Publications

• Urate oxidase from the rust Puccinia recondita is a heterotetramer with two different-sized monomers.

  Aguilar M., Montalbini P., Pineda M.

  Uricase (urate: oxygen oxidoreductase; EC 1.7.3.3) from the rust Puccinia recondita was purified to electrophoretic homogeneity. Preparations with a specific activity of 8.4 U/mg were used for characterization of the enzyme, which showed a strong similarity to other plant and fungal urate oxidases ... >> More

  Uricase (urate: oxygen oxidoreductase; EC 1.7.3.3) from the rust Puccinia recondita was purified to electrophoretic homogeneity. Preparations with a specific activity of 8.4 U/mg were used for characterization of the enzyme, which showed a strong similarity to other plant and fungal urate oxidases. The enzyme had a pH optimum of 9.0, a K(m) of 35 microM for urate, and it was inhibited only by oxonate and xanthine. A molecular mass of 152 kDa was estimated for the native protein. SDS-PAGE analysis revealed a striking difference to most urate oxidases, since two different-sized subunits were detected. These results suggest that P. recondita uricase is a tetramer with two types of subunits. << Less

  Curr. Microbiol. 44:257-261(2002) [PubMed] [EuropePMC]

• Spectroscopic characterization of intermediates in the urate oxidase reaction.

  Kahn K., Tipton P.A.

  The oxidation of urate catalyzed by soybean urate oxidase was studied under single-turnover conditions using stopped-flow absorbance and fluorescence spectrophotometry. Two discrete enzyme-bound intermediates were observed; the first intermediate to form had an absorbance maximum at 295 nm and was ... >> More

  The oxidation of urate catalyzed by soybean urate oxidase was studied under single-turnover conditions using stopped-flow absorbance and fluorescence spectrophotometry. Two discrete enzyme-bound intermediates were observed; the first intermediate to form had an absorbance maximum at 295 nm and was assigned to a urate dianion species; the second intermediate had an absorbance maximum at 298 nm and is believed to be urate hydroperoxide. These data are consistent with a catalytic mechanism that involves formation of urate hydroperoxide from O2 and the urate dianion, collapse of the peroxide to form dehydrourate, and hydration of dehydrourate to form the observed product, 5-hydroxyisourate. The rate of formation of the first intermediate was too fast to measure accurately at 20 degreesC; the second intermediate formed with a rate constant of 32 s-1 and decayed with a rate constant of 6.6 s-1. The product of the reaction, 5-hydroxyisourate, is fluorescent, and its release from the active site occurred with a rate constant of 31 s-1. << Less

  Biochemistry 37:11651-11659(1998) [PubMed] [EuropePMC]

• The role of sex chromosomes in black fly evolution.

  Feraday R.M., Leonhardt K.G., Brockhouse C.L.

  Sex chromosomes have been repeatedly implicated in the process of speciation of black flies and other nemotocerans. Arguments are presented here against the case that frequent differences between species in their sex chromosomes are based on (i) different average rates of differentiation of sex-li ... >> More

  Sex chromosomes have been repeatedly implicated in the process of speciation of black flies and other nemotocerans. Arguments are presented here against the case that frequent differences between species in their sex chromosomes are based on (i) different average rates of differentiation of sex-linked and autosomal loci or (ii) the fact that the X and Y chromosomes are less numerous than autosomal chromosomes and so are more subject to the effects of drift and the random fixation of chromosome rearrangements. The argument is made that speciation in black flies and many other groups is an adaptive process and that differentiated sex-chromosome systems play a role in this process. << Less

  Genome 32:538-542(1989) [PubMed] [EuropePMC]

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

  Gabison L., Prange T., Colloc'h N., El Hajji M., Castro B., Chiadmi M.

  <h4>Background</h4>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 dime ... >> More

  <h4>Background</h4>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.<h4>Results</h4>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.<h4>Conclusion</h4>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 A 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. << Less

  BMC Struct. Biol. 8:32-32(2008) [PubMed] [EuropePMC]

• The all2 gene is required for the induction of the purine deamination pathway in Schizosaccharomyces pombe.

  Fluri R., Kinghorn J.R.

  Five mutants were isolated at the all2 gene on the basis of their inability to utilize hypoxanthine as a sole source of nitrogen. These mutants failed to utilize the purines adenine, hypoxanthine, xanthine, uric acid, allantoin and allantoic acid, although they could utilize urea and ammonium. The ... >> More

  Five mutants were isolated at the all2 gene on the basis of their inability to utilize hypoxanthine as a sole source of nitrogen. These mutants failed to utilize the purines adenine, hypoxanthine, xanthine, uric acid, allantoin and allantoic acid, although they could utilize urea and ammonium. The all2 mutants appeared to be defective in purine induction of uricase, allantoinase, allantoicase and ureidoglycollase activities but retained wild-type activity of the constitutively synthesized urease. The all2 mutations were recessive. << Less

  J. Gen. Microbiol. 131:527-532(1985) [PubMed] [EuropePMC]

• Oxygen pressurized X-ray crystallography: probing the dioxygen binding site in cofactorless urate oxidase and implications for its catalytic mechanism.

  Colloc'h N., Gabison L., Monard G., Altarsha M., Chiadmi M., Marassio G., Sopkova-de Oliveira Santos J., El Hajji M., Castro B., Abraini J.H., Prange T.

  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 ... >> More

  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. << Less

  Biophys. J. 95:2415-2422(2008) [PubMed] [EuropePMC]