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N-{trans-3-[(5-Cyano-6-methylpyridin-2-yl)oxy]-2,2,4,4-tetramethylcyclobutyl}imidazo[1,2-a]pyrimidine-3-carboxamide (1) was recently identified as a full antagonist of the androgen receptor, demonstrating excellent in vivo tumor growth inhibition in castration-resistant prostate cancer (CRPC). However, the imidazo[1,2-a]pyrimidine moiety is rapidly metabolized by aldehyde oxidase (AO). The present paper describes a number of medicinal chemistry strategies taken to avoid the AO-mediated oxidation of this particular system. Guided by an AO protein structure-based model, our investigation revealed the most probable site of AO oxidation and the observation that altering the heterocycle or blocking the reactive site are two of the more effective strategies for reducing AO metabolism. These strategies may be useful for other drug discovery programs.

Aldehyde oxidases are molybdoflavoenzymes with broad substrate specificity, oxidizing different types of aldehydes, and heterocyclic rings. The physiological function of aldehyde oxidases is largely unknown, although the enzymes play an important role in the metabolism of numerous compounds of medicinal and toxicological interest, as they oxidize a wide range of aldehydes and heterocyclic compounds. In this article, we review the significance of aldehyde oxidases for the design and development of new drugs and discuss associated problems. These include species-specific differences in the complement of isoenzymes synthesized and general difficulties in studying the enzymatic characteristics of purified or recombinant aldehyde oxidases. We highlight the potential offered by human aldehyde oxidase targeting for the development of new pharmacological agents, limiting our attention to the realms of antiobesity and anti-cancer drugs. The last point is discussed in the context of the design of novel anticancer drugs, selectively activated or inactivated by this enzyme, with the final aim of achieving organ and/or tumor selectivity.

Aldehyde oxidase is a widely distributed enzyme that is involved in the metabolism of an extensive range of aldehydes and N-heterocyclic compounds with physiological, pharmacological, and toxicological relevance. In the present study, a highly sensitive RP-HPLC-fluorescence method based on the oxidation of phenanthridine to phenanthridinone has been developed and validated to assay aldehyde oxidase activity in biological samples. Determination of phenanthridinone was achieved on a C18 column using 10 mmol/L phosphate buffer (pH 5.0) containing 0.1 mmol/L EDTA-acetonitrile (40 + 60, v/v) as the mobile phase. The fluorescence intensity of phenanthridinone was measured at 364 nm with excitation at 236 nm. The proposed method was precise, accurate, specific and rapid (analysis time, approximately 8 min) with a mean RSD of 2.54%. Peak responses were linear from 0.5 to 100 nmol/L, with an LOD of 0.125 nmol/L. The applicability of the method was demonstrated by measurement of aldehyde oxidase activity in rat liver, kidney, ovary, and heart fractions.

Aldehyde oxidase 1 (AOX1) is a major member of the xanthine oxidase family belonging to the class of complex molybdo-flavoenzymes and plays an important role in the nucleophilic oxidation of N-heterocyclic aromatic compounds and various aldehydes. The enzyme has been well known to show remarkable species differences. Comparing the rabbit and monkey enzymes, the former showed extremely high activity toward cinchonidine and methotrexate, but the latter exhibited only marginal activities. In contrast, monkey had several times greater activity than did rabbit toward zonisamide and (+)-4-(4-cyanoanilino)-5,6-dihydro-7-hydroxy-7H-cyclopenta[d]-pyrimidine [(S)-RS-8359]. In this report, we tried to confer high cinchonidine oxidation activity comparable with that of rabbit AOX1 to monkey AOX1. The chimera proteins prepared by restriction enzyme digestion and recombination methods between monkey and rabbit AOX1s indicated that the sequences from Asn993 to Ala1088 of rabbit AOX1 are essential for the activity. The kinetic parameters were then measured using monkey AOX1 mutants prepared by site-directed mutagenesis. The monkey V1085A mutant acquired the high cinchonidine oxidation activity. Inversely, the reciprocal rabbit A1081V mutant lost the activity entirely: amino acid 1081 of rabbit AOX1 corresponding to amino acid 1085 of monkey AOX1. Thus, cinchonidine oxidation activity was drastically changed by mutation of a single residue in AOX1. However, this might be true for bulky substrates such as cinchonidine but not for small substrates. The mechanism of substrate-dependent species differences in AOX1 activity toward bulky substrates is discussed.

