosynthesis protein MobA. J. Biol. Chem. 278, 25302–25307 (2003)

    Article  CAS  Google Scholar 

  30. Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet. 11, 281–296 (1991)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank R. N. Pau for inspiring discussion; T. Otte and F. Koenig for technical assistance; the staff at beamlines BW6 at DESY and PSF-BL2 at BESSY; and V. Wray for critically reading the manuscript. This work was supported by grants from the Deutsche Forschungsgemeinschaft (to H.J.H., R.R.M. and G.S.), and the Fonds der Chemischen Industrie and the Fritz Thyssen Stiftung (to R.R.M.).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Günter Schwarz.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Figure 1

Coordination of Cu-MPT and Cu-MPT-AMP. (DOC 196 kb)

Supplementary Figure 2

Structural comparison between Cnx1G and S583A. (DOC 867 kb)

Supplementary Figure 3

Structural comparison of MPT-AMP. (DOC 210 kb)

Supplementary Figure 4

Structural comparison between S583A and the Cnx1E-homologous MoeA. (DOC 1155 kb)

Supplementary Table 1

Data collection and refinement statistics. (DOC 32 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kuper, J., Llamas, A., Hecht, HJ. et al. Structure of the molybdopterin-bound Cnx1G domain links molybdenum and copper metabolism. Nature 430, 803–806 (2004). https://doi.org/10.1038/nature02681

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature02681

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