Avian and canine aldehyde oxidases. Novel insights into the biology and evolution of molybdo-flavoenzymes

Aldehyde oxidases are molybdo-flavoenzymes structurally related to xanthine oxidoreductase. They catalyze the oxidation of aldehydes or N-heterocycles of physiological, pharmacological, and toxicological relevance. Rodents are characterized by four aldehyde oxidases as follows: AOX1 and aldehyde oxidase homologs 1-3 (AOH1, AOH2, and AOH3). Humans synthesize a single functional aldehyde oxidase, AOX1. Here we define the structure and the characteristics of the aldehyde oxidase genes and proteins in chicken and dog. The avian genome contains two aldehyde oxidase genes, AOX1 and AOH, mapping to chromosome 7. AOX1 and AOH are structurally very similar and code for proteins whose sequence was deduced from the corresponding cDNAs. AOX1 is the ortholog of the same gene in mammals, whereas AOH represents the likely ancestor of rodent AOH1, AOH2, and AOH3. The dog genome is endowed with two structurally conserved and active aldehyde oxidases clustering on chromosome 37. Cloning of the corresponding cDNAs and tissue distribution studies demonstrate that they are the orthologs of rodent AOH2 and AOH3. The vestiges of dog AOX1 and AOH1 are recognizable upstream of AOH2 and AOH3 on the same chromosome. Comparison of the complement and the structure of the aldehyde oxidase and xanthine oxidoreductase genes in vertebrates and other animal species indicates that they evolved through a series of duplication and inactivation events. Purification of the chicken AOX1 protein to homogeneity from kidney demonstrates that the enzyme possesses retinaldehyde oxidase activity. Unlike humans and most other mammals, dog and chicken are devoid of liver aldehyde oxidase activity.     

Purification of the aldehyde oxidase homolog 1 (AOH1) protein and cloning of the AOH1 and aldehyde oxidase homolog 2 (AOH2) genes. Identification of a novel molybdo-flavoprotein gene cluster on mouse chromosome 1

We report the cloning of the AOH1 and AOH2 genes, which encode two novel mammalian molybdo-flavoproteins. We have purified the AOH1 protein to homogeneity in its catalytically active form from mouse liver. Twenty tryptic peptides, identified or directly sequenced by mass spectrometry, confirm the primary structure of the polypeptide deduced from the AOH1 gene. The enzyme contains one molecule of FAD, one atom of molybdenum, and four atoms of iron per subunit and shows spectroscopic features similar to those of the prototypic molybdo-flavoprotein xanthine oxidoreductase. The AOH1 and AOH2 genes are 98 and 60 kilobases long, respectively, and consist of 35 coding exons. The AOH1 gene has the potential to transcribe an extra leader non-coding exon, which is located downstream of exon 26, and is transcribed in the opposite orientation relative to all the other exons. AOH1 and AOH2 map to chromosome 1 in close proximity to each other and to the aldehyde oxidase gene, forming a molybdo-flavoenzyme gene cluster. Conservation in the position of exon/intron junctions among the mouse AOH1, AOH2, aldehyde oxidase, and xanthine oxidoreductase loci indicates that these genes are derived from the duplication of an ancestral precursor.     

Regulation and biochemistry of mouse molybdo-flavoenzymes. The DBA/2 mouse is selectively deficient in the expression of aldehyde oxidase homologues 1 and 2 and represents a unique source for the purification and characterization of aldehyde oxidase

Mouse molybdo-flavoenzymes consist of xanthine oxidoreductase, aldehyde oxidase (AOX1), and two recently identified proteins, AOH1 and AOH2 (aldehyde oxidase homologues 1 and 2). Here we demonstrate that CD-1, C57BL/6, 129/Sv, and other mouse strains synthesize high levels of AOH1 in the liver and AOH2 in the skin. By contrast, the DBA/2 and CBA strains are unique, having a selective deficit in the expression of the AOH1 and AOH2 genes. DBA/2 animals synthesize trace amounts of a catalytically active AOH1 protein. However, relative to CD-1 animals, an over 2 log reduction in the steady-state levels of liver AOH1 mRNA, protein, and enzymatic activity is observed in basal conditions and following administration of testosterone. The DBA/2 mouse represents a unique opportunity to purify AOX1 and compare its enzymatic characteristics to those of the AOH1 protein. The spectroscopy and biochemistry of AOX1 are very similar to those of AOH1 except for a differential sensitivity to the non-competitive inhibitory effect of norharmane. AOX1 and AOH1 oxidize an overlapping set of aldehydes and heterocycles. For most compounds, the substrate efficiency (V(max)/K(m)) of AOX1 is superior to that of AOH1. Alkylic alcohols and acetaldehyde, the toxic metabolite of ethanol, are poor substrates of both enzymes. Consistent with this, the levels of acetaldehyde in the livers of ethanol administered CD-1 and DBA/2 mice are similar, indicating that neither enzyme is involved in the in vivo biotransformation of acetaldehyde.     

