Publications

• Human xanthine oxidase changes its substrate specificity to aldehyde oxidase type upon mutation of amino acid residues in the active site: roles of active site residues in binding and activation of purine substrate.

  Yamaguchi Y., Matsumura T., Ichida K., Okamoto K., Nishino T.

  Xanthine oxidase (oxidoreductase; XOR) and aldehyde oxidase (AO) are similar in protein structure and prosthetic group composition, but differ in substrate preference. Here we show that mutation of two amino acid residues in the active site of human XOR for purine substrates results in conversion ... >> More

  Xanthine oxidase (oxidoreductase; XOR) and aldehyde oxidase (AO) are similar in protein structure and prosthetic group composition, but differ in substrate preference. Here we show that mutation of two amino acid residues in the active site of human XOR for purine substrates results in conversion of the substrate preference to AO type. Human XOR and its Glu803-to-valine (E803V) and Arg881-to-methionine (R881M) mutants were expressed in an Escherichia coli system. The E803V mutation almost completely abrogated the activity towards hypoxanthine as a substrate, but very weak activity towards xanthine remained. On the other hand, the R881M mutant lacked activity towards xanthine, but retained slight activity towards hypoxanthine. Both mutants, however, exhibited significant aldehyde oxidase activity. The crystal structure of E803V mutant of human XOR was determined at 2.6 A resolution. The overall molybdopterin domain structure of this mutant closely resembles that of bovine milk XOR; amino acid residues in the active centre pocket are situated at very similar positions and in similar orientations, except that Glu803 was replaced by valine, indicating that the decrease in activity towards purine substrate is not due to large conformational change in the mutant enzyme. Unlike wild-type XOR, the mutants were not subject to time-dependent inhibition by allopurinol. << Less

  J. Biochem. 141:513-524(2007) [PubMed] [EuropePMC]

• Inhibition studies of bovine xanthine oxidase by luteolin, silibinin, quercetin, and curcumin.

  Pauff J.M., Hille R.

  Xanthine oxidoreductase (XOR) is a molybdenum-containing enzyme that under physiological conditions catalyzes the final two steps in purine catabolism, ultimately generating uric acid for excretion. Here we have investigated four naturally occurring compounds that have been reported to be inhibito ... >> More

  Xanthine oxidoreductase (XOR) is a molybdenum-containing enzyme that under physiological conditions catalyzes the final two steps in purine catabolism, ultimately generating uric acid for excretion. Here we have investigated four naturally occurring compounds that have been reported to be inhibitors of XOR in order to examine the nature of their inhibition utilizing in vitro steady-state kinetic studies. We find that luteolin and quercetin are competitive inhibitors and that silibinin is a mixed-type inhibitor of the enzyme in vitro, and, unlike allopurinol, the inhibition is not time-dependent. These three natural products also decrease the production of superoxide by the enzyme. In contrast, and contrary to previous reports in the literature based on in vivo and other nonmechanistic studies, we find that curcumin did not inhibit the activity of purified XO nor its superoxide production in vitro. << Less

  J Nat Prod 72:725-731(2009) [PubMed] [EuropePMC]

• Mammalian xanthine oxidoreductase - mechanism of transition from xanthine dehydrogenase to xanthine oxidase.

  Nishino T., Okamoto K., Eger B.T., Pai E.F., Nishino T.

  Reactive oxygen species are generated by various biological systems, including NADPH oxidases, xanthine oxidoreductase, and mitochondrial respiratory enzymes, and contribute to many physiological and pathological phenomena. Mammalian xanthine dehydrogenase (XDH) can be converted to xanthine oxidas ... >> More

  Reactive oxygen species are generated by various biological systems, including NADPH oxidases, xanthine oxidoreductase, and mitochondrial respiratory enzymes, and contribute to many physiological and pathological phenomena. Mammalian xanthine dehydrogenase (XDH) can be converted to xanthine oxidase (XO), which produces both superoxide anion and hydrogen peroxide. Recent X-ray crystallographic and site-directed mutagenesis studies have revealed a highly sophisticated mechanism of conversion from XDH to XO, suggesting that the conversion is not a simple artefact, but rather has a function in mammalian organisms. Furthermore, this transition seems to involve a thermodynamic equilibrium between XDH and XO; disulfide bond formation or proteolysis can then lock the enzyme in the XO form. In this review, we focus on recent advances in our understanding of the mechanism of conversion from XDH to XO. << Less

  FEBS J 275:3278-3289(2008) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• QM/MM studies of xanthine oxidase: variations of cofactor, substrate, and active-site Glu802.

  Metz S., Thiel W.

  In continuation of our previous QM/MM study on the reductive half-reaction of wild-type xanthine oxidase, we consider the effects of variations in the cofactor, the substrate, and the active-site Glu802 residue on the reaction mechanism. Replacement of the sulfido ligand in the natural cofactor by ... >> More

  In continuation of our previous QM/MM study on the reductive half-reaction of wild-type xanthine oxidase, we consider the effects of variations in the cofactor, the substrate, and the active-site Glu802 residue on the reaction mechanism. Replacement of the sulfido ligand in the natural cofactor by an oxo ligand leads to a substantial increase in the computed barriers, consistent with the experimentally observed inactivity of this modified cofactor, whereas the selenido form is predicted to have lower barriers and hence higher activity. For the substrate 2-oxo-6-methylpurine, the calculated pathways for three different tautomers show great similarity to those found previously for xanthine, contrary to claims in the literature that the mechanisms for these two substrates are different. Compared with the wild-type enzyme, the conversion of xanthine to uric acid follows a somewhat different pathway in the Glu802 --> Gln mutant which exhibits a lower overall activity, in agreement with recently published kinetic data. The present results confirm the basic stepwise reaction mechanism and the orientation of the substrate that has been proposed in our previous QM/MM work on aldehyde oxidoreductase and xanthine oxidase. << Less

  J Phys Chem B 114:1506-1517(2010) [PubMed] [EuropePMC]

• Crystal structures of urate bound form of xanthine oxidoreductase: substrate orientation and structure of the key reaction intermediate.