Aldehyde oxidase (AO) is a cytosolic enzyme with an important role in drug and xenobiotic metabolism. Although AO has structural similarity to bacterial nitrite reductases, it is unknown whether AO-catalyzed nitrite reduction can be an important source of NO. The mechanism, magnitude, and quantitative importance of AO-mediated nitrite reduction in tissues have not been reported. To investigate this pathway and its quantitative importance, EPR spectroscopy, chemiluminescence NO analyzer, and immunoassays of cGMP formation were performed. The kinetics and magnitude of AO-dependent NO formation were characterized. In the presence of typical aldehyde substrates or NADH, AO reduced nitrite to NO. Kinetics of AO-catalyzed nitrite reduction followed Michaelis-Menten kinetics under anaerobic conditions. Under physiological conditions, nitrite levels are far below its measured K(m) value in the presence of either the flavin site electron donor NADH or molybdenum site aldehyde electron donors. Under aerobic conditions with the FAD site-binding substrate, NADH, AO-mediated NO production was largely maintained, although with aldehyde substrates oxygen-dependent inhibition was seen. Oxygen tension, substrate, and pH levels were important regulators of AO-catalyzed NO generation. From kinetic data, it was determined that during ischemia hepatic, pulmonary, or myocardial AO and nitrite levels were sufficient to result in NO generation comparable to or exceeding maximal production by constitutive NO synthases. Thus, AO-catalyzed nitrite reduction can be an important source of NO generation, and its NO production will be further increased by therapeutic administration of nitrite.

Neonicotinoid insecticide metabolism involves considerable substrate specificity and regioselectivity of the relevant CYP450, aldehyde oxidase, and phase II enzymes. Human CYP450 recombinant enzymes carry out the following conversions: CYP3A4, 2C19, and 2B6 for thiamethoxam (TMX) to clothianidin (CLO); 3A4, 2C19, and 2A6 for CLO to desmethyl-CLO; 2C19 for TMX to desmethyl-TMX. Human liver aldehyde oxidase reduces the nitro substituent of CLO to nitroso much more rapidly than it does that of TMX. Imidacloprid (IMI), CLO, and several of their metabolites do not give detectable N-glucuronides but 5-hydroxy-IMI, 4,5-diol-IMI, and 4-hydroxythiacloprid are converted to O-glucuronides in vitro with mouse liver microsomes and UDP-glucuronic acid or in vivo in mice. Mouse liver cytosol with S-adenosylmethionine converts desmethyl-CLO to CLO but not desmethyl-TMX to TMX. Two organophosphorus CYP450 inhibitors partially block IMI, thiacloprid, and CLO metabolism in vivo in mice, elevating brain and liver levels of the parent compounds while reducing amounts of the hydroxylated metabolites.

Aldehyde oxidase (AO) plays important role in plant hormone biosynthetic pathways, such as abscisic acid (ABA) and indole-3-acetic acid (IAA). The enzyme catalyzes the last step of the pathways. In this study a full-length cDNA encoding an aldyhyde oxidase was cloned and sequenced from leaves of peanut by RT-PCR, RACE-PCR and genomic DNA walking methods. The full-length cDNA, designated as Arachis hygogaea L. aldehyde oxidase 1 (AhAO1), consists of an open reading frame of 4131 bp, a 326 bp 5' untranslated region and a 128 bp 3' untranslated region including a poly (A) tail of 21 nucleotides. The gene encodes a polypeptide of 1377 amino acids with a calculated molecular weight of 150 kDa and an isoelectric point (pl) of 6.99. Analysis of amino acid sequence of AhAO1 shows that it had 61%, 59% and 55% identity with the AOs from tomato, Arabidopsis and maize, respectively The peanut AO polypeptide contains consensus sequences for iron-sulfur centers and a molybdenum cofactor (MoCo)-binding domain. Semi-quantitative RT-PCR analysis showed that AhAO1 expression was higher in leaves than in roots of peanut.

