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.

  • Article
  • Published:

Crystal structure of a plant catechol oxidase containing a dicopper center

Nature Structural Biology volume 5, pages 1084–1090 (1998)Cite this article

Abstract

Catechol oxidases are ubiquitous plant enzymes containing a dinuclear copper center. In the wound-response mechanism of the plant they catalyze the oxidation of a broad range of ortho-diphenols to the corresponding o-quinones coupled with the reduction of oxygen to water. The crystal structures of the enzyme from sweet potato in the resting dicupric Cu(II)-Cu(II) state, the reduced dicuprous Cu(I)-Cu(I) form, and in complex with the inhibitor phenylthiourea were analyzed. The catalytic copper center is accommodated in a central four-helix-bundle located in a hydrophobic pocket close to the surface. Both metal binding sites are composed of three histidine ligands. His 109, ligated to the CuA site, is covalently linked to Cys 92 by an unusual thioether bond. Based on biochemical, spectroscopic and the presented structural data, a catalytical mechanism is proposed in which one of the oxygen atoms of the diphenolic substrate binds to CuB of the oxygenated enzyme.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Ribbon drawings of catechol oxidase.
Figure 2: A sample of 2 |Fo| - |Fc|Φcalc electron density, for the oxidized catalytic dinuclear copper site.
Figure 3: Active site region of catechol oxidase.
Figure 4: Superposition of the dinuclear copper center of sweet potato catechol oxidase with bound phenylthiourea (PTU) with the oxygenated form of Limulus polyphemus hemocyanin19.
Figure 5: Proposed reaction pathway of catechol oxidase, based on biochemical, spectroscopic, and structural data.
Figure 6: Sequence alignment of sweet potato catechol oxidase (ibCO), human tyrosinase (hsTYR), Neurospora crassa tyrosinase (ncTYR), Panulirus interruptus hemocyanin (piHC), Limulus polyphemus (lpHC), Helix pomatia hemocyanin (hpHC), and the subunit g of Octopus dofleini hemocyanin (odgHC).

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Karlin, K. D. Metalloenzymes, structural motifs, and inorganic models. Science 261, 701–708 (1993).

    Article  CAS  Google Scholar 

  2. Solomon, E. I., Sundaram, U. M. & Machonkin, T. E. Multicopper oxidases and oxygenases. Chem. Rev. 96, 2563–2605 (1996).

    Article  CAS  Google Scholar 

  3. Magnus, K. A., Ton-That, H. & Carpenter, J. E. Recent structural work on the oxygen transport protein hemocyanin. Chem. Rev. 94, 727–735 (1994).

    Article  CAS  Google Scholar 

  4. Eicken, C., Zippel, F., Büldt-Karentzopoulos, K. & Krebs, B. Biochemical and spectroscopic characterization of catechol oxidase from sweet potatoes (Ipomoea batatas) containing a type-3 dicopper center. FEBS Lett. 436, 293–299 (1998).

    Article  CAS  Google Scholar 

  5. Lerch, K. Neurospora tyrosinase: structural, spectroscopic and catalytic properties. Mol. Cell. Biochem. 52, 125–138 (1983).

    Article  CAS  Google Scholar 

  6. Cary, J. W., Lax, A. R. & Flurkey, W. H. Cloning and characterization of cDNAs coding for Vicia faba polyphenol oxidase. Plant Mol. Biol. 20, 245–253 (1992).

    Article  CAS  Google Scholar 

  7. Dervall, B. J. Phenolase and pectic enzyme activity in the chocolate spot disease of beans. Nature 189, 311 (1961).

    Article  Google Scholar 

  8. Rompel, A. et al. Spectroscopic and EXAFS studies on catechol oxidases with dinuclear copper centers of type 3: evidence for μ-η2: η2-peroxo-intermediates during the reaction with catechol. J. Inorg. Biochem. 59,–715 (1995).

    Google Scholar 

  9. Ito, N. et al. Novel thioether bond revealed by a 1.7 Å crystal structure of galactose oxidase. Nature 350, 87–90 (1991).

    Article  CAS  Google Scholar 

  10. Lerch, K. Primary structure of tyrosinase from Neurospora crassa. II. Complete amino acid sequence and chemical structure of a tripeptide containing an unusual thioether. J. Biol. Chem. 257, 6414–6419 (1982).

    CAS  PubMed  Google Scholar 

  11. Gielens, C. et al. Evidence for a cysteine-histidine thioether bridge in functional units of molluscan haemocyanins and location of the disulfide bridges in functional units d and g of the betaC-haemocyanin of Helix pomatia. Eur. J. Biochem. 248, 879–888 (1997).

    Article  CAS  Google Scholar 

  12. Miller, K. I. et al. Sequence of the Octopus dofleini hemocyanin subunit: structural and evolutionary implications. J. Mol. Biol. 278, 827–842 (1998).

    Article  CAS  Google Scholar 

  13. Cuff, M. E., Miller, K. I., van Holde, K. E. & Hendrickson, W.A. Crystal structure of a functional unit from octopus hemocyanin. J. Mol. Biol. 278, 855–870 (1998).

