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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The native flavin, FAD, was removed from chicken liver xanthine dehydrogenase and milk xanthine oxidase by incubation with CaCl2. The deflavoenzymes, still retaining their molybdopterin and iron-sulfur prosthetic groups, were reconstituted with a series of FAD derivatives containing chemically reactive or environmentally sensitive substituents in the isoalloxazine ring system. The reconstituted enzymes containing these artificial flavins were all catalytically active. With both the chicken liver dehydrogenase and the milk oxidase, the flavin 8-position was found to be freely accessible to solvent. The flavin 6-position was also freely accessible to solvent in milk xanthine oxidase, but was significantly less exposed to solvent in the chicken liver dehydrogenase. Pronounced differences in protein structure surrounding the bound flavin were indicated by the spectral properties of the two enzymes reconstituted with flavins containing ionizable -OH or -SH substituents at the flavin 6- or 8-positions. Milk xanthine oxidase either displayed no preference for binding of the neutral or anionic flavin (8-OH-FAD) or a slight preference for the anionic form of the flavin (6-hydroxy-FAD, 6-mercapto-FAD, and possibly 8-mercapto-FAD). On the other hand, the chicken liver dehydrogenase had a dramatic preference for binding the neutral (protonated) forms of all four flavins, perturbing the pK of the ionizable substituent greater than or equal to 4 pH units. These results imply the existence of a strong negative charge in the flavin binding site of the dehydrogenase, which is absent in the oxidase.
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PMID:Differences in protein structure of xanthine dehydrogenase and xanthine oxidase revealed by reconstitution with flavin active site probes. 273 38

Affinity labeling of the NAD-binding site of chicken liver xanthine dehydrogenase by 5'-p-fluorosulfonylbenzoyladenosine (5'-FSBA) caused spectral perturbation around 450 nm in the same way as NAD. Reductive titration with xanthine of native xanthine dehydrogenase in the presence of NAD showed that redox potentials of the FAD/FADH. and FADH./FADH2 couples were shifted positive by NAD binding to the enzyme. The redox potentials of these couples were also shifted to some extent by modification of the NAD-binding site with 5'-FSBA. These results provide further evidence that binding of NAD to chicken liver xanthine dehydrogenase modulates the reactivity of the enzyme by shifting the redox potential of FAD. Proteolytic cleavage of the [14C]-5'-FSBA-modified enzyme yielded several domain peptides, only one of which contained radioactivity. The isolated radioactive peptide was further digested with Staphylococcus aureus protease and the 14C-labeled peptide was purified by two steps of high performance liquid chromatography. The amino acid sequence of the peptide was determined, and a reactive tyrosine residue was identified.
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PMID:The nicotinamide adenine dinucleotide-binding site of chicken liver xanthine dehydrogenase. Evidence for alteration of the redox potential of the flavin by NAD binding or modification of the NAD-binding site and isolation of a modified peptide. 292 14

Crude and purified xanthine dehydrogenase preparations from rat liver were examined for the existence of a naturally occurring inactive form. Reduction of the purified enzyme by xanthine under anaerobic conditions proceeded in two phases. The enzyme was inactivated by cyanide, which caused the release of a sulfur atom from the molybdenum center as thiocyanate. The amount of thiocyanate released was almost in parallel with the initial specific activity. The active and inactive enzymes could be resolved by affinity chromatography on Sepharose 4B/folate gel. These results provided evidence that the purified enzyme preparation from rat liver contained an inactive form. A method for the determination of the active and inactive enzymes in crude enzyme preparations from rat liver was devised based on the fact that only active enzyme could react with [14C]allopurinol and both active and inactive enzymes could be immunoprecipitated quantitatively by excess specific antibody to xanthine dehydrogenase. The amount of [14C]alloxanthine (derived from [14C]allopurinol) bound to the active sulfo enzyme in crude rat liver extracts was about 0.5 mol/mol of FAD. As this content is closely similar to that in the purified enzyme, these results suggest the existence of an inactive desulfo form in vivo.
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PMID:The presence of desulfo xanthine dehydrogenase in purified and crude enzyme preparations from rat liver. 345 93