Mouse aldehyde oxidase (mAOX1) forms a homodimer and belongs to the xanthine oxidase family of molybdoenzymes which are characterized by an essential equatorial sulfur ligand coordinated to the molybdenum atom. In general, mammalian AOs are characterized by broad substrate specificity and an yet obscure physiological function. To define the physiological substrates and the enzymatic characteristics of mAOX1, we established a system for the heterologous expression of the enzyme in Escherichia coli. The recombinant protein showed spectral features and a range of substrate specificity similar to the native protein purified from mouse liver. The EPR data of recombinant mAOX1 were similar to those of AO from rabbit liver, but differed from the homologous xanthine oxidoreductase enzymes. Site-directed mutagenesis of amino acids Val806, Met884 and Glu1265 at the active site resulted in a drastic decrease in the oxidation of aldehydes with no increase in the oxidation of purine substrates. The double mutant V806E/M884R and the single mutant E1265Q were catalytically inactive enzymes regardless of the aldehyde or purine substrates tested. Our results show that only Glu1265 is essential for the catalytic activity by initiating the base-catalyzed mechanism of substrate oxidation. In addition, it is concluded that the substrate specificity of molybdo-flavoenzymes is more complex and not only defined by the three characterized amino acids in the active site.

Aldehyde oxidase (AO) is a homodimer with a subunit molecular mass of approximately 150 kDa. Each subunit consists of about 20 kDa 2Fe-2S cluster domain storing reducing equivalents, about 40 kDa flavine adenine dinucleotide (FAD) domain and about 85 kDa molybdenum cofactor (MoCo) domain containing a substrate binding site. In order to clarify the properties of each domain, especially substrate binding domain, chimeric cDNAs were constructed by mutual exchange of 2Fe-2S/FAD and MoCo domains between monkey and rat. Chimeric monkey/rat AO was referred to one with monkey type 2Fe-2S/FAD domains and a rat type MoCo domain. Rat/monkey AO was vice versa. AO-catalyzed 2-oxidation activities of (S)-RS-8359 were measured using the expressed enzyme in Escherichia coli. Substrate inhibition was seen in rat AO and chimeric monkey/rat AO, but not in monkey AO and chimeric rat/monkey AO, suggesting that the phenomenon might be dependent on the natures of MoCo domain of rat. A biphasic Eadie-Hofstee profile was observed in monkey AO and chimeric rat/monkey AO, but not rat AO and chimeric monkey/rat AO, indicating that the biphasic profile might be related to the properties of MoCo domain of monkey. Two-fold greater V(max) values were observed in monkey AO than in chimeric rat/monkey AO, and in chimeric monkey/rat AO than in rat AO, suggesting that monkey has the more effective electron transfer system than rat. Thus, the use of chimeric enzymes revealed that 2Fe-2S/FAD and MoCo domains affect the velocity and the quantitative profiles of AO-catalyzed (S)-RS-8359 2-oxidation, respectively.

DFT calculations support a concerted mechanism for xanthine oxidase and aldehyde oxidase hydride displacement from the sp(2) carbon of 6-substituted 4-quinazolinones. The variations in transition state structure show that C-O bond formation is nearly complete in the transition state and the transition state changes are anti-Hammond with the C-H and C-O bond lengths being more product-like for the faster reactions. The C-O bond length in the transition state is around 90% formed. However, the C-H bond is only about 80% broken. This leads to a very tetrahedral transition state with an O-C-N angle of 109 degrees. Thus, while the mechanism is concerted, the antibonding orbital of the C-H bond that is broken is not directly attacked by the nucleophile and instead hydride displacement occurs after almost complete tetrahedral transition state formation. In support of this the C=N bond is lengthened in the transition state indicating that attack on the electrophilic carbon occurs by addition to the C=N bond with negative charge increasing on the nitrogen. Differences in experimental reaction rates are accurately reproduced by these calculations and tend to support this mechanism.

