Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Previous work from this laboratory has shown that the spectral and functional properties of a prokaryotic xanthine dehydrogenase from Comamonas acidovorans show some similarities to those of the well-characterized eukaryotic enzymes isolated from bovine milk and from chicken liver [Xiang, Q. & Edmondson, D.E. (1996) Biochemistry35, 5441-5450]. Therefore, this system was chosen to study the factors involved in the expression of functional recombinant enzyme in Escherichia coli to provide insights into the assembly of the functional Mo-pyranopterin center. Genes xdhA and xdhB (encoding the two known subunits of the native enzyme) and putative genes xprA and ssuABC were sequenced. Heterologous expression of the xdhAB genes in E. coli JM109(DE3) produced active enzyme. The Mo content was 0.11-0.16 mol per alphabeta protomer, while the Fe and FAD levels were at stoichiometries similar to that of the native enzyme. The XDH activity increased sixfold when the culture was grown under conditions of low aeration (6 L.min-1) as compared with high aeration (12 L.min-1). Co-expression of the xdhAB genes with the Pseudomonas aeruginosa PA1522 (xdhC) gene increased the level of Mo incorporated into the expressed enzyme to a 1 : 1 stoichiometry. Under these conditions, high levels of functional protein (2.284 U.mg-1 and 8.039 mg.L-1 of culture) were obtained independently of the level of culture aeration. Therefore, the assembly of a functional Mo-pyranopterin center in XDH requires the presence of a functional xdhC gene product. The purified, recombinant XDH shows spectral and kinetic properties identical to those of the native enzyme.
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PMID:Factors involved in the assembly of a functional molybdopyranopterin center in recombinant Comamonas acidovorans xanthine dehydrogenase. 1462 63

There is substantial evidence that oxidative stress participates in the pathophysiology of cardiovascular disease. Biochemical, molecular and pharmacological studies further implicate xanthine oxidoreductase (XOR) as a source of reactive oxygen species in the cardiovascular system. XOR is a member of the molybdoenzyme family and is best known for its catalytic role in purine degradation, metabolizing hypoxanthine and xanthine to uric acid with concomitant generation of superoxide. Gene expression of XOR is regulated by oxygen tension, cytokines and glucocorticoids. XOR requires molybdopterin, iron-sulphur centres, and FAD as cofactors and has two interconvertible forms, xanthine oxidase and xanthine dehydrogenase, which transfer electrons from xanthine to oxygen and NAD(+), respectively, yielding superoxide, hydrogen peroxide and NADH. Additionally, XOR can generate superoxide via NADH oxidase activity and can produce nitric oxide via nitrate and nitrite reductase activities. While a role for XOR beyond purine metabolism was first suggested in ischaemia-reperfusion injury, there is growing awareness that it also participates in endothelial dysfunction, hypertension and heart failure. Importantly, the XOR inhibitors allopurinol and oxypurinol attenuate dysfunction caused by XOR in these disease states. Attention to the broader range of XOR bioactivity in the cardiovascular system has prompted initiation of several randomised clinical outcome trials, particularly for congestive heart failure. Here we review XOR gene structure and regulation, protein structure, enzymology, tissue distribution and pathophysiological role in cardiovascular disease with an emphasis on heart failure.
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PMID:Xanthine oxidoreductase and cardiovascular disease: molecular mechanisms and pathophysiological implications. 1469 47

