The neutron structure of urate oxidase resolves a long-standing mechanistic conundrum and reveals unexpected changes in protonation

PLoS One. 2014 Jan 23;9(1):e86651. doi: 10.1371/journal.pone.0086651. eCollection 2014.

Abstract

Urate oxidase transforms uric acid to 5-hydroxyisourate without the help of cofactors, but the catalytic mechanism has remained enigmatic, as the protonation state of the substrate could not be reliably deduced. We have determined the neutron structure of urate oxidase, providing unique information on the proton positions. A neutron crystal structure inhibited by a chloride anion at 2.3 Å resolution shows that the substrate is in fact 8-hydroxyxanthine, the enol tautomer of urate. We have also determined the neutron structure of the complex with the inhibitor 8-azaxanthine at 1.9 Å resolution, showing the protonation states of the K10-T57-H256 catalytic triad. Together with X-ray data and quantum chemical calculations, these structures allow us to identify the site of the initial substrate protonation and elucidate why the enzyme is inhibited by a chloride anion.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aspergillus flavus / enzymology*
  • Catalysis
  • Crystallography
  • Crystallography, X-Ray
  • Models, Molecular
  • Neutrons*
  • Protein Conformation
  • Protons*
  • Urate Oxidase / chemistry*
  • Urate Oxidase / metabolism
  • Xanthines / chemistry
  • Xanthines / metabolism*

Substances

  • Protons
  • Xanthines
  • Urate Oxidase

Grants and funding

EO and MBS acknowledge the Finnish Academy for a post-doctoral fellowship to EO and the European Molecular Biology Organisation for a Long-term Fellowship to EO. EO acknowledges the Federation of European Biochemical Societies for a Short-term Fellowship for a research visit to Lund University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.