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

Azide inhibition of urate oxidase

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Etude du mécanisme de l'urate oxydase par diffraction des rayons X et spectroscopie RPE

L'urate oxydase est une enzyme-clé de la voie de dégradation des purines. Cette protéine, dépourvue de cofacteur (organique ou métallique), dégrade en présence d'oxygène moléculaire l'urate (mono-anion de l'acide urique) en 5-hydroxyisourate avec formation de peroxyde d'hydrogène. Le 5-hydroxyisourate conduit finalement à l'allantoïne, spontanément, ou via deux protéines spécifiques. Ce mécanisme enzymatique, étudié depuis le début du XXeme siècle, n'a cependant toujours pas été complètement élucidé. L'utilisation de la diffraction des rayons X et la spectroscopie RPE permettent d'apporter un nouvel éclairage sur le mécanisme de l'urate oxydase. La spectroscopie RPE met en évidence le caractère radicalaire du mécanisme et amène à proposer l'état électronique de certains intermédiaires. Les études tridimensionnelles par diffraction des rayons X des complexes enzyme-inhibiteur à haute résolution, suggèrent une déprotonation de l'urate lors de la première étape du mécanisme, pour donner un intermédiaire di-déprotoné sur les atomes N3 et N7 de l'acide urique. Les différentes approches pour bloquer le substrat naturel dans le site actif, ont confirmé cet état de déprotonation, et ont également permis de proposer le dehydrourate comme intermédiaire réactionnel stable. Finalement les différentes tentatives infructueuses pour détecter l'intermédiaire hypothétique 5-hydroperoxyisourate, mis en avant dans la littérature, ont conduit à formuler l'hypothèse soit un passage direct de l'urate radicalaire au dehydrourate, soit un passage indirect via le 4-hydroperoxyisourate. Le dehydrourate est ensuite hydroxylé et libéré du site actif. A partir de l ensemble de ces résultats, deux nouvelles alternatives du mécanisme sont suggéréesUrate oxidase is a key enzyme of the purine degradation pathway. This protein work without cofactor (metallic nor organic) and catalyse in presence of molecular oxygen, the degradation of urate (uric acid mono-anion) into 5-hydroxyisourate with release of hydrogen peroxide. 5-hydroxyisourate finally decomposes spontaneously or via two specific proteins to allantoïne. Urate oxidase mechanism is studied since the beginning of the century and it still remains nottotally understood. X-Ray diffraction and EPR spectroscopy allow bringing new lighting on urate oxidas mechanism. EPR spectroscopy shows the radical character of the mechanism and brings to propose the electronic state of some intermediate. Tridimensional studies by x-ray diffraction of high resolution inhibitor-enzyme complexes suggest urate déprotonation for the first step of the mechanism to give di-deprotonated intermediate on N3 and N7 atoms of uric acid. Several approach to block the natural substrate in the active site confirm deprotonated state and also permit to propose dehydrourate as stable relational intermediate. Finally different fruitless attempts to detect the hypothetic 5-hydroperoxyisourate intermediate from literature drove to formulate hypothesis of a direct passage to dehydrourate or generation of 4-hydroperoxyisourate. Dehydrourate is the hydroxyled and un-complexe from the active site. From all the result two news alternative mechanism were suggestedPARIS-BIUP (751062107) / SudocSudocFranceF

X-ray, ESR, and quantum mechanics studies unravel a spin well in the cofactor-less urate oxidase

International audienceUrate oxidase (EC 1.7.3.3 or UOX) catalyzes the conversion of uric acid using gaseous molecular oxygen to 5-hydroxyisourate and hydrogen peroxide in absence of any cofactor or transition metal. The catalytic mechanism was investigated using X-ray diffraction, electron spin resonance spectroscopy (ESR), and quantum mechanics calculations. The X-ray structure of the anaerobic enzyme-substrate complex gives credit to substrate activation before the dioxygen fixation in the peroxo hole, where incoming and outgoing reagents (dioxygen, water, and hydrogen peroxide molecules) are handled. ESR spectroscopy establishes the initial monoelectron activation of the substrate without the participation of dioxygen. In addition, both X-ray structure and quantum mechanic calculations promote a conserved base oxidative system as the main structural features in UOX that protonates/deprotonates and activate the substrate into the doublet state now able to satisfy the Wigner's spin selection rule for reaction with molecular oxygen in its triplet ground state

A Pressure-response analysis of anesthetic gases xenon and nitrous oxide on urate oxidase: a crystallographic study.

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Oxygen Pressurized X-Ray Crystallography: Probing the Dioxygen Binding Site in Cofactorless Urate Oxidase and Implications for Its Catalytic Mechanism

The localization of dioxygen sites in oxygen-binding proteins is a nontrivial experimental task and is often suggested through indirect methods such as using xenon or halide anions as oxygen probes. In this study, a straightforward method based on x-ray crystallography under high pressure of pure oxygen has been developed. An application is given on urate oxidase (UOX), a cofactorless enzyme that catalyzes the oxidation of uric acid to 5-hydroxyisourate in the presence of dioxygen. UOX crystals in complex with a competitive inhibitor of its natural substrate are submitted to an increasing pressure of 1.0, 2.5, or 4.0 MPa of gaseous oxygen. The results clearly show that dioxygen binds within the active site at a location where a water molecule is usually observed but does not bind in the already characterized specific hydrophobic pocket of xenon. Moreover, crystallizing UOX in the presence of a large excess of chloride (NaCl) shows that one chloride ion goes at the same location as the oxygen. The dioxygen hydrophilic environment (an asparagine, a histidine, and a threonine residues), its absence within the xenon binding site, and its location identical to a water molecule or a chloride ion suggest that the dioxygen site is mainly polar. The implication of the dioxygen location on the mechanism is discussed with respect to the experimentally suggested transient intermediates during the reaction cascade