Structural and conformational analysis of the oxidase to dehydrogenase conversion of xanthine oxidoreductase

Xanthine oxidoreductase (XOR) is a 300-kDa homodimer that can exist as an NAD+-dependent dehydrogenase (XD) or as an O2-dependent oxidase (XO) depending on the oxidation state of its cysteine thiols. Both XD and XO undergo limited cleavage by chymotrypsin and trypsin. Trypsin selectively cleaved both enzyme forms at Lys184, while chymotrypsin cleaved XD primarily at Met181 but cleaved XO at Met181 and at Phe560. Chymotrypsin, but not trypsin, cleavage also prevented the reductive conversion of XO to XD; thus the region surrounding Phe560 appears to be important in the interconversion of the two forms. Size exclusion chromatography showed that disulfide bond formation reduced the hydrodynamic volume of the enzyme, and two-dimensional gel electrophoresis of chymotrypsin-digested XO showed significant, disulfide bond-mediated, conformational heterogeneity in the N-terminal third of the enzyme but no evidence of disulfide bonds between the N-terminal and C-terminal regions or between XOR subunits. These results indicate that intrasubunit disulfide bond formation leads to a global conformational change in XOR that results in the exposure of the region surrounding Phe560. Conformational changes within this region in turn appear to play a critical role in the interconversion between the XD and XO forms of the enzyme.     

Mechanism of transition from xanthine dehydrogenase to xanthine oxidase: effect of guanidine-HCL or urea on the activity

Mammalian xanthine oxidoreductase can be converted from the dehydrogenase to the oxidase form, either reversibly by formation of disulfide bridges or irreversibly by proteolytic cleavage within the xanthine oxidoreductase protein molecule. A tightly packed amino acid cluster stabilizes the dehydrogenase form, and disruption of this cluster is accompanied with rearrangement of the active site loop. Here, we show that the conversion occurs in the presence of guanidine-HCl or urea. We propose that xanthine dehydrogenase and oxidase are in a thermodynamic equilibrium that can be shifted by disruption of the amino acid cluster with a denaturant.     

Two mutations convert mammalian xanthine oxidoreductase to highly superoxide-productive xanthine oxidase

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.     

Sulfhydryl oxidase-catalyzed conversion of xanthine dehydrogenase to xanthine oxidase

Xanthine oxidase may be isolated from various mammalian tissues as one of two interconvertible forms, viz., a dehydrogenase (NAD⁺ dependent, form D) or an oxidase (O₂ utilizing, form O). A crude preparation of rat liver xanthine dehydrogenase (form D) was treated with an immobilized preparation of crude bovine sulfhydryl oxidase. Comparison of the rates of conversion of xanthine dehydrogenase to the O form in the presence and absence of the immobilized enzyme indicated that sulfhydryl oxidase catalyzes such conversion. These results were substantiated in a more definitive study in which purified bovine milk xanthine oxidase, which had been converted to the D form by treatment with dithiothreitol, was incubated with purified bovine milk sulfhydryl oxidase. Comparison of measured rates of conversion (in the presence and absence of active sulfhydryl oxidase and in the presence of thermally denatured sulfhydryl oxidase) revealed that sulfhydryl oxidase enzymatically catalyzes the conversion of type D activity to type O activity in xanthine oxidase with the concomitant disappearance of its sulfhydryl groups. It is possible that the presence or absence of sulfhydryl oxidase in a given tissue may be an important factor in determining the form of xanthine-oxidizing activity found in that tissue.     

Rat liver xanthine oxidoreductase: effect of adenine on the oxidase and dehydrogenase activities

Xanthine oxidase (XO) and total oxidase plus dehydrogenase (XO+XDH) activities from rat liver were measured in the presence or absence of adenine in extracts prepared with or without DTT/PMSF in homogenization buffer. Presence of adenine in extracts, prepared with or without DTT/PMSF, caused a 45-60% decrease in XO and XO+XDH activities. Removal of adenine by dialysis from extracts prepared with or without DTT/PMSF resulted in the recovery of XO and XO+XDH activities to almost their pre-dialysis control levels. Enzyme activity after 24hr storage at -20 degrees C depended on the presence or absence of DTT/PMSF and adenine, with both XO and XO+XDH activities being lower in extracts with the combined presence of DTT/PMSF and adenine. Incubation of extracts at 37 degrees C for 30 minutes resulted in increased XO and XO+XDH activities, however, adenine-treated samples did not differ from their pre-incubation activities. The molecular mass of the enzyme from control and adenine-treated extracts was unchanged (300 kDa). Adenine-treated extracts prepared with or without DTT/PMSF showed higher D/O ratios in all post-dialysis samples when compared with their pre-dialysis ratios. The results suggest that adenine may play a role in preventing the dehydrogenase to oxidase conversion during extract preparation, storage, overnight dialysis and heat treatment.     

Myocardial xanthine oxidase/dehydrogenase

High-energy phosphates in heart muscle deprived of oxygen are rapidly broken down to purine nucleosides and oxypurines. We studied the role of xanthine oxidase/dehydrogenase (EC 1.2.3.2/EC 1.2.1.37) in this process with novel high-pressure liquid chromatographic techniques. Under various conditions, including ischemia and anoxia, the isolated perfused rat heart released adenosine, inosine and hypoxanthine, and also substantial amounts of xanthine and urate. Allopurinol, an inhibitor of xanthine oxidase, greatly enhanced the release of hypoxanthine. From the purine release we calculated that the rat heart contained about 18 mU xanthine oxidase per g wet weight. Subsequently, we measured a xanthine oxidase activity of 9 mU/g wet wt. in rat-heart homogenate. When endogenous low molecular weight inhibitors were removed by gel-filtration, the activity increased to 31 mU/g wet wt. Rat myocardial xanthine oxidase seems to be present mainly in the dehydrogenase form, which upon storage at -20 degrees C is converted to the oxidase form.     

Xanthine oxidoreductase and xanthine oxidase in human cornea

Xanthine oxidoreductase (xanthine dehydrogenase + xanthine oxidase) is a complex enzyme that catalyzes the oxidation of hypoxanthine to xanthine, subsequently producing uric acid. The enzyme complex exists in separate but interconvertible forms, xanthine dehydrogenase and xanthine oxidase, which generate reactive oxygen species (ROS), a well known causative factor in ischemia/reperfusion injury and also in some other pathological states and diseases. Because the enzymes had not been localized in human corneas until now, the aim of this study was to detect xanthine oxidoreductase and xanthine oxidase in the corneas of normal post-mortem human eyes using histochemical and immunohistochemical methods. Xanthine oxidoreductase activity was demonstrated by the tetrazolium salt reduction method and xanthine oxidase activity was detected by methods based on cerium ion capture of hydrogen peroxide. For immunohistochemical studies. we used rabbit antibovine xanthine oxidase antibody, rabbit antihuman xanthine oxidase antibody and monoclonal mouse antihuman xanthine oxidase/xanthine dehydrogenase/aldehyde oxidase antibody. The results show that the enzymes are present in the corneal epithelium and endothelium. The activity of xanthine oxidoreductase is higher than that of xanthine oxidase, as clearly seen in the epithelium. Further studies are necessary to elucidate the role of these enzymes in the diseased human cornea. Based on the findings obtained in this study (xanthine oxidoreductase/xanthine oxidase activities are present in normal human corneas), we hypothesize that during various pathological states, xanthine oxidase-generated ROS might be involved in oxidative eye injury.     

Lack of conversion of xanthine dehydrogenase to xanthine oxidase during warm renal ischemia

Irreversible transformation of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) during ischemia was determined measuring XDH and total enzyme activity in kidneys before and after 60 min of clamp of the renal pedicle. Tissue levels of adenine nucleotides, xanthine and hypoxanthine were used as indicators of ischemia. After 60 min of clamping, ATP levels decreased by 72% with respect to controls whereas xanthine and hypoxanthine progressively reached tissue concentrations of 732 +/- 49 and 979 +/- 15 nmol.g tissue-1, respectively. Both total and XDH activities in ischemic kidneys (30 +/- 15 and 19 +/- 1 nmol.min-1.g tissue-1) were significantly lower than in controls when expressed on a tissue weight basis. The fraction of enzyme in the XDH form was however unchanged indicating that the reduction of the nucleotide pool is not accompanied by induction of the type-O activity of xanthine oxidase.     

Comparative study of chicken liver xanthine dehydrogenase and bovine liver xanthine oxidase. dehydrogenase activity of xanthine oxidase (author's transl)]

A method to purify bovine liver xanthine oxidase in described, with which samples of 256-fold specific activity with respect to the initial homogenate are obtained. Bovine liver xanthine oxidase and chicken liver xanthine dehydrogenase with oxygen as electron acceptor exhibit similar profile in pKM and log V versus pH plots. With NAD+ as electron acceptor a different profile in the pKM xanthine plot is obtained for chicken liver xanthine dehydrogenase. However three inflection points at the same pH values appear in all plots. Both enzymes are irreversibly inhibited by pCMB and reversibly by N-ethylmaleimide and by iodoacetamide, with competitive and uncompetitive type inhibitions respectively. These results suggest that NAD+ alters the enzymatic action since its binding to the enzyme antecedes the binding of xanthine to the xanthine oxidase molecule, without undergoing itself any modification. 0.15 M DDT of DTE treatment of bovine liver xanthine oxidase gives to the enzyme a permanent activity with NAD+ without modifying its activity with oxygen. The enzyme thus treated produces parallel straight lines in Lineweaver-Burk plots.     

Post-irradiation free radical generation: evidence from the conversion of xanthine dehydrogenase into xanthine oxidase

The xanthine oxidoreductase (XOR) system which consists of xanthine dehydrogenase (XDH) and xathine oxidase (XO), is one of the major sources of free radicals in biological systems. The XOR system is pre-dominantly present as XDH in normal tissues and converts into the free radical generating XO-form in the damaged tissue. Therefore, the XO-form of the XOR system is expected to be mainly found in radiolytically damaged tissues. In such an event, XO may catalyze the generation of free radicals and potentiate radiation effects in the post-irradiation period. Recent findings on the effect of ionizing radiation on the XOR system in the liver of mice, peroxidative damage and lactate dehydrogenase support this possibility. From these results it has been hypothesized that free radical generating systems could be activated in the radiolytically damaged cell and in turn contribute to the cause and complications of late effects and their persistence in post-irradiation period. This aspect may have great significance in the understanding of radiation-induced damages. It may also have serious implication in various fields like radiation therapy, health physics, carcinogenesis, space travelling radiation exposures and post nuclear accident care. Further, it is suggested that efforts need to be made to search more system(s) which could be activated particularly at lower doses of radiation to generate free radicals in the post-exposure period.     

Phosphorylation of xanthine dehydrogenase/oxidase in hypoxia

The enzyme xanthine oxidase (XO) has been implicated in the pathogenesis of several disease processes, such as ischemia-reperfusion injury, because of its ability to generate reactive oxygen species. The expression of XO and its precursor xanthine dehydrogenase (XDH) is regulated at pre- and posttranslational levels by agents such as lipopolysaccharide and hypoxia. Posttranslational modification of the protein, for example through thiol oxidation or proteolysis, has been shown to be important in converting XDH to XO. The possibility of posttranslational modification of XDH/XO through phosphorylation has not been adequately investigated in mammalian cells, and studies have reported conflicting results. The present report demonstrates that XDH/XO is phosphorylated in rat pulmonary microvascular endothelial cells (RPMEC) and that phosphorylation is greatly increased ( approximately 50-fold) in response to acute hypoxia (4 h). XDH/XO phosphorylation appears to be mediated, at least in part, by casein kinase II and p38 kinase as inhibitors of these kinases partially prevent XDH/XO phosphorylation. In addition, the results indicate that p38 kinase, a stress-activated kinase, becomes activated in response to hypoxia (an approximately 4-fold increase after 1 h of exposure of RPMEC to hypoxia) further supporting a role for this kinase in hypoxia-stimulated XDH/XO phosphorylation. Finally, hypoxia-induced XDH/XO phosphorylation is accompanied by a 2-fold increase in XDH/XO activity, which is prevented by inhibitors of phosphorylation. In summary, this study shows that XDH/XO is phosphorylated in hypoxic RPMEC through a mechanism involving p38 kinase and casein kinase II and that phosphorylation is necessary for hypoxia-induced enzymatic activation.     

Distribution of xanthine oxidase and xanthine dehydrogenase specificity types among bacteria.

A diverse collection of xanthine-metabolizing bacteria was examined for xanthine-, 1-methylxanthine-, and 3-methylxanthine-oxidizing activity. Both particulate and soluble fractions of extracts from aerobically grown gram-negative bacteria exhibited oxidation of all three substrates; however, when facultative gram-negative bacteria were grown anaerobically, low particulate and 3-methylxanthine activities were detected. Gram-positive and obligately anaerobic bacteria showed no particulate activity or 3-methylxanthine oxidation. Substrate specificity studies indicate two types of enzyme distributed among the bacteria along taxonomic lines, although other features indicate diversity of the enzyme within these two major groups. The soluble and particulate enzymes from Pseudomonas putida and the enzyme from Arthrobacter S-2 were examined as type examples with a series of purine and analogues differing in the number and position of oxygen groups. Each preparation was active with a variety of compounds, but the compounds and position attacked by each enzyme was different, both from the other enzymes examined and from previously investigated enzymes. The soluble enzyme from Pseudomonas was inhibited in a competitive manner by uric acid, whereas the Arthrobacter enzyme was not. This was correlated with the ability of Pseudomonas, but not Arthrobacter, to incorporate radioactivity from [2-14C]uric acid into cellular material.     

Electrophoretic analyses of alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, sorbitol dehydrogenase and xanthine oxidase from mouse tissues.

1. Cellulose acetate zymograms of alcohol dehydrogenase (ADH), aldehyde dehydrogenase, sorbitol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase extracted from tissues of inbred mice were examined. 2. ADH isozymes were differentially distributed in mouse tissues: A2--liver, kidney, adrenals and intestine; B2--all tissues examined; C2--stomach, adrenals, epididymis, ovary, uterus, lung. 3. Two NAD+-specific aldehyde dehydrogenase isozymes were observed in liver and kidney and differentially distributed in other tissues. Alcohol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase were also stained when aldehyde dehydrogenase was being examined. 4. Two aldehyde oxidase isozymes exhibited highest activities in liver. 5. "Phenazine oxidase" was widely distributed in mouse tissues whereas xanthine oxidase exhibited highest activity in intestine and liver extracts. 6. Genetic variants for ADH-C2 established its identity with a second form of sorbitol dehydrogenase observed in stomach and other tissues. The major sorbitol dehydrogenase was found in high activity in liver, kidney, pancreas and male reproductive tissues.     

Nonlinear kinetics of the xanthine oxidase reaction catalyzed by chicken liver xanthine dehydrogenase

The xanthine oxidase reaction catalyzed by chicken liver xanthine dehydrogenase has been shown to give nonlinear kinetics of the type which has been identified as substrate activation. When a very wide range of substrate (pteridine) concentrations were studied, it was found that a downward deflection in reciprocal plots (substrate activation) occurs in the high region and an upward deflection in the very low region. When product (isoxanthopterin) was included in reaction mixtures, the upward deflection was enhanced and shifted to higher substrate concentration ranges. In addition, reciprocal plots with a second substrate (oxygen) and a product (isoxanthopterin) were nonlinear.     

Interrelation of xanthine oxidase and dehydrogenase and L-gulonolactone oxidase in animal tissues

There is a correlation between phylogeny and the activities of L-gulonolactone oxidase (LGO), the key enzyme responsible for ascorbic acid (AH2) synthesis in animals and total xanthine oxidase and dehydrogenase [XOD(D/O)], the enzyme responsible for the production of endogenous superoxide radical (O2-.). LGO appears in the kidneys of amphibians and reptiles but livers of mammals. XOD(D/O) also is present mainly in the kidneys of amphibians and reptiles and livers of mammals. AH2 is a potential scavenger of O2-. and it appears that tissue specific expression of LGO takes place to counteract the endogenous O2-. toxicity. The interrelation of XOD(D/O) and LGO was also observed in the liver of rats during prenatal to postnatal development.     

Electrophoretic analyses of alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde reductase, aldehyde oxidase and xanthine oxidase from horse tissues

Cellulose acetate zymograms of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (AHD), aldehyde reductase (AHR), aldehyde oxidase (AOX) and xanthine oxidase (XOX) extracted from horse tissues were examined. Five ADH isozymes were resolved: three corresponded to the previously reported class I ADHs (EE, ES and SS) (Theorell, 1969); a single form of class II ADH (designated ADH-C2) and of class III ADH (designated ADH-B2) were also observed. The latter isozyme was widely distributed in horse tissues whereas the other enzymes were found predominantly in liver. Four AHD isozymes were differentially distributed in subcellular preparations of horse liver: AHD-1 (large granules); AHD-3 (small granules); and AHD-2, AHD-4 (cytoplasm). AHD-1 was more widely distributed among the horse tissues examined. Liver represented the major source of activity for most AHDs. A single additional form of NADPH-dependent AHR activity (identified as hexonate dehydrogenase), other than the ADHs previously described, was observed in horse liver. Single forms of AOX and XOX were observed in horse tissue extracts, with highest activities in liver.     

Comparative localization of aldehyde oxidase and xanthine oxidoreductase activity in rat tissues

The distribution of aldehyde oxidase activity was evaluated in unfixed cryostat sections from tissues of male Wistar rats using a tissue protectant, polyvinyl alcohol, with Tetranitro BT as a final electron acceptor. The distribution of aldehyde oxidase activity was compared with that of xanthine oxidoreductase. The enzyme histochemical method demonstrated aldehyde oxidase activity in the epithelium of the tongue, renal tubules and bronchioles, as well as in the cytoplasm of liver cells. Such activity was not detected in oesophagus, stomach, spleen, adrenal glands, small or large intestine or skeletal and heart muscle fibres. In contrast, xanthine oxidoreductase activity was demonstrated in the tongue, renal tubules, bronchioles, oesophageal, gastric, small and large intestinal epithelial cells, adrenal glands, spleen and liver cytoplasm but not in skeletal and heart muscle fibres. The significance of the ubiquitous distribution of aldehyde oxidase activity, especially in surface epithelial cells from various tissues, except for the gastrointestinal tract, is unclear. However, aldehyde oxidase may possess some physiological activity other than in the metabolism of N-heterocyclics or of certain drugs. © 1998 Chapman & Hall