Complexed and ligand-free high-resolution structures of urate oxidase (Uox) from<i>Aspergillus flavus</i>: a reassignment of the active-site binding mode

Retailleau, Pascal; Colloc’h, N.; Vivarès, Denis; Bonneté, Françoise; Castro, Bertrand; El-Hajji, Mohamed; Mornon, J.‐P.; Monard, Gérald; Prangé, Thierry

doi:10.1107/s0907444903029718

Cited by 90 publications

(118 citation statements)

References 26 publications

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“…UOX crystals were grown either by batch and hangingdrop methods using a 10-15 mg/ml solution of UOX, with an excess of 8-azaxanthine (purchased from Sigma-Aldrich, Lyon, France), in 50 mM Tris/acetate (pH 8) in the presence of 5-8% PEG 8000. This led to crystal in space group I222 with 1 monomer per asymmetric unit [7].…”

Section: Crystallization and Data Collectionsmentioning

confidence: 99%

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Functional relevance of the internal hydrophobic cavity of urate oxidase

Colloc’h

Prangé

2014

FEBS Letters

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a b s t r a c tUrate oxidase from Aspergillus flavus is a 135 kDa homo-tetramer which has a hydrophobic cavity buried within each monomer and located close to its active site. Crystallographic studies under moderate gas pressure and high hydrostatic pressure have shown that both gas presence and high pressure would rigidify the cavity leading to an inhibition of the catalytic activity. Analysis of the cavity volume variations and functional modifications suggest that the flexibility of the cavity would be an essential parameter for the active site efficiency. This cavity would act as a connecting vessel to give flexibility to the neighboring active site, and its expansion under pure oxygen pressure reveals that it might serve as a transient reservoir on its pathway to the active site.

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Section: Crystallization and Data Collectionsmentioning

confidence: 99%

“…The active site is located at the interface between two subunits folded around a tunnel. 8-azaxanthine is locked in the active site by a Arg 176-Gln 228 molecular tweezers [6,7].…”

Section: Introductionmentioning

confidence: 99%

Functional relevance of the internal hydrophobic cavity of urate oxidase

Colloc’h

Prangé

2014

FEBS Letters

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“…Crystallization has the inherent advantages of providing higher final purity yields, not denaturing the protein of interest and often providing some stabilization effects, but it requires a good knowledge of the phase diagram and a substantial amount of the protein to be crystallized. The structure of the urate oxidase from A. flavus has been solved in the absence (Retailleau et al, 2004) as well as in the presence of different inhibitors (Retailleau et al, 2005). It is a homotetrameric enzyme of 135kDa with a subunit consisting of 301 amino acids.…”

Section: A Protein Of Pharmaceutical Interest: Urate Oxidasementioning

confidence: 99%

Macromolecular Crystallization Controlled by Colloidal Interactions: The Case of Urate Oxidase

Bonneté¹

2012

Crystallization - Science and Technology

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“…Uric acid oxidase has been crystallized in the presence of several substrate analogues, including 9-methyluric acid [15]. In that case, the compound binds as the 2,6,8-tri-keto species where both N-3 and N-7 are deprotonated.…”

Section: Lack Of Binding By Related Compoundsmentioning

confidence: 99%

Characterization of active site variants of xanthine hydroxylase from Aspergillus nidulans

Li¹,

Müller²,

Fraser³

et al. 2008

Archives of Biochemistry and Biophysics

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Xanthine/α-ketoglutarate (αKG) dioxygenase (XanA) is a non-heme mononuclear Fe II enzyme that decarboxylates αKG to succinate and CO 2 while hydroxylating xanthine to generate uric acid. In the absence of a XanA crystal structure, a homology model was used to target several putative active site residues for mutagenesis. Wild-type XanA and ten enzyme variants were purified from recombinant Escherichia coli cells and characterized. The H149A and D151A variants were inactive and the H340A variant exhibited only 0.17 % of the wild-type enzyme activity, consistent with the proposed role of His149, Asp151, and His340 as Fe ligands. The K122A variant led to a 2-fold increase in the K d of αKG as measured by fluorescence quenching analysis, in agreement with Lys122 acting to stabilize the binding of αKG. The N358A variant exhibited a 23-fold decrease in k cat /K m compared to wild-type XanA, pointing to a key role of Asn358 in catalysis. 9-Methylxanthine was exploited as an alternate substrate, and the C357A, E137A, and D138A variants were found to exhibit relatively enhanced activity consistent with Cys357, Glu137, and Asp138 being proximal to N-9 or involved in its proper positioning. 6,8-Dihydroxypurine was identified as a slow-binding competitive inhibitor of XanA, and significant decreases (E137A and D138A) or increases (Q356A and N358A) in K i app of the variants were interpreted in terms of distinct interactions between this compound and the corresponding active site side chains. Further support for Cys357 residing at the active site was obtained using thiol-specific reagents that inactivated wild-type enzyme (with partial protection by substrate), whereas the C357A variant was resistant to these reagents. The Q101A, Q356A, and C357A variants showed elevated ferroxidase activity in the absence of substrates, pointing to the presence of the corresponding side chains at the active site. These results confirm most aspects of the homology model and provide additional insight into the enzyme reactivity. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. , and His340 residues of the protein, (ii) Lys122 and Arg352 stabilize the binding of αKG (which by analogy to other systems chelates the metal via its C-1 carboxylate and C-2 keto group) by hydrogen bonding to the C-1 carboxylate and salt bridge formation with the C-5 carboxylate, and (iii) the active site pocket is lined with potential hydrogen bond donors or acceptors (Gln99, Gln101, Lys122, Glu137, Asp138, Gln356, Cys357, and Asn358) whereas aromatic residues are not important for binding substrate. Keywords NIH Public ...

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Complexed and ligand-free high-resolution structures of urate oxidase (Uox) fromAspergillus flavus: a reassignment of the active-site binding mode

Cited by 90 publications

References 26 publications

Functional relevance of the internal hydrophobic cavity of urate oxidase

Functional relevance of the internal hydrophobic cavity of urate oxidase

Macromolecular Crystallization Controlled by Colloidal Interactions: The Case of Urate Oxidase

Characterization of active site variants of xanthine hydroxylase from Aspergillus nidulans

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