Xanthine-NAD and NADH-methylene blue oxidoreductase activities of chicken liver xanthine dehydrogenase were inactivated by incubation with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (5'-FSBA), an active site directed reagent for nucleotide binding sites. The inactivation reaction displayed pseudo-first-order kinetics. A double-reciprocal plot of inactivation velocity vs. 5'-FSBA concentration showed that 5'-FSBA and enzyme formed a complex prior to inactivation. NAD protected the enzyme from inactivation by 5'-FSBA in a competitive fashion. The modified enzyme had the same xanthine-dichlorophenolindophenol and xanthine-O2 oxidoreductase activities as the native enzyme, and on addition of xanthine to the modified enzyme, bleaching of the spectrum occurred in the visible region. The amount of radioactivity incorporated into the enzyme by incubation with [14C]-5'-FSBA was parallel to the loss of xanthine-NAD oxidoreductase activity, and the stoichiometry was 1 mol/mol of enzyme-bound FAD for complete inactivation. These results indicated that 5'-FSBA modified specifically the binding site for NAD of chicken liver xanthine dehydrogenase. The incorporated radioactivity was released slowly from 14C-labeled enzyme by incubation with dithiothreitol with concomitant restoration of catalytic activity. The modified residue responsible for inactivation was identified as a tyrosine.
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PMID:Evidence for the existence of a tyrosyl residue in the nicotinamide adenine dinucleotide binding site of chicken liver xanthine dehydrogenase. 347 29

Rabbit liver aldehyde oxidase (AO), like milk xanthine oxidase (XO) and chicken liver xanthine dehydrogenase (XDH), possesses the following prosthetic groups: FAD, a functional Mo center, and two spectroscopically distinct iron-sulfur centers, one with gav less than 2.0 (termed Fe/S I) and the other with gav greater than 2.0 (termed Fe/S II) in the reduced enzyme. EPR spectra for the Mov species were found to be nearly identical in AO and XO for a number of enzyme complexes, and the midpoint reduction potentials for functional MoVI/MoV (-359 mV) and MoV/MoVI (-351 mV) were nearly the same in all three enzymes (50 mM phosphate, pH 7.8). A strong magnetic interaction between MoV and reduced Fe/S I, previously detected in XO and XDH, was also found in AO. No MoV-Fe/S II interaction could be detected in AO (nor in XO). In contrast, the order of reduction of Fe/S I and Fe/S II, as measured from their midpoint potentials, is reversed in AO (Em = -207 and -310 mV, respectively) as compared to XO (Em = -280 and -245 mV, respectively) in phosphate buffer at pH 7.8. The oxidized-reduced extinction coefficients at 450 and 550 nm for the two centers are also apparently reversed in AO and XO. Although magnetic interaction between FAD and one or both reduced Fe/S centers has been detected in both AO and XO, no magnetic interaction between the two reduced Fe/S centers themselves was found in AO (although such interaction has been seen in XO). The average FAD reduction potential is substantially more positive in AO (Em for FAD/FADH., -258 mV; FADH./FADH2, -212 mV at pH 7.8) than in XO or XDH. It can be concluded that although the properties and immediate environment of the functional Mo center are conserved in the three Mo hydroxylase enzymes, and all three enzymes possess the same set of prosthetic groups, the properties of the groups which transfer electrons from the Mo to the ultimate electron acceptor can vary substantially in AO, XO, and XDH.
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PMID:Properties of the prosthetic groups of rabbit liver aldehyde oxidase: a comparison of molybdenum hydroxylase enzymes. 628 79

Although mammalian xanthine oxidase exists originally as a dehydrogenase form in freshly prepared samples, it is converted to an oxidase form during purification, either irreversibly by proteolysis or reversibly by sulfhydryl oxidation of the protein molecule. However, avoiding proteolysis the mammalian enzyme can be purified as an interconvertible form and thus can be used to compare directly the properties of xanthine dehydrogenase and the oxidase derived from the same enzyme molecule. The cDNAs encoding the enzyme have been cloned from several sources, and structural information is becoming available. The most significant difference between the two forms is the protein conformation around FAD, which changes the redox potential of the flavin and the reactivity of FAD with the electron acceptors, NAD and molecular oxygen. The flavin semiquinone is thermodynamically stable in xanthine dehydrogenase, but is unstable in xanthine oxidase. Detailed analyses by stopped-flow techniques suggest that the flavin semiquinone reacts with oxygen to form superoxide anion while the fully reduced flavin reacts to form hydrogen peroxide. Although xanthine dehydrogenase can produce greater amounts of superoxide anion than xanthine oxidase during xanthine-oxygen turnover, it seems to be physiologically insignificant because NAD inhibits almost completely the formation of superoxide anion. Although the involvement of this enzyme in reperfusion injury has been proposed, this seems to be more complex than originally envisaged and still remains to be established.
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PMID:The conversion of xanthine dehydrogenase to xanthine oxidase and the role of the enzyme in reperfusion injury. 779 66

Pseudomonas thermocarboxydovorans strain C2 is capable of using carbon monoxide as the sole source of carbon and energy. The key enzyme for CO utilisation is the molybdenum containing iron-flavoprotein carbon monoxide dehydrogenase (CODH). This paper reports the DNA sequencing of a 4.7 kb region of the C2 genome which appears to encode the CODH enzyme. The genes for the three subunits of CODH, which we have named cut A, B and C, have been identified and they appear to form an operon. The predicted protein sequences of the three subunits have homology to the structurally related protein, xanthine dehydrogenase, from Drosophila melanogaster. By comparison with xanthine dehydrogenase it can be predicted that the molybdenum cofactor binds to the large subunit of CODH, the small subunit of CODH contains the iron-sulphur centers and the medium subunit binds FAD/NAD+.
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PMID:DNA sequence of the cut A, B and C genes, encoding the molybdenum containing hydroxylase carbon monoxide dehydrogenase, from Pseudomonas thermocarboxydovorans strain C2. 780 57