The enzyme catalyzing the conversion of N(alpha)-benzyloxycarbonyl-L-aminoadipic-delta-semialdehyde (N(alpha)-Z-l-AASA) to N(alpha)-benzyloxycarbonyl-L-aminoadipic acid (N(alpha)-Z-L-AAA) in Aspergillus niger AKU 3302 was identified, and its characteristics were revealed. This reaction was catalyzed by an oxidase with a molecular mass of 215 kDa and pI of 4.1. The enzyme exhibited oxidase activity on N(alpha)-Z-L-AASA but not on short-chain aliphatic aldehydes, aromatic aldehydes or alcohols. The apparent K(m) value for N(alpha)-Z-L-AASA was estimated to be 7.0 mM. Thus, N(alpha)-Z-L-lysine was converted to N(alpha)-Z-L-AAA via N(alpha)-Z-L-AASA by a combination of an amine oxidase with broad substrate specificity and an aldehyde oxidase specific to N(alpha)-Z-L-AASA in A. niger AKU 3302.

An aldehyde oxidase exhibiting high activity on glyoxal was purified to an electrophoretically homogenous state from Pseudomonas sp. AIU 362, which was isolated from a soil sample using a methoxyethanol medium. The enzyme oxidized not only glyoxal but also short-chain aliphatic aldehydes and aromatic aldehydes. Thus, this enzyme was classified into the aldehyde oxidase (ALOD) group. However, it was composed of four identical subunits with a molecular mass of 27 kDa, whereas other microbial ALODs were composed of three hetero subunits, and ALODs from plant and animals were composed of two identical subunits. The NH(2)-terminal sequence also showed no similarity to that of other ALODs. These results indicate that ALOD from Pseudomonas sp. AIU 362 is a new aldehyde oxidase. This ALOD was induced by 2-methoxyethanol, methanol or isopropanol.

Pseudomonas sp. MX-058 produces aldehyde oxidase catalysing glyoxal to glyoxylic acid. Two aldehyde oxidases (F10 and F13) were purified to homogeneity from Pseudomonas sp. MX-058. F10 and F13 had subunit structures, a heterotetramer and heteropentamer respectively. The N-terminal amino acid sequences of all subunits were highly homologous to amino acid sequences of the putative oxidoreductases of Pseudomonas strains. All of these homologous oxidoreductases have a heterotrimer structure consisting of 85-88 (α), 37-39 (β) and 18-23 (γ) kDa subunits. However, the α-subunits of F10 and F13 might have decomposed into two [80 (α(1)) and 9 kDa (α(2))] and three [58 (α(1')), 22 (α(1″)) and 9 (α(2)) kDa] subunits, respectively, while the β- and γ-subunits remained intact. Both F10 and F13 show high activity toward several aliphatic and aromatic aldehydes. The aldehyde oxidases of Pseudomonas sp. MX-058 has unique protein structures, α(1)α(2)βγ for F10 and α(1')α(1″)α(2)βγ for F13, a heterotetramer and heteropentamer respectively. The enzymes exhibit significantly low activity toward glyoxylic acid compared with glyoxal, which is an advantageous property for glyoxylic acid production from glyoxal., (© 2008 The Authors. Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Recent studies have shown that nitrite is an important storage form and source of NO in biological systems. Controversy remains, however, regarding whether NO formation from nitrite occurs primarily in tissues or in blood. Questions also remain regarding the mechanism, magnitude, and contributions of several alternative pathways of nitrite-dependent NO generation in biological systems. To characterize the mechanism and magnitude of NO generation from nitrite, electron paramagnetic resonance spectroscopy, chemiluminescence NO analyzer, and immunoassays of cGMP formation were performed. The addition of nitrite triggered a large amount of NO generation in tissues such as heart and liver, but only trace NO production in blood. Carbon monoxide increased NO release from blood, suggesting that hemoglobin acts to scavenge NO not to generate it. Administration of the xanthine oxidase (XO) inhibitor oxypurinol or aldehyde oxidase (AO) inhibitor raloxifene significantly decreased NO generation from nitrite in heart or liver. NO formation rates increased dramatically with decreasing pH or with decreased oxygen tension. Isolated enzyme studies further confirm that XO and AO, but not hemoglobin, are critical nitrite reductases. Overall, NO generation from nitrite mainly occurs in tissues not in the blood, with XO and AO playing critical roles in nitrite reduction, and this process is regulated by pH, oxygen tension, nitrite, and reducing substrate concentrations.

