Near-atomic resolution structures of urate oxidase complexed with its substrate and analogues: the protonation state of the ligand.
Urate oxidase (uricase; EC 1.7.3.3; UOX) from Aspergillus flavus catalyzes the oxidation of uric acid in the presence of molecular oxygen to 5-hydroxyisourate in the degradation cascade of purines; intriguingly, catalysis proceeds using neither a metal ion (Fe, Cu etc.) nor a redox cofactor. UOX is a tetrameric enzyme with four active sites located at the interface of two subunits; its structure was refined at atomic resolution (1 A) using new crystal data in the presence of xanthine and at near-atomic resolution (1.3-1.7 A) in complexes with the natural substrate (urate) and two inhibitors: 8-nitroxanthine and 8-thiouric acid. Three new features of the structural and mechanistic behaviour of the enzyme were addressed. Firstly, the high resolution of the UOX-xanthine structure allowed the solution of an old structural problem at a contact zone within the tetramer; secondly, the protonation state of the substrate was determined from both a halochromic inhibitor complex (UOX-8-nitroxanthine) and from the H-atom distribution in the active site, using the structures of the UOX-xanthine and the UOX-uric acid complexes; and thirdly, it was possible to extend the general base system, characterized by the conserved catalytic triad Thr-Lys-His, to a large water network that is able to buffer and shuttle protons back and forth between the substrate and the peroxo hole along the reaction pathway.
Gabison
Laure
L
Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS, France.
Chiadmi
Mohamed
M
El Hajji
Mohamed
M
Castro
Bertrand
B
Colloc'h
Nathalie
N
Prangé
Thierry
T
D001231:Aspergillus flavus / Q000201:enzymology*; D018360:Crystallography, X-Ray; D008024:Ligands; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011522:Protons; D013379:Substrate Specificity; D014503:Urate Oxidase / Q000737:chemistry* / Q000378:metabolism; D014527:Uric Acid / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism
D008024:Ligands; D011522:Protons; D014527:Uric Acid; D014503:Urate Oxidase