Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P47989 (xanthine oxidase)
 8,633 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transcriptional control of the nitrogen fixation (nif) genes in response to oxygen in Azotobacter vinelandii is mediated by nitrogen fixation regulatory protein L (NifL), a regulatory flavoprotein that modulates the activity of the transcriptional activator nitrogen fixation regulatory protein A (NifA). CD spectra of purified NifL indicate that FAD is bound to NifL in an asymmetric environment and the protein is predominantly alpha-helical. The redox potential of NifL is -226 mV at pH 8 as determined by the enzymic reduction of NifL by xanthine oxidase/xanthine in the presence of appropriate mediators. The reduction of NifL by xanthine oxidase prevented NifL from acting as an inhibitor of NifA. In the absence of electron mediators NifL could also be reduced by Escherichia coli flavohaemoprotein (Hmp) with NADH as reductant. Hmp contains a globin-like domain with haem B as prosthetic group and an FAD-containing oxidoreductase module. The carboxyferrohaem form of Hmp was competent to reduce NifL, suggesting that electron donation to NifL originates from the flavin in Hmp rather than by direct electron transfer from the haem. Spinach ferredoxin:NAD(P) oxidoreductase, which adopts a folding similar to the FAD- and NAD-binding domains of Hmp, also reduced NifL with NADH as reductant. Re-oxidation of NifL occurs rapidly in the presence of air, raising the possibility that NifL might sense intracellular oxygen. We propose a physiological redox cycle in which the oxidation of NifL by oxygen and hence the activation of its inhibitory properties occurs rapidly, in contrast with the switch from the active to the reduced form of NifL, which occurs more slowly.
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PMID:Electron donation to the flavoprotein NifL, a redox-sensing transcriptional regulator. 960 Oct 70

Xanthine oxidase (XO) is conventionally known as a generator of reactive oxygen species (ROS) which contribute to hypoxic-reperfusion injury in tissues. However, this role for human XO is disputed due to its distinctive lack of activity towards xanthine, and the failure of allopurinol to suppress reperfusion injury. In this paper, we have employed native gel electrophoresis together with activity staining to investigate the role human xanthine dehydrogenase (XD) and XO in hypoxic reperfusion injury. This approach has provided information which cannot be obtained by conventional spectrophotometric assays. We found that both XD and XO of human umbilical vein endothelial cells (HUVECs) and lymphoblastic leukaemic cells (CEMs) catalysed ROS generation by oxidising NADH, but not hypoxanthine. The conversion of XD to XO was observed in both HUVECs and CEMs in response to hypoxia, although the level of conversion varied. Purified human milk XD generated ROS more efficiently in the presence of NADH than in the presence of hypoxanthine. This NADH oxidising activity was blocked by the FAD site inhibitor, diphenyleneiodonium (DPI), but was not suppressible by the molybdenum site inhibitor, allopurinol. However, in the presence of both DPI and allopurinol the activities of XD/XO were completely blocked with either NADH or hypoxanthine as substrates. We conclude that both human XD and XO can oxidise NADH to generate ROS. Therefore, the conversion of XD to XO is not necessary for post-ischaemic ROS generation. The hypoxic-reperfusion injury hypothesis should be reappraised to take into account the important role played by XD and XO in oxidising NADH to yield ROS.
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PMID:A reappraisal of xanthine dehydrogenase and oxidase in hypoxic reperfusion injury: the role of NADH as an electron donor. 964 92

2-Hydroxyisonicotinate dehydrogenase from Mycobacterium sp. INA1 was purified 26-fold to apparent homogeneity. The enzyme is involved in isonicotinate degradation by Mycobacterium sp. INA1 and catalyzes the conversion of 2-hydroxyisonicotinate to 2,6-dihydroxypyridine-4-carboxylate. The purified protein exhibited a native molecular mass of 300 kDa and subunits of 97, 31 and 17 kDa, respectively, indicating an alpha 2 beta 2 gamma 2 structure. The absorption spectrum of the homogeneous enzyme was characteristic for an iron/sulfur flavoprotein, 3.8 mol of iron, 3.7 mol of acid labile sulfur, 0.94 mol of FAD and 0.75 mol of molybdenum were determined per mol of protomer. The molybdenum cofactor was identified as molybdopterin cytosine dinucleotide. 2-Hydroxyisonicotinate dehydrogenase was inactivated in the presence of cyanide. According to these basic properties the protein seems to belong to the class of molybdo-iron/sulfur flavoproteins of the xanthine oxidase family.
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PMID:2-Hydroxyisonicotinate dehydrogenase isolated from Mycobacterium sp. INA1. 968 80

Nitric oxide (NO) synthesis is well-known to result from the oxidation of L-arginine by a family of NO synthases (NOS). However, under hypoxic conditions this mechanism of NO synthesis may be impaired and NO is formed by a NOS independent mechanism. This study was designed to examine the reduction of nitrite to NO by xanthine oxidase (XO) under hypoxia, because the bacterial nitrate/nitrite reductases have structural similarity to XO. We found that both purified and tissue containing XO catalyze the reduction of nitrite to NO, as demonstrated using a chemiluminescent NO meter. This redox reaction requires NADH as an electron donor, and is oxygen independent. The inhibitory profiles suggest that reduction of nitrite takes place at the molybdenum center of XO whilst NADH is oxidized at the FAD center. Heparin binding of XO caused an increase in the catalysis of nitrite reduction. The XO-catalyzed generation of NO may be important in redistribution of blood flow to ischaemic tissue as a supplement to NOS, since both nitrite and NADH have been shown to be elevated in hypoxic tissue.
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PMID:Generation of nitric oxide by a nitrite reductase activity of xanthine oxidase: a potential pathway for nitric oxide formation in the absence of nitric oxide synthase activity. 973 Dec 11

Xanthine oxidoreductase from bovine milk can be prepared in two interconvertible forms, xanthine oxidase (XO) and xanthine dehydrogenase (XDH), depending on the number of protein cysteines versus cystines. Enzyme forms differ in respect to their oxidizing substrates; XDH prefers NAD to molecular oxygen, whereas XO only reacts significantly with oxygen. The preference for oxidizing substrate is partially explained by thermodynamics. Unlike XDH, the midpoint potential of the FAD, the center at which oxygen and NAD react, is too high in XO to efficiently reduce NAD (Hunt, J., Massey, V., Dunham, W.R., and Sands, R.H. (1993) J. Biol. Chem. 268, 18685-18691). To distinguish between changes in thermodynamics and in substrate binding, samples of both XO and XDH have been prepared in which the native FAD has been replaced with an FAD analog of different redox potential, 1-deaza-FAD or 8-CN-FAD. Reductive titrations indicate that both 1-deaza-XO and 1-deaza-XDH have a flavin midpoint potential similar to native XDH and that 8-CN-XO and 8-CN-XDH each have a flavin potential higher than XO. Both the low potential 1-deaza-XO and the high potential 8-CN-XDH contain essentially no xanthine/NAD activity. However, 1-deaza-XDH does exhibit xanthine/NAD activity, and 8-CN-XO has normal xanthine/oxygen activity. The binding of NAD to oxidized XO and XDH was investigated by ultrafiltration and isothermal titration calorimetry. The Kd for the binding of NAD to XDH was determined to be 280 +/- 145 microM by ultrafiltration and 160 +/- 40 microM by isothermal titration calorimetry. No evidence for the binding of NAD to XO by either method could be obtained. A low flavin midpoint potential is necessary but not sufficient for dehydrogenase activity.
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PMID:Role of the flavin midpoint potential and NAD binding in determining NAD versus oxygen reactivity of xanthine oxidoreductase. 998 90

In order to investigate the effects of trace elements on different metabolic pathways, the thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius (DSM 639) has been cultivated on various carbon substrates in the presence and absence of molybdate. When grown on glucose (but neither on glutamate nor casein hydrolysate) as sole carbon source, the lack of molybdate results in serious growth inhibition. By analysing cytosolic fractions of glucose adapted cells for molybdenum containing compounds, an aldehyde oxidoreductase was detected that is present in the cytosol to at least 0.4% of the soluble protein. With Cl2Ind (2,6-dichlorophenolindophenol) as artificial electron acceptor, the enzyme exhibits oxidizing activity towards glyceraldehyde, glyceraldehyde-3-phosphate, isobutyraldehyde, formaldehyde, acetaldehyde and propionaldehyde. At its pH-optimum (6.7), close to the intracellular pH of Sulfolobus, the glyceraldehyde-oxidizing activity is predominant. The protein has an apparent molecular mass of 177 kDa and consists of three subunits of 80.5 kDa (alpha), 32 kDa (beta) and 19.5 kDa (gamma). It contains close to one Mo, four Fe, four acid-labile sulphides and four phosphates per protein molecule. Methanol extraction revealed the existence of 1 FAD per molecule and 1 molybdopterin per molecule, which was identified as molybdopterin guanine dinucleotide on the basis of perchloric acid cleavage and thin layer chromatography. EPR-spectra of the aerobically prepared enzyme exhibit the so-called 'desulpho-inhibited'-signal, known from chemically modified forms of molybdenum containing proteins. Anaerobically prepared samples show both, the signals arising from the active molybdenum-cofactor as well as from the two [2Fe-2S]-clusters. According to metal-, cofactor-, and subunit-composition, the enzyme resembles the members of the xanthine oxidase family. Nevertheless, the melting point and long-term thermostability of the protein are outstanding and perfectly in tune with the growth temperature of S. acidocaldarius (80 degrees C). The findings suggest the enzyme to function as a glyceraldehyde oxidoreductase in the course of the nonphosphorylated Entner-Doudoroff pathway and thereby may attribute a new physiological role to this class of enzyme.
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PMID:The strict molybdate-dependence of glucose-degradation by the thermoacidophile Sulfolobus acidocaldarius reveals the first crenarchaeotic molybdenum containing enzyme--an aldehyde oxidoreductase. 1009 93