Mammalian aldehyde oxidases are a small group of proteins belonging to the larger family of molybdo-flavoenzymes along with xanthine oxidoreductase and other bacterial enzymes. The two general types of reactions catalyzed by aldehyde oxidases are the hydroxylation of heterocycles and the oxidation of aldehydes into the corresponding carboxylic acids. Different animal species are characterized by a different complement of aldehyde oxidase genes. Humans contain a single active gene, while marsupials and rodents are characterized by four such genes clustering at a short distance on the same chromosome. At present, little is known about the physiological relevance of aldehyde oxidases in humans and other mammals, although these enzymes are known to play a role in the metabolism of drugs and compounds of toxicological importance in the liver. The present article provides an overview of the current knowledge of genetics, evolution, structure, enzymology, tissue distribution and regulation of mammalian aldehyde oxidases.

We previously demonstrated the existence of a minor 130 kDa subunit in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blot analysis of monkey liver cytosol and expressed monkey aldehyde oxidase (AO) in Escherichia coli. In contrast, the 130 kDa subunit was not observed in rat AO. In the current study, the properties of the 130 kDa subunit were investigated from the viewpoint of species differences in the presence of the subunit and AO activity. Monkey AO with His-tag at the N- and C-terminus were expressed, and were immunoanalyzed with anti-AO and anti-His-tag antisera. The results revealed that the minor 130 kDa subunit was produced by cleavage at the N-terminal side of the 150 kDa subunit. The cleavage point was shown to be located between 188Leu and 189Pro of 150 kDa AO subunit by the Edman degradation method. The two amino acids related to the cleavage are contained in the linkage between the 2Fe-2S and FAD domains in AO of human and monkey, but not in AO of rat and mouse. As a fact, the 130 kDa subunit was observed in AO of human and monkey, but not in AO of rat and mouse, suggesting the two amino acids might be one reason of a species difference in the formation of the 130 kDa subunit. However, the existence of the 130 kDa subunit is not associated with the species differences in AO activity, because the cleavage results in the loss of 2Fe-2S cluster domain essential for exertion of AO activity.

Signal inactivation is a crucial step in the dynamic of olfactory process and involves various Odorant-Degrading Enzymes. In the silkworm Bombyx mori, one of the best models for studying olfaction in insects, the involvement of an antennal-specific aldehyde oxidase in the degradation of the sex pheromone component bombykal has been demonstrated over the three past decades by biochemical studies. However, the corresponding enzyme has never been characterized at the molecular level. Bioinformatic screening of B. mori genome and molecular approaches have been used to isolate several candidate sequences of aldehyde oxidases. Two interesting antennal-expressed genes have been further characterized and their putative functions are discussed in regard to their respective expression pattern and to our knowledge on aldehyde oxidase properties. Interestingly, one gene appeared as specifically expressed in the antennae of B. mori and associated in males with the bombykal-sensitive sensilla, strongly suggesting that it could encode for the previously biochemically characterized enzyme.