The First Mammalian Aldehyde Oxidase Crystal Structure: INSIGHTS INTO SUBSTRATE SPECIFICITY*

Aldehyde oxidases (AOXs) are homodimeric proteins belonging to the xanthine oxidase family of molybdenum-containing enzymes. Each 150-kDa monomer contains a FAD redox cofactor, two spectroscopically distinct [2Fe-2S] clusters, and a molybdenum cofactor located within the protein active site. AOXs are characterized by broad range substrate specificity, oxidizing different aldehydes and aromatic N-heterocycles. Despite increasing recognition of its role in the metabolism of drugs and xenobiotics, the physiological function of the protein is still largely unknown. We have crystallized and solved the crystal structure of mouse liver aldehyde oxidase 3 to 2.9 Å. This is the first mammalian AOX whose structure has been solved. The structure provides important insights into the protein active center and further evidence on the catalytic differences characterizing AOX and xanthine oxidoreductase. The mouse liver aldehyde oxidase 3 three-dimensional structure combined with kinetic, mutagenesis data, molecular docking, and molecular dynamics studies make a decisive contribution to understand the molecular basis of its rather broad substrate specificity.     

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.     

Alcohol-induced breast cancer: a proposed mechanism

Alcohol consumption increases the risk for breast cancer in women by still undefined means. Alcohol metabolism is known to produce reactive oxygen species (ROS), and breast cancer is associated with high levels of hydroxyl radical (^·OH) modified DNA, point mutations, single strand nicks, and chromosome rearrangement. Furthermore, ROS modification of DNA can produce the mutations and DNA damage found in breast cancer. Alcohol dehydrogenase (ADH) and xanthine oxidoreductase (XOR) are expressed and regulated in breast tissues and aldehyde oxidase (AOX) may be present as well. Mammary gland XOR is an efficient source of ROS. Recently, hepatic XOR and AOX were found to generate ROS in two ways from alcohol metabolism: by acetaldehyde consumption and by the intrinsic NADH oxidase activity of both XOR and AOX. The data obtained suggests that: (1) expression of ADH and XOR or AOX in breast tissue provides the enzymes that generate ROS; (2) metabolism of alcohol produces acetaldehyde and NADH that can both be substrates for XOR or AOX and thereby result in ROS formation; and (3) ROS generated by XOR or AOX can induce the carcinogenic mutations and DNA damage found in breast cancer. Accumulation of iron coupled with diminished antioxidant defenses in breast tissue with advancing age provide additional support for this hypothesis because both result in elevated ROS damage that may exacerbate the risk for ROS-induced breast cancer.     

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.     

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.     

Identification of the candidate ALS2 gene at chromosome 2q33 as a human aldehyde oxidase gene

Denver, Tokyo, and Salt Lake City investigators recently published different complimentary deoxyribonucleic acid (cDNA) sequences for human liver xanthine dehydrogenase/xanthine oxidase (XD/XO). The gene encoding the Denver cDNA was subsequently linked to juvenile familial amyotrophic lateral sclerosis (JFALS) at chromosome 2q33 and has been proposed as the ALS2 locus. The present investigation was undertaken to elucidate the differences between the three cDNA sequences, and we provide evidence that the Denver cDNA encodes aldehyde oxidase (AO): first, the Denver cDNA sequence diverged significantly from the Tokyo and Salt Lake City cDNA sequences which were very similar; second, the deduced protein sequence from the Denver cDNA was very similar to the amino acid sequence of purified rabbit liver AO protein; third, the deduced Denver protein sequence was 76% identical to the encoded 101 amino acid long peptides from partial cDNAs for rabbit and rat AO and 81.7% identical to 300 amino acids from an incomplete cDNA encoding bovine AO; fourth, the Denver gene was expressed in liver, kidney, lung, pancreas, prostate, testes, and ovary while the Tokyo XD gene was expressed predominantly in liver and small intestine; fifth, the Denver gene was previously mapped to chromosome 2q33 which is syntenic to the mouse AO locus on chromosome 1. Our results have revealed dramatic similarities in protein and DNA sequence in the human molybdenum hydroxylases, have uncovered unanticipated complexity in the human molybdenum hydroxylase genes, and advance the potential for AO derived oxygen radicals in JFALS and other human diseases.     

