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

Nature volume 430, pages 803806 (12 August 2004) | Download Citation

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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.

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References

  1. 1.

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

  2. 2.

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

  3. 3.

    , , , & Structure of a hyperthermophilic tungstopterin, aldehyde ferredoxin oxidoreductase. Science 267, 1463–1469 (1995)

  4. 4.

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

  5. 5.

    Molybdenum bolsters the bioinorganic brigade. Science 272, 1599–1600 (1996)

  6. 6.

    & Molybdenum enzymes and sulfur metabolism. Met. Ions Biol. Syst. 39, 621–654 (2002)

  7. 7.

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

  8. 8.

    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)

  9. 9.

    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)

  10. 10.

    , , , & The active site of the molybdenum cofactor biosynthetic protein domain Cnx1G. Arch. Biochem. Biophys. 411, 36–46 (2003)

  11. 11.

    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)

  12. 12.

    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)

  13. 13.

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

  14. 14.

    , , & 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)

  15. 15.

    , , & Mechanism of ubiquitin activation revealed by the structure of a bacterial MoeB–MoaD complex. Nature 414, 325–329 (2001)

  16. 16.

    , , , & Crystal structure of DMSO reductase: redox-linked changes in molybdopterin coordination. Science 272, 1615–1621 (1996)

  17. 17.

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

  18. 18.

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

  19. 19.

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

  20. 20.

    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)

  21. 21.

    , , & The crystal structure of Escherichia coli MoeA and its relationship to the multifunctional protein Gephyrin. Structure 9, 299–310 (2001)

  22. 22.

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

  23. 23.

    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)

  24. 24.

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

  25. 25.

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

  26. 26.

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

  27. 27.

    , , & 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)

  28. 28.

    , & Refinement of macromolecular structures by the maximum likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

  29. 29.

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

  30. 30.

    , & Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet. 11, 281–296 (1991)

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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

Author notes

    • Jochen Kuper

    Present address: EMBL Hamburg outstation, DESY, D-22603 Hamburg, Germany

Affiliations

  1. Department of Plant Biology, Technical University, Spielmannstrasse 7, D-38106 Braunschweig, Germany

    • Jochen Kuper
    • , Angel Llamas
    • , Ralf R. Mendel
    •  & Günter Schwarz
  2. German Research Center for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany

    • Hans-Jürgen Hecht

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Competing interests

The authors declare that they have no competing financial interests.

Corresponding author

Correspondence to Günter Schwarz.

Supplementary information

Word documents

  1. 1.

    Supplementary Figure 1

    Coordination of Cu-MPT and Cu-MPT-AMP.

  2. 2.

    Supplementary Figure 2

    Structural comparison between Cnx1G and S583A.

  3. 3.

    Supplementary Figure 3

    Structural comparison of MPT-AMP.

  4. 4.

    Supplementary Figure 4

    Structural comparison between S583A and the Cnx1E-homologous MoeA.

  5. 5.

    Supplementary Table 1

    Data collection and refinement statistics.

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DOI

https://doi.org/10.1038/nature02681

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