In addition to the many articles reporting on the marked differences in species and large differences in rat strains in response to aldehyde oxidase (AO), individual differences in some rat strains have also been reported. However, little has been clarified about any related molecular biological mechanisms. We previously revealed that nucleotide substitutions of 377G>A and 2604C>T in the AO gene might be responsible for individual differences in AO activity in Donryu strain rats. By using native polyacrylamide gel electrophoresis/Western blotting in this study, the lack of formation of the AO dimer protein, which is essential for catalytic activity, was shown in poor metabolizer Donryu rats, and this could be a major reason for the individual differences. Rat strain differences were also verified from the same perspectives of nucleotide substitutions and expression levels of a dimer protein. Rat strains with high AO activity showed nucleotide sequences of (377G, 2604C) and a dimer protein. In the case of those with low AO activity, the nucleotide at position 2604 was fixed at T, but varied at position 377, such as G, G/A, and A. An AO dimer was detected in the liver cytosols of rat strains with (377G, 2604T), whereas a monomer was observed in those with (377A, 2604T). These results suggest that the lack of formation of a dimer protein leading to loss of catalytic activity might be due to 377G>A nucleotide substitution. Individual and strain differences in AO activity in rats could be explained by this 377G>A substitution, at least in the rat strains used in this study.

Aldehyde oxidase is a molybdenum hydroxylase that catalyzes the oxidation of aldehydes and nitrogen-containing heterocycles. The enzyme plays a dual role in the metabolism of physiologically important endogenous compounds and the biotransformation of xenobiotics. Using density functional theory methods, geometry optimization of tetrahedral intermediates of drugs and druglike compounds was examined to predict the likely metabolites of aldehyde oxidase. The calculations suggest that the lowest energy tetrahedral intermediate resulting from the initial substrate corresponds to the observed metabolite >or=90% of the time. Additional calculations were performed on a series of heterocyclic compounds where the products resulting from metabolism by xanthine oxidase and aldehyde oxidase differ in many instances. Again, the lowest energy tetrahedral intermediate corresponded to the observed product of aldehyde oxidase metabolism >or=90% for the compounds examined, while the observed products of xanthine oxidase were not well predicted.

Aldehyde oxidase (AO) is a homodimer with a molecular weight of 300 kDa. To clarify the reasons for the well-known differences in rat strains, we set out to study the relationship between AO activity and the expression levels of its dimer. AO-catalyzed 2-oxidation activity of (S)-RS-8359 was measured in liver cytosols from ten rat strains. The expression levels of AO dimeric protein were evaluated by the native-PAGE/Western blot. Rat strains with low AO activity showed only a monomer, whereas strains with high activity overwhelmingly exhibited a dimer. Exceptionally, one strain in the high AO activity group displayed complex mixed expression patterns of low and high AO activity groups. However, there was a good relationship between AO activity and the expression levels of a dimer, but not of a monomer. The results suggest that rat strains with low AO activity lack the ability to produce a dimer necessary for catalytic activity, and AO differences in rat strains should be discussed in terms of the expression levels of the dimer itself.

In this study, we investigated the properties of monkey liver aldehyde oxidase directed toward the clarification of species differences. The aldehyde oxidase preparation purified from male cynomolgus monkey liver cytosol showed a major 150 kDa Coomassie brilliant blue (CBB)-stained band together with a minor 130 kDa band using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both bands were identified as being aldehyde oxidase by a database search of the MS data obtained with nano-liquid chromatography, quardrupole time of flight, mass spectrometry (nano-LC Q/TOF MS). Based on the sequence coverage, the 130 kDa protein was presumed to be deficient in 20-30 kDa mass from the N-terminus. Full male cynomolgus monkey aldehyde oxidase cDNA was cloned and sequenced with the four degenerate primers designed by considering the peptide sequences containing the amino acids specific for monkey aldehyde oxidase. The deduced amino acid sequences had 96% amino acid identity with those of human enzyme. The aldehyde oxidase expressed in Escherichia coli also exhibited two immunoreactive bands on SDS-PAGE/Western blot analysis. Further, the biphasic pattern was observed for Eadie-Hofstee plots of the (S)-enantiospecific 2-oxidation activity of RS-8359 with the expressed and cytosolic monkey liver aldehyde oxidase. The results suggested that two forms of aldehyde oxidase in monkey were the expression products by a single gene. In contrast, the similarly expressed rat aldehyde oxidase showed only one immunoreactive protein and monophasic pattern. The biphasic phenomenon could be caused by the existence of two aldehyde oxidase isoforms or two active sites in a single enzyme or some other reasons. Further studies on the problems of the biphasic pattern and species differences in aldehyde oxidase are needed.

