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.
Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS, France.
Laure
L
Gabison
Mohamed
M
Chiadmi
Mohamed
M
El Hajji
Bertrand
B
Castro
Nathalie
N
Colloc'h
Thierry
T
Prangé
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