  Okamoto K., Kawaguchi Y., Eger B.T., Pai E.F., Nishino T.

  Two contradictory models have been proposed for the binding mode of the substrate xanthine to and its activation mechanism by xanthine oxidoreductase. In an effort to distinguish between the two models, we determined the crystal structures of the urate complexes of the demolybdo-form of the D428A ... >> More

  Two contradictory models have been proposed for the binding mode of the substrate xanthine to and its activation mechanism by xanthine oxidoreductase. In an effort to distinguish between the two models, we determined the crystal structures of the urate complexes of the demolybdo-form of the D428A mutant of rat xanthine oxidoreductase at 1.7 Å and of the reduced bovine milk enzyme at 2.1 Å, the latter representing a reaction intermediate. The results clearly indicate the catalytically relevant binding mode of the substrate xanthine. << Less

  J Am Chem Soc 132:17080-17083(2010) [PubMed] [EuropePMC]

• Substrate orientation and catalytic specificity in the action of xanthine oxidase: the sequential hydroxylation of hypoxanthine to uric acid.

  Cao H., Pauff J.M., Hille R.

  Xanthine oxidase is a molybdenum-containing enzyme catalyzing the hydroxylation of a sp(2)-hybridized carbon in a broad range of aromatic heterocycles and aldehydes. Crystal structures of the bovine enzyme in complex with the physiological substrate hypoxanthine at 1.8 A resolution and the chemoth ... >> More

  Xanthine oxidase is a molybdenum-containing enzyme catalyzing the hydroxylation of a sp(2)-hybridized carbon in a broad range of aromatic heterocycles and aldehydes. Crystal structures of the bovine enzyme in complex with the physiological substrate hypoxanthine at 1.8 A resolution and the chemotherapeutic agent 6-mercaptopurine at 2.6 A resolution have been determined, showing in each case two alternate orientations of substrate in the two active sites of the crystallographic asymmetric unit. One orientation is such that it is expected to yield hydroxylation at C-2 of substrate, yielding xanthine. The other suggests hydroxylation at C-8 to give 6,8-dihydroxypurine, a putative product not previously thought to be generated by the enzyme. Kinetic experiments demonstrate that >98% of hypoxanthine is hydroxylated at C-2 rather than C-8, indicating that the second crystallographically observed orientation is significantly less catalytically effective than the former. Theoretical calculations suggest that enzyme selectivity for the C-2 over C-8 of hypoxanthine is largely due to differences in the intrinsic reactivity of the two sites. For the orientation of hypoxanthine with C-2 proximal to the molybdenum center, the disposition of substrate in the active site is such that Arg(880) and Glu(802), previous shown to be catalytically important for the conversion of xanthine to uric acid, play similar roles in hydroxylation at C-2 as at C-8. Contrary to the literature, we find that 6,8-dihydroxypurine is effectively converted to uric acid by xanthine oxidase. << Less

  J Biol Chem 285:28044-28053(2010) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Purification, crystallization and preliminary X-ray diffraction studies of xanthine dehydrogenase and xanthine oxidase isolated from bovine milk.

  Eger B.T., Okamoto K., Enroth C., Sato M., Nishino T., Pai E.F., Nishino T.

  Xanthine dehydrogenase catalyzes the oxidation of hypoxanthine to xanthine and the further oxidation of xanthine to uric acid. The enzyme is the target of the anti-gout drug allopurinol and its involvement in postischemic reperfusion injury is presently being defined. Each subunit of the homodimer ... >> More

  Xanthine dehydrogenase catalyzes the oxidation of hypoxanthine to xanthine and the further oxidation of xanthine to uric acid. The enzyme is the target of the anti-gout drug allopurinol and its involvement in postischemic reperfusion injury is presently being defined. Each subunit of the homodimeric 290 kDa enzyme contains four cofactors: one Mo-pterin, two [2Fe-2S] clusters and one FAD. Both the dehydrogenase (XDH) and the proteolytically modified oxidase form (XO) of the enzyme from bovine milk have been crystallized. XO crystals belong to space group C222(1), with unit-cell parameters a = 116.3, b = 164.4, c = 153.2 A at room temperature and a = 117.8, b = 165.4, c = 154.5 A when flash-frozen. They allow data collection to 3.3 and 2.5 A, respectively. In addition, a data set was collected from frozen XDH crystals and processed to 2.1 A. These crystals belong to space group C2, with unit-cell parameters a = 169.9, b = 124.8, c = 148.6 A, beta = 90.9 degrees. The unit-cell volumes and Matthews parameters are similar for the two crystal forms. There is one monomer per asymmetric unit in the XO crystals and a complete native dimer per asymmetric unit in the XDH crystals. << Less

  Acta Crystallogr D Biol Crystallogr 56:1656-1658(2000) [PubMed] [EuropePMC]