Xanthine oxidase (oxidoreductase; XOR) and aldehyde oxidase (AO) are similar in protein structure and prosthetic group composition, but differ in substrate preference. Here we show that mutation of two amino acid residues in the active site of human XOR for purine substrates results in conversion of the substrate preference to AO type. Human XOR and its Glu803-to-valine (E803V) and Arg881-to-methionine (R881M) mutants were expressed in an Escherichia coli system. The E803V mutation almost completely abrogated the activity towards hypoxanthine as a substrate, but very weak activity towards xanthine remained. On the other hand, the R881M mutant lacked activity towards xanthine, but retained slight activity towards hypoxanthine. Both mutants, however, exhibited significant aldehyde oxidase activity. The crystal structure of E803V mutant of human XOR was determined at 2.6 A resolution. The overall molybdopterin domain structure of this mutant closely resembles that of bovine milk XOR; amino acid residues in the active centre pocket are situated at very similar positions and in similar orientations, except that Glu803 was replaced by valine, indicating that the decrease in activity towards purine substrate is not due to large conformational change in the mutant enzyme. Unlike wild-type XOR, the mutants were not subject to time-dependent inhibition by allopurinol.

Aldehyde oxidase, a molybdoflavoenzyme that plays an important role in aldehyde biotransformation, requires oxygen as substrate and produces reduced oxygen species. However, little information is available regarding its importance in cellular redox stress. Therefore, studies were undertaken to characterize its superoxide and hydrogen peroxide production. Aldehyde oxidase was purified to >98% purity and exhibited a single band at approximately 290 kDa on native polyacrylamide gradient gel electrophoresis. Superoxide generation was measured and quantitated by cytochrome c reduction and EPR spin trapping with p-dimethyl aminocinnamaldehyde as reducing substrate. Prominent superoxide generation was observed with an initial rate of 295 nmol min(-1) mg(-1). Electrochemical measurements of oxygen consumption and hydrogen peroxide formation yielded values of 650 and 355 nmol min(-1) mg(-1). In view of the ubiquitous distribution of aldehydes in tissues, aldehyde oxidase can be an important basal source of superoxide that would be enhanced in disease settings where cellular aldehyde levels are increased.

(3-Tert-butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)pyrazolo[1,5-d] [1,2,4]triazine was recently identified as a functionally selective, inverse agonist at the benzodiazepine site of GABA(A) alpha5-containing receptors, which enhances performance in animal models of cognition. The routes of metabolism of this compound in rat, dog, rhesus monkey and human in vitro systems, and in vivo in rat, dog and rhesus monkey have been characterized. The current study demonstrates that both a cytosolic oxidative reaction and cytochrome P450 play important roles in the metabolism of the compound. Chemical inhibition studies showed the oxidation in human cytosol to be catalysed predominantly by aldehyde oxidase rather than the related enzyme, xanthine oxidase. The aldehyde oxidase-mediated metabolites were present in vitro and in vivo in both rat and rhesus monkey, and also in vitro in man. They were absent both in vitro and in vivo in dog.