An improved procedure is described for the high-level expression of Comamonas acidovorans XDH in Pseudomonas aeruginosa PAO1-LAC. The level of functional expression (56 mg protein/L culture) is found to be 7-fold higher than that observed in Escherichia coli and 30-fold higher than that induced in C. acidovorans. Co-expression of the xdhC gene is required for maximal level of functional expression. Comparison of purified preparations of XDH expressed in the absence of xdhC (XDH(AB)) with that expressed in its presence (XDH(ABC)) shows the increased level of activity due to the level of Mo incorporation. The Fe and FAD contents of expressed enzymes are independent of xdhC co-expression. Electron paramagnetic resonance spectroscopy, circular dichroism spectroscopy, metal analysis, and kinetic properties of recombinant purified XDH(ABC) are identical with those exhibited by the native enzyme. This expression system should serve as a valuable tool for further biophysical and mechanistic investigations of xanthine dehydrogenase by site-directed mutagenesis. A method is also described to evaluate the suitability of P. aeruginosa and other organisms as potential expression hosts for five different sources of xdh genes.
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PMID:High-level expression and characterization of a highly functional Comamonas acidovorans xanthine dehydrogenase in Pseudomonas aeruginosa. 1529 83

The plant molybdenum-cofactor (Moco) and flavin-containing enzymes, xanthine dehydrogenase (XDH; EC 1.2.1.37) and aldehyde oxidase (AO; EC 1.2.3.1) are thought to play important metabolic roles in purine metabolism and hormone biosynthesis, respectively. Their animal counterparts contribute to reactive oxygen species (ROS) production in numerous pathologies and here we examined these enzymes as potential sources of ROS in plants. Novel in-gel assay techniques and Moco sulfurase mutants, lacking a sulfur ligand in their Moco active center, were employed to demonstrate that the native tomato and Arabidopsis XDHs are capable of producing O, but not H2O2, while the animal counterpart was shown to produce both, O and H2O2. Superoxide production was dependent on Moco sulfuration when using hypoxanthine/xanthine but not NADH as substrates. The activity was inhibited by diphenylene iodonium (DPI), a suicide inhibitor of FAD containing enzymes. Analysis of XDH in an Arabidopsis Atxdh1 T-DNA insertion mutant and RNA interference lines revealed loss of O activity, providing direct molecular evidence that plant XDH generates superoxides. Contrary to XDH, AO activity produced only H2O2 dissimilar to native animal AO, that can produce O as well. Surprisingly, H2O2 accumulation was not sensitive to DPI. Plant ROS production and transcript levels of AO and XDH were rapidly upregulated by application of abscisic acid and in water-stressed leaves and roots. These results, supported by in vivo measurement of ROS accumulation, indicate that plant AO and XDH are possible novel sources for ROS increase during water stress.
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PMID:The plant Mo-hydroxylases aldehyde oxidase and xanthine dehydrogenase have distinct reactive oxygen species signatures and are induced by drought and abscisic acid. 1594 99

The anaerobic soil bacterium Eubacterium barkeri catabolizes nicotinate to pyruvate and propionate via a unique fermentation. A full molecular characterization of nicotinate fermentation in this organism was accomplished by the following results: (i) A 23.2-kb DNA segment with a gene cluster encoding all nine enzymes was cloned and sequenced, (ii) two chiral intermediates were discovered, and (iii) three enzymes were found, completing the hitherto unknown part of the pathway. Nicotinate dehydrogenase, a (nonselenocysteine) selenium-containing four-subunit enzyme, is encoded by ndhF (FAD subunit), ndhS (2 x [2Fe-2S] subunit), and by the ndhL/ndhM genes. In contrast to all enzymes of the xanthine dehydrogenase family, the latter two encode a two-subunit molybdopterin protein. The 6-hydroxynicotinate reductase, catalyzing reduction of 6-hydroxynicotinate to 1,4,5,6-tetrahydro-6-oxonicotinate, was purified and shown to contain a covalently bound flavin cofactor, one [2Fe-2S](2+/1+) and two [4Fe-4S](2+/1+) clusters. Enamidase, a bifunctional Fe-Zn enzyme belonging to the amidohydrolase family, mediates hydrolysis of 1,4,5,6-tetrahydro-6-oxonicotinate to ammonia and (S)-2-formylglutarate. NADH-dependent reduction of the latter to (S)-2-(hydroxymethyl)glutarate is catalyzed by a member of the 3-hydroxyisobutyrate/phosphogluconate dehydrogenase family. A [4Fe-4S]-containing serine dehydratase-like enzyme is predicted to form 2-methyleneglutarate. After the action of the coenzyme B(12)-dependent 2-methyleneglutarate mutase and 3-methylitaconate isomerase, an aconitase and isocitrate lyase family pair of enzymes, (2R,3S)-dimethylmalate dehydratase and lyase, completes the pathway. Genes corresponding to the first three enzymes of the E. barkeri nicotinate catabolism were identified in nine Proteobacteria.
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PMID:Molecular and functional analysis of nicotinate catabolism in Eubacterium barkeri. 1689 75

Reactive oxygen species are generated by various systems, including NADPH oxidases, xanthine oxidoreductase (XOR) 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 in a molar ratio of about 1:3, depending upon the conditions. Here, we present a mutant of rat XOR that displays mainly XO activity with a superoxide:hydrogen peroxide production ratio of about 6:1. In the mutant, tryptophan 335, which is a component of the amino acid cluster crucial for switching from the XDH to the XO conformation, was replaced with alanine, and phenylalanine 336, which modulates FAD's redox potential through stacking interactions with the flavin cofactor, was changed to leucine. When the mutant was expressed in Sf9 cells, it was obtained in the XO form, and dithiothreitol treatment only partially restored the pyridine nucleotide-binding capacity. The crystal structure of the dithiothreitol-treated mutant at 2.3 Angstroms resolution showed the enzyme's two subunits to be quite similar, but not identical: the cluster involved in conformation-switching was completely disrupted in one subunit, but remained partly associated in the other one. The chain trace of the active site loop in this mutant is very similar to that of the bovine XO form. These results are consistent with the idea that the XDH and XO forms of the mutant are in an equilibrium that greatly favours the XO form, but the equilibrium is partly shifted towards the XDH form upon incubation with dithiothreitol.
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PMID:Two mutations convert mammalian xanthine oxidoreductase to highly superoxide-productive xanthine oxidase. 1730 Oct 76

The oxidation of xanthine by xanthine oxidase (XO) or xanthine dehydrogenase represents an important source of reactive oxygen species (ROS), which contribute to the damaging consequences of cerebral ischemia, inflammation, and neurodegenerative disorders. However, both enzymes are also able to act on reduced nicotinamide adenine dinucleotide (NADH). The FAD binding site to which NADH binds is distinct from that of the xanthine binding site. We report that the combination of xanthine oxidase and NADH is toxic to cultures of cerebellar granule neurons. Protection by superoxide dismutase (Cu,Zn-SOD or Mn-SOD) or catalase indicates mediation of the toxicity by superoxide and hydrogen peroxide. In addition, pre-incubating XO with EDTA at concentrations as low as 2 microM, prevented the toxicity, indicating that a metal contaminating XO is involved in producing the toxic effects of XO/NADH. It is possible that such a metal might play a role in the toxicity of XO in vivo.
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PMID:Xanthine oxidase-induced neuronal death via the oxidation of NADH: prevention by micromolar EDTA. 1945 May 65

The reducing substrates 4-thiolumazine and 2,4-dithiolumazine have been used to form Mo(IV)-product complexes with xanthine oxidase (XO) and xanthine dehydrogenase. These Mo(IV)-product complexes display an intense metal-to-ligand charge-transfer (MLCT) band in the near-infrared region of the spectrum. Optical pumping into this MLCT band yields resonance Raman spectra of the Mo site that are devoid of contributions from the highly absorbing FAD and 2Fe2S clusters in the protein. The resonance Raman spectra reveal in-plane bending modes of the bound product and low-frequency molybdenum dithiolene and pyranopterin dithiolene vibrational modes. This work provides keen insight into the role of the pyranopterin dithiolene in electron-transfer reactivity.
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PMID:Pyranopterin dithiolene distortions relevant to electron transfer in xanthine oxidase/dehydrogenase. 2497 5


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