    Article  CAS  Google Scholar 

  14. Gaykema, W. P. J et al. 3.2 Å structure of the copper-containing, oxygen-carrying protein Panulirus interruptus haemocyanin. Nature 309, 23–29 (1984).

    Article  CAS  Google Scholar 

  15. Volbeda, A. & Hol, W. G. J. Crystal structure of hexameric haemocyanin from Panulirus interruptus refined at 3.2 A resolution. J. Mol. Biol. 209, 249–279 (1989).

    Article  CAS  Google Scholar 

  16. Hazes, B. et al. Crystal structure of deoxygenated Limulus polyphemus subunit II hemocyanin at 2.18 A resolution: clues for a mechanism for allosteric regulation. Prot. Sci. 2, 576–619 (1993).

    Google Scholar 

  17. Wilcox, D. E. et al. Substrate analogue binding to the coupled binuclear copper active site in tyrosinase. J. Am. Chem. Soc. 107, 4015–4027 (1985).

    Article  CAS  Google Scholar 

  18. Solomon, E. I., Sundaram, U. M. & Machonkin, T. E. Multicopper oxidases and oxygenases. Chem. Rev. 96, 2563–2605 (1996).

    Article  CAS  Google Scholar 

  19. Magnus, K.A. et al. Crystallographic analysis of oxgyenated and deoxygenated states of arthropod hemocyanin shows unusual differences. Proteins 19, 302–309 (1994).

    Article  CAS  Google Scholar 

  20. Himmelwright, R. S., Eickman, N. C., LuBien, C. D. & Lerch, K. and Solomon, E. I. Chemical and spectroscopic studies of the binuclear copper active site of Neurospora tyrosinase: comparison to hemocyanins. J. Am. Chem. Soc. 102, 7339–7344 (1980).

    Article  CAS  Google Scholar 

  21. Jackman, M.P., Hajnal, A. & Lerch, K. Albino mutants of Streptomyces glaucescens tyrosinase. Biochem. J. 274, 707–713 (1991).

    Article  CAS  Google Scholar 

  22. Otwinowski, Z. Oscillation Data Reduction Program. In Proceedings of the CCP4 study weekend: data collection and processing (eds Sawyer, L., Isaacs, N., Bailey, S.) 56–62 (SERC Daresbury Laboratory, Warrington, UK; 1993).

    Google Scholar 

  23. Furey,W. & Swaminathan, S. PHASES. A program package for processing and analysis of diffraction data from macromolecules. Meth. Enz. 277, 590–620 (1995).

    Article  Google Scholar 

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

  25. de La Fortelle, E. & Bricogne, G. Meth. Enz. 276, 472–494.

  26. Cowtan, K. D. & Main, P. Phase combination and cross validation in iterated density-modification calculations. Acta Crystallogr. D 52, 43–48 (1996).

    Article  CAS  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. Brünger, A. T., Kuriyan, J. & Karplus, M. Crystallographic R factor refinement by molecular dynamics. Science 235, 458–460 (1987).

    Article  Google Scholar 

  29. Navaza, J. AMoRe: an automated package for molecular replacement. Acta Crystallogr. A 50, 157–163 (1994).

    Article  Google Scholar 

  30. Evans, S. V. SETOR: hardware lighted three-dimensional solid model representations of macromolecules. J. Mol. Graphics 11, 134–138 (1993).

    Article  CAS  Google Scholar 

  31. Christopher, J. A. SPOCK: The structural properties observation and calculation kit (program manual). the center for macromolecular design. (Texas A&M University, College Station, Texas, USA;1998).

  32. Brünger, A. T. Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. Nature 355, 472–475 (1992).

    Article  Google Scholar 

Download references

Acknowledgements

Supported by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, the Welch Foundation, and the NIH. T.K. thanks the Deutsche Forschungsgemeinschaft for a postdoctoral fellowship. We thank N. Sträter for reviewing the manuscript and V. L. Pecoraro for many helpful discussions. We all express our appreciation to the late Herbert Witzel for his commitment and contributions to this science. This study is in partial fulfillment of the requirements of C.E. for the degree of Dr. rer. nat. at the Westfälische Wilhelms-Universität Münster.

Author information

Author notes

  1. Thomas Klabunde, Christoph Eicken and Bernt Krebs: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Texas A&M University, College Station, 77843-2128, Texas, USA

    Thomas Klabunde & James C. Sacchettini

  2. Anorganisch-Chemisches Institut, Universität Münster, Münster, 48149, Germany

    Christoph Eicken & Bernt Krebs

Authors
  1. Thomas Klabunde
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christoph Eicken
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. James C. Sacchettini
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bernt Krebs
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to James C. Sacchettini or Bernt Krebs.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Klabunde, T., Eicken, C., Sacchettini, J. et al. Crystal structure of a plant catechol oxidase containing a dicopper center. Nat Struct Mol Biol 5, 1084–1090 (1998). https://doi.org/10.1038/4193

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/4193

This article is cited by

Search

Advanced 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