The nitroguanidine or nitromethylene moiety of the newest major class of insecticides, the neonicotinoids, is important for potency at insect nicotinic receptors and selectivity relative to mammalian receptors. Aldehyde oxidase (AOX) was recently identified as the imidacloprid nitroreductase of mammalian liver, producing both nitrosoguanidine and aminoguanidine metabolites. The present study considers the ability of AOX, partially purified from rabbit liver, to reduce five commercial nitroguanidine (i.e., imidacloprid, thiamethoxam, clothianidin, and dinotefuran) and nitromethylene (i.e., nitenpyram) neonicotinoid insecticides and three derivatives thereof (i.e., the N-methyl and nitromethylene analogues of imidacloprid and desmethylthiamethoxam). LC/MS/MS was used to demonstrate that AOX reduces nitroguanidines to both nitroso- and aminoguanidines, while nitromethylenes are reduced only to the corresponding nitroso metabolites. Additionally, nitrosonitenpyram was found to spontaneously dehydrate to form a 2-cyanoamidine metabolite, mimicking a predominant photoreaction. The substrate specificity of AOX was characterized as follows: Neonicotinoids with a tertiary nitrogen (N-methylimidacloprid and thiamethoxam) are poor substrates; nitroguanidines are metabolized faster than nitromethylenes; and clothianidin is the most rapidly reduced. Kinetic constants were measured for reduction of three nitroguanidines at two concentrations of AOX. At 2 mg protein/mL, only nitroso metabolites were detected, with Km values of 1.03, 2.99, and 2.41 mM and Vmax values of 5.13, 2.54, and 0.98 nmol/min/mg protein measured for clothianidin, imidacloprid, and dinotefuran, respectively. At 5 mg protein/mL, both amino and nitroso metabolites were detected. However, with each nitroguanidine, the formation of nitroso metabolites did not saturate at substrate levels up to 4 mM, whereas amino metabolite formation exhibited Km values of 0.052, 0.16, and 0.084 mM with corresponding Vmax values of 0.80, 1.24, and 0.79 nmol/min/mg protein for clothianidin, imidacloprid, and dinotefuran, respectively. These in vitro observations show large structural differences in the rates of AOX-catalyzed reduction and help to interpret the extensive studies on in vivo metabolism of neonicotinoid insecticides.

Signal termination is a crucial step in the dynamic of the olfactory process. It involves different classes of odorant-degrading enzymes. Whereas aldehyde oxidase enzymatic activities have been demonstrated in insect antennae by previous biochemical studies, the corresponding enzymes have never been characterized at the molecular level. In the cabbage armyworm Mamestra brassicae, we isolated for the first time an aldehyde oxidase partial cDNA specifically expressed in chemosensory organs, with the strongest expression in antennae of both sexes. In these organs, expression was restricted to the olfactory sensilla. Our results suggest that the corresponding enzyme could degrade aldehyde odorant compounds, such as pheromones or plant's volatiles.

Imidacloprid (IMI), the prototypical neonicotinoid insecticide, is used worldwide for crop protection and flea control on pets. It is both oxidatively metabolized by cytochrome P450 enzymes and reduced at the nitroguanidine moiety by a previously unidentified cytosolic "neonicotinoid nitroreductase", the subject of this investigation. Two major metabolites are detected on incubation of IMI with rabbit liver cytosol: the nitrosoguanidine (IMI-NO) and the aminoguanidine (IMI-NH2). Three lines of evidence identify the molybdo-flavoenzyme aldehyde oxidase (AOX, EC 1.2.3.1) as the neonicotinoid nitroreductase. First, classical AOX electron donor substrates (benzaldehyde, 2-hydroxypyrimidine, and N-methylnicotinamide) dramatically increase the rate of formation of IMI metabolites. Allopurinol and diquat are also effective electron donors, while NADPH and xanthine are not. Second, AOX inhibitors (potassium cyanide, menadione, and promethazine) inhibit metabolite formation when N-methylnicotinamide is utilized as an electron donor. Without the addition of an electron donor, rabbit liver cytosol reduces IMI only to IMI-NO at a slow rate. This reduction is also inhibited by potassium cyanide, menadione, and promethazine, as well as by additional AOX inhibitors, cimetidine and chlorpromazine. Finally, IMI nitroreduction by AOX is sensitive to an aerobic atmosphere, but to a much lesser extent than cytochrome P450 2D6. Large species differences are observed in the IMI nitroreductive activity of liver cytosol. While rabbit and monkey (Cynomolgus) give the highest levels of total metabolite formation, human, mouse, cow, and rat also metabolize IMