Structural analysis of urate oxidase in complex with its natural substrate inhibited by cyanide: Mechanistic implications

<p>Abstract</p> <p>Background</p> <p>Urate oxidase (EC 1.7.3.3 or UOX) catalyzes the conversion of uric acid and gaseous molecular oxygen to 5-hydroxyisourate and hydrogen peroxide, in the absence of cofactor or particular metal cation. The functional enzyme is a homo-tetramer with four active sites located at dimeric interfaces.</p> <p>Results</p> <p>The catalytic mechanism was investigated through a ternary complex formed between the enzyme, uric acid, and cyanide that stabilizes an intermediate state of the reaction. When uric acid is replaced by a competitive inhibitor, no complex with cyanide is formed.</p> <p>Conclusion</p> <p>The X-ray structure of this compulsory ternary complex led to a number of mechanistic evidences that support a sequential mechanism in which the two reagents, dioxygen and a water molecule, process through a common site located 3.3 Å above the mean plane of the ligand. This site is built by the side chains of Asn 254, and Thr 57, two conserved residues belonging to two different subunits of the homo-tetramer. The absence of a ternary complex between the enzyme, a competitive inhibitor, and cyanide suggests that cyanide inhibits the hydroxylation step of the reaction, after the initial formation of a hydroperoxyde type intermediate.</p

Analyse qualitative des surfaces moleculaires a l'aide de fonctions de lissage B-spline et etudes topologiques quantitatives : application aux proteines

CNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueSIGLEFRFranc

Crystallographic studies under high hydrostatic pressure and under moderate gas pressure : examples of urate oxidase and neuroglobin

CERVOxyNational audienc

X-ray crystallography of proteins and gas: methods and interpretation

CERVOxyInternational audienc

Exploration des caractéristiques tridimensionnelles des amas protéiques hydrophobes issus du formalisme "Hydrophobic Cluster Analysis" (HCA) (modélisation de formes oligomériques solubles du peptide Ab impliqué dans la maladie d'Alzheimer, et identification d'un 'point chaud" commun à différentes protéines amyloïdes)

Nous avons d abord exploré les caractéristiques 3D des amas d acides aminés hydrophobes issus de la méthode "Hydrophobic Cluster Analysis" (HCA), au travers d une représentation originale en tesselation de Voronoï. Chaque amas peut être ainsi caractérisé dans ses conformations ou et ses affinités préférentielles pour d autres amas peuvent être décrites. Le repliement protéique peut alors être décrit comme l assemblage de ces amas HCA 3D. Nous avons ensuite construit, en utilisant des outils d alignement tels que HCA, des modèles 3D des formes oligomériques solubles du peptide A . Ce peptide, impliqués dans la maladie d Alzheimer, serait neurotoxique sous cette forme soluble. Ce travail nous a permis de proposer une hypothèse expliquant le comportement différencié de p3, sans la région N-terminale d A . Enfin, nous avons pu mettre en évidence la présence d un ou plusieurs "points chauds" communs aux protéines amyloïdes, pouvant être à l origine de leur propriété à former des fibres.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Azide inhibition of urate oxidase

International audienc

Sequence-Based Modeling of Ab42 Soluble Oligomers

International audienceAb fibrils, which are central to the pathology of Alzheimer's disease, form a cross-b-structure that contains likely parallel b-sheets with a salt bridge between residues Asp23 and Lys28. Recent studies suggest that soluble oligomers of amyloid peptides have neurotoxic effects in cell cultures, raising the interest in studying the structures of these intermediate forms. Here, we present three models of possible soluble Ab forms based on the sequences similarities, assumed to support local structural similarities, of the Ab peptide with fragments of three proteins (adhesin, Semliki Forest virus capsid protein, and transthyretin). These three models share a similar structure in the C-terminal region composed of two b-strands connected by a loop, which contain the Asp23-Lys28 salt bridge. This segment is also structurally well conserved in Ab fibril forms. Differences between the three monomeric models occur in the N- terminal region and in the C-terminal tail. These three models might sample some of the most stable conformers of the soluble Ab peptide within oligomeric assemblies, which were modeled here in the form of dimers, trimers, tetramers, and hexamers. The consistency of these models is discussed with respect to available experimental and theorical data

Exploring hydrophobic sites in proteins with xenon or krypton

International audienceX-ray diffraction is used to study the binding of xenon and krypton to a variety of crystallised proteins: porcine pancreatic elastase; subtilisin Carlsberg from Bacillus licheniformis; cutinase from Fusarium solani; collagenase from Hypoderma lineatum; hen egg lysozyme, the lipoamide dehydrogenase domain from the outer membrane protein P64k from Neisseria meningitidis; urate-oxidase from Aspergillus flavus, mosquitocidal delta-endotoxin CytB from Bacillus thuringiensis and the ligand-binding domain of the human nuclear retinoid-X receptor RXR-alpha. Under gas pressures ranging from 8 to 20 bar, xenon is able to bind to discrete sites in hydrophobic cavities, ligand and substrate binding pockets, and into the pore of channel-like structures. These xenon complexes can be used to map hydrophobic sites in proteins, or as heavy-atom derivatives in the isomorphous replacement method of structure determination

Urate oxidase from Aspergillus flavus : new crystal-packing contacts in relation to the content of the active site

International audienceUrate oxidase from Aspergillus flavus (uricase or Uox; EC 1.7.3.3) is a 135 kDa homotetramer with a subunit consisting of 301 amino acids. It catalyses the first step of the degradation of uric acid into allantoin. The structure of the extracted enzyme complexed with a purine-type inhibitor (8-aza-xanthin) had been solved from high-resolution X-ray diffraction of I222 crystals. Expression of the recombinant enzyme in Saccharomyces cerevisiae followed by a new purification procedure allowed the crystallization of both unliganded and liganded enzymes utilizing the same conditions but in various crystal forms. Here, four different crystal forms of Uox are analyzed. The diversity of the Uox crystal forms appears to depend strongly on the chemicals used as inhibitors. In the presence of uracil and 5,6-diaminouracil crystals usually belong to the trigonal space group P3(1)21, the asymmetric unit (AU) of which contains one tetramer of Uox (four subunits). Chemical oxidation of 5,6-diaminouracil within the protein may occur, leading to the canonical (I222) packing with one subunit per AU. Coexistence of two crystal forms, P2(1) with two tetramers per AU and I222, was found in the same crystallization drop containing another inhibitor, guanine. Finally, a fourth form, P2(1)2(1)2 with one tetramer per AU, resulted fortuitously in the presence of cymelarsan, an additive. Of all the reported forms, the I222 crystal forms present by far the best X-ray diffraction resolution (similar to1.6 Angstrom resolution compared with 2.3-3.2 Angstrom for the other forms). The various structures and contacts in all crystalline lattices are compared. The backbones are essentially conserved except for the region near the active site. Its location at the dimer interface is thus likely to be at the origin of the crystal contact changes as a response to the various bound inhibitors