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

• Published in: PloS one 2014-01, Vol.9 (1), p.e86651-e86651
• Main Authors: Oksanen, Esko, Blakeley, Matthew P, El-Hajji, Mohamed, Ryde, Ulf, Budayova-Spano, Monika
• Format: Article
• Language: English
• Subjects: Anions; Aspergillus flavus; Aspergillus flavus - enzymology; Automation; Biochemistry, Molecular Biology; Biology; Catalysis; Chemical Sciences; Chemistry; Chlorides; Cofactors; Crystal structure; Crystallography; Crystallography, X-Ray; Enzymes; Hydrogen; Kemi; Life Sciences; Models, Molecular; Natural Sciences; Naturvetenskap; Neutrons; Oxidase; Oxidases; Physics; Physiology; Protein Conformation; Protonation; Protons; Quantum chemistry; Structural Biology; Studies; Substrates; Teoretisk kemi; Theoretical Chemistry; Urate Oxidase; Urate Oxidase - chemistry; Urate Oxidase - metabolism; Uric acid; Xanthines; Xanthines - chemistry; Xanthines - metabolism
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0086651

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.