The genes of nicotine dehydrogenase (NDH) were identified, cloned and sequenced from the catabolic plasmid pAO1 of Arthrobacter nicotinovorans. In immediate proximity to this gene cluster is the beginning of the 6-hydroxy-L-niotine oxidase (6-HLNO) gene. NDH is composed of three subunits (A, B and C) of M(r) 30,011, 14,924 and 87,677. It belongs to a family of bacterial hydroxylases with a similar subunit structure; they have molybdopterin dinucleotide, FAD and Fe-S clusters as cofactors. Here the first complete primary structure of a bacterial hydroxylase is provided. Sequence alignments of each of the NDH subunits show similarities to the sequences of eukaryotic xanthine dehydrogenase (XDH) but not to other known molybdenum-containing bacterial enzymes. Based on alignment with XDH it is inferred that the smallest subunit (NDHB) carries an iron-sulphur cluster, that the middle-sized subunit (NDHA) binds FAD, and that the largest NDH subunit (NDHC) corresponds to the molybdopterin-binding domain of XDH. Expression of both the ndh and the 6-hino genes required the presence of nicotine and molybdenum in the culture medium. Tungsten inhibited enzyme activity but not the synthesis of the enzyme protein. The enzyme was found in A. nicotinovorans cells in a soluble form and in a membrane-associated form. In the presence of tungsten the fraction of membrane-associated NDH increased.
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PMID:Structural analysis and molybdenum-dependent expression of the pAO1-encoded nicotine dehydrogenase genes of Arthrobacter nicotinovorans. 781 50

We have cloned and sequenced the hxA gene coding for the xanthine dehydrogenase (purine hydroxylase I) of Aspergillus nidulans. The gene codes for a polypeptide of 1363 amino acids. The sequencing of a nonsense mutation, hxA5, proves formally that the clones isolated correspond to the hxA gene. The gene sequence is interrupted by three introns. Similarity searches reveal two iron-sulfur centers and a NAD/FAD-binding domain and have enabled a consensus sequence to be determined for the molybdenum cofactor-binding domain. The A. nidulans sequence is a useful outclass for the other known sequences, which are all from metazoans. In particular, it gives added significance to the missense mutations sequenced in Drosophila melanogaster and leads to the conclusion that while one of the recently sequenced human genes codes for a xanthine dehydrogenase, the other one must code for a different molybdenum-containing hydroxylase, possibly an aldehyde oxidase. The transcription of the hxA gene is induced by the uric acid analogue 2-thiouric acid and repressed by ammonium. Induction necessitates the product of the uaY regulatory gene.
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PMID:Cloning and molecular characterization of hxA, the gene coding for the xanthine dehydrogenase (purine hydroxylase I) of Aspergillus nidulans. 787 88

The reduction of milk xanthine dehydrogenase by salicylate anion radical (SL-), nicotinamide adenine dinucleotide radical (NAD.), and 1-methylnicotinamide (NMA) radicals was investigated by the use of pulse radiolysis. Reduction of the dehydrogenase with SL- proceeded via two phases. From the kinetic difference spectra obtained, the faster and slower phases of reduction represent that of one of the iron-sulfur centers and of FAD, respectively. The rate constant of the faster phase increased with the concentration of the enzyme, suggesting that the reduction follows a bimolecular reaction of SL- with the iron-sulfur center. In contrast, the rate constant of the slower phase (510 s-1) was independent of the concentration of the enzyme at pH 7.5. In order to elucidate the contribution of the molybdenum site in the reaction, a similar reaction was performed with enzyme modified with oxipurinol. In the modified enzyme, the slower phase was lost, whereas the faster phase was not affected. These results suggest that the slower phase is due to intramolecular electron transfer from the molybdenum center to FAD. On the other hand, NAD. reacted predominantly with FAD of the dehydrogenase to form the neutral semiquinone of FAD with a second order rate constant of 1.4 x 10(7) M-1 s-1 at pH 7.5, whereas a similar reaction in the oxidase, which was converted from xanthine dehydrogenase by proteolytical cleavage, was not observed. This suggests that NAD. transfers an electron via the binding site for NAD+ on the dehydrogenase. In contrast, NMA radical reduced only an iron-sulfur center of the dehydrogenase with a second order rate constant of 6.5 x 10(7) M-1 s-1 at pH 7.5.
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PMID:Electron transfer process in milk xanthine dehydrogenase as studied by pulse radiolysis. 822 23


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