In Klebsiella pneumoniae, NifL modulates the activity of the transcriptional activator NifA in response to combined nitrogen or external molecular oxygen. We recently showed that K. pneumoniae NifL is a flavoprotein which apparently senses oxygen through a redox-sensitive, conformational change. In order to study whether the nitrogen signal might be transmitted to NifA through a stable modification of NifL we characterized the redox properties of NifL synthesized in Escherichia coli in the presence of different nitrogen sources. FAD analyses showed that purified NifL carried FAD as cofactor independent of nitrogen and oxygen availability. The redox potential of NifL synthesized in the presence of ammonium was -277+/-5 mV at pH 8.0 and 25 degrees C, as determined by reduction with dithionite or with enzymatic reduction by xanthine oxidase in the presence of methyl viologen as redox mediator. When synthesized under nitrogen-limiting conditions, NifL showed a redox potential of -274+/-6 mV at pH 8.0 and 25 degrees C. Fully reduced NifL fractions, synthesized under either condition listed above, reoxidized rapidly in the presence of molecular oxygen. These results indicate that for NifL synthesized in E. coli, the redox potential of the NifL-bound FAD is not influenced by the nitrogen source. The two NifL fractions differed, however, in that a non-flavin specific absorbance at 420 nm was found only in NifL synthesized in the presence of ammonium.
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PMID:NifL of Klebsiella pneumoniae: redox characterization in relation to the nitrogen source. 1035 Jun 21

A specific dehydrogenase, different from nicotinic acid hydroxylase, was induced during growth of Eubacterium barkeri on xanthine. The protein designated as xanthine dehydrogenase was enriched 39-fold to apparent homogeneity using a three-step purification scheme. It exhibited an NADP-dependent specific activity of 164 micromol xanthine oxidized per min and per mg of protein. In addition it showed an NADPH-dependent oxidase and diaphorase activity. A molecular mass of 530 kDa was determined for the native enzyme and SDS/PAGE revealed three types of subunits with molecular masses of 17.5, 30 and 81 kDa indicating a dodecameric native structure. Molybdopterin was identified as the molybdenum-complexing cofactor using activity reconstitution experiments and fluorescence measurements after KI/I2 oxidation. The molecular mass of the cofactor indicated that it is of the dinucleotide type. The enzyme contained iron, acid-labile sulfur, molybdenum, tungsten, selenium and FAD at molar ratios of 17.5, 18.4, 2.3, 1.1, 0.95 and 2.8 per mol of native enzyme. Xanthine dehydrogenase was inactivated upon incubation with arsenite, cyanide and different purine analogs. Reconstitution experiments of xanthine dehydrogenase activity by addition of selenide and selenite performed with cyanide-inactivated enzyme and with chloramphenicol-treated cells, respectively, indicated that selenium is not attached to the protein in a covalently bound form such as selenocysteine.
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PMID:Selenium-containing xanthine dehydrogenase from Eubacterium barkeri. 1049 Nov 34

Xanthine oxidase (XO) was shown to catalyze the reduction of nitrite to nitric oxide (NO), under anaerobic conditions, in the presence of either NADH or xanthine as reducing substrate. NO production was directly demonstrated by ozone chemiluminescence and showed stoichiometry of approximately 2:1 versus NADH depletion. With xanthine as reducing substrate, the kinetics of NO production were complicated by enzyme inactivation, resulting from NO-induced conversion of XO to its relatively inactive desulfo-form. Steady-state kinetic parameters were determined spectrophotometrically for urate production and NADH oxidation catalyzed by XO and xanthine dehydrogenase in the presence of nitrite under anaerobic conditions. pH optima for anaerobic NO production catalyzed by XO in the presence of nitrite were 7.0 for NADH and </=6.0 for xanthine. Involvement of the molybdenum site of XO in nitrite reduction was shown by the fact that alloxanthine inhibits xanthine oxidation competitively with nitrite. Strong preference for Mo=S over Mo=O was shown by the relatively very low NADH-nitrite reductase activity shown by desulfo-enzyme. The FAD site of XO was shown not to influence nitrite reduction in the presence of xanthine, although it was clearly involved when NADH was the reducing substrate. Apparent production of NO decreased with increasing oxygen tensions, consistent with reaction of NO with XO-generated superoxide. It is proposed that XO-derived NO fulfills a bactericidal role in the digestive tract.
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PMID:Reduction of nitrite to nitric oxide catalyzed by xanthine oxidoreductase. 1071 88

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.
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PMID:Purification, crystallization and preliminary X-ray diffraction studies of xanthine dehydrogenase and xanthine oxidase isolated from bovine milk. 1109 37


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