Studies on the specificity toward aldehyde substrates and steady-state kinetics of xanthine oxidase

The aldehyde specificity of xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) has been reinvestigated. The biogenic aldehydes and succinate semialdehyde are reasonable substrates for xanthine oxidase. Pyrophosphate, which binds to xanthine oxidase, does not seem to affect significantly the enzyme's catalytic activity. The steady-state parameters for the oxidation of several substrates by xanthine oxidase and oxygen have been determined. Formaldehyde differs from xanthine and other aldehydes in phi 2, the parameter describing the reaction with oxygen. Substrate inhibition has been studied at high concentrations of xanthine with oxygen as the electron acceptor. The inhibition is hyperbolic and uncompetitive with respect to oxygen. This is possibly due to rate-limiting product release from molybdenum(IV) being slower than from molybdenum(VI).     

Iron regulates xanthine oxidase activity in the lung

The iron chelator deferoxamine has been reported to inhibit both xanthine oxidase (XO) and xanthine dehydrogenase activity, but the relationship of this effect to the availability of iron in the cellular and tissue environment remains unexplored. XO and total xanthine oxidoreductase activity in cultured V79 cells was increased with exposure to ferric ammonium sulfate and inhibited by deferoxamine. Lung XO and total xanthine oxidoreductase activities were reduced in rats fed an iron-depleted diet and increased in rats supplemented with iron, without change in the ratio of XO to total oxidoreductase. Intratracheal injection of an iron salt or silica-iron, but not aluminum salts or silica-zinc, significantly increased rat lung XO and total xanthine oxidoreductase activities, immunoreactive xanthine oxidoreductase, and the concentration of urate in bronchoalveolar fluid. These results suggest the possibility that the production of uric acid, a major chelator of iron in extracellular fluid, is directly influenced by iron-mediated regulation of the expression and/or activity of its enzymatic source, xanthine oxidase.     

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     

Analysis of the electron paramagnetic resonance properties of the [2Fe-2S]1+ centers in molybdenum enzymes of the xanthine oxidase family: assignment of signals I and II

Molybdoenzymes of the xanthine oxidase family contain two [2Fe-2S](1+,2+) clusters that are bound to the protein by very different cysteine motifs. In the X-ray crystal structure of Desulfovibrio gigas aldehyde oxidoreductase, the cluster ligated by a ferredoxin-type motif is close to the protein surface, whereas that ligated by an unusual cysteine motif is in contact with the molybdopterin [Romao, M. J., Archer, M., Moura, I., Moura, J. J. G., LeGall, J., Engh, R., Schneider, M., Hof, P., and Huber, R. (1995) Science 270, 1170-1176]. These two clusters display distinct electron paramagnetic resonance (EPR) signals: the less anisotropic one, called signal I, is generally similar to the g(av) approximately 1.96-type signals given by ferredoxins, whereas signal II often exhibits anomalous properties such as very large g values, broad lines, and very fast relaxation properties. A detailed comparison of the temperature dependence of the spin-lattice relaxation time and of the intensity of these signals in D. gigas aldehyde oxidoreductase and in milk xanthine oxidase strongly suggests that the peculiar EPR properties of signal II arise from the presence of low-lying excited levels reflecting significant double exchange interactions. The issue raised by the assignment of signals I and II to the two [2Fe-2S](1+) clusters was solved by using the EPR signal of the Mo(V) center as a probe. The temperature dependence of this signal could be quantitatively reproduced by assuming that the Mo(V) center is coupled to the cluster giving signal I in xanthine oxidase as well as in D. gigas aldehyde oxidoreductase. This demonstrates unambiguously that, in both enzymes, signal I arises from the center which is closest to the molybdenum cofactor.     

Identification of Crucial Amino Acids in Mouse Aldehyde Oxidase 3 That Determine Substrate Specificity

In order to elucidate factors that determine substrate specificity and activity of mammalian molybdo-flavoproteins we performed site directed mutagenesis of mouse aldehyde oxidase 3 (mAOX3). The sequence alignment of different aldehyde oxidase (AOX) isoforms identified variations in the active site of mAOX3 in comparison to other AOX proteins and xanthine oxidoreductases (XOR). Based on the structural alignment of mAOX3 and bovine XOR, differences in amino acid residues involved in substrate binding in XORs in comparison to AOXs were identified. We exchanged several residues in the active site to the ones found in other AOX homologues in mouse or to residues present in bovine XOR in order to examine their influence on substrate selectivity and catalytic activity. Additionally we analyzed the influence of the [2Fe-2S] domains of mAOX3 on its kinetic properties and cofactor saturation. We applied UV-VIS and EPR monitored redox-titrations to determine the redox potentials of wild type mAOX3 and mAOX3 variants containing the iron-sulfur centers of mAOX1. In addition, a combination of molecular docking and molecular dynamic simulations (MD) was used to investigate factors that modulate the substrate specificity and activity of wild type and AOX variants. The successful conversion of an AOX enzyme to an XOR enzyme was achieved exchanging eight residues in the active site of mAOX3. It was observed that the absence of the K889H exchange substantially decreased the activity of the enzyme towards all substrates analyzed, revealing that this residue has an important role in catalysis.     

Liver aldehyde oxidase and xanthine oxidase genetics in the mouse

A 'null' activity variant for the major liver isozyme of aldehyde oxidase (AOX-1) in adult male mice and an electrophoretically distinct, high activity variant of the second liver isozyme (AOX-2) were used to examine the segregation of the genetic loci encoding these enzymes (Aox-1 and Aox-2 respectively) in breeding studies. A single recombinant between these loci was observed among the 147 backcross progeny examined, which confirms a previous report (Holmes, 1979) for close linkage and genetic distinctness of the two loci. An activity variant for mouse liver xanthine oxidase (XOX) is also reported which behaved as though controlled by codominant alleles at a single locus (designated Xox-1 ). Genetic analyses showed that the Xox-1 locus segregated independently of the multiple- A ox loci.     

The role of aldehyde oxidase in ethanol-induced hepatic lipid peroxidation in the rat.

Hepatic lipid peroxidation has been implicated in the pathogenesis of alcohol-induced liver injury, but the mechanism(s) by which ethanol metabolism or resultant free radicals initiate lipid peroxidation is not fully defined. The role of the molybdenum-containing enzymes aldehyde oxidase and xanthine oxidase in the generation of such free radicals was investigated by measuring alkane production (lipoperoxidation products) in isolated rat hepatocytes during ethanol metabolism. Inhibition of aldehyde oxidase and xanthine oxidase (by feeding tungstate at 100 mg/day per kg) decreased alkane production (80-95%), whereas allopurinol (20 mg/kg by mouth), a marked inhibitor of xanthine oxidase, inhibited alkane production by only 35-50%. Addition of acetaldehyde (0-100 microM) (in the presence of 50 microM-4-methylpyrazole) increased alkane production in a dose-dependent manner (Km of aldehyde oxidase for acetaldehyde 1 mM); menadione, an inhibitor of aldehyde oxidase, virtually inhibited alkane production. Desferrioxamine (5-10 microM) completely abolished alkane production induced by both ethanol and acetaldehyde, indicating the importance of catalytic iron. Thus free radicals generated during the metabolism of acetaldehyde by aldehyde oxidase may be a fundamental mechanism in the initiation of alcohol-induced liver injury.     

The molybdoenzymes xanthine oxidase and aldehyde oxidase contain fast- and slow-DTNB reacting sulphydryl groups

The reactivities with an excess of 5-5-dithiobis (2-nitrobenzoic) acid (DTNB) of sulphydryl residues present in xanthine oxidase and aldehyde oxidase were studied and compared. The results show that two classes of sulphydryl groups with quite different reactivities exist in both enzymes either native or denatured. Some of the available sulphydryl residues thus react instantaneously with the DTNB, whereas the others react very slowly following pseudo-first-order kinetics. The number of sulphydryl residues of each class and the rate constant of slowly reacting groups are, respectively, 1.7 and 0.8 in native xanthine oxidase and 1.6 and 1.7 in native aldehyde oxidase. In denatured enzymes, the number of fast- and slow-reacting sulphydryl residues obtained are, respectively, 13.9 and 7.9 in xanthine oxidase and 5.7 and 5.4 in aldehyde oxidase. Analogously, the rate constant for the slowly reacting groups is similar for the two native enzymes, but in denatured aldehyde oxidase it is double that of denatured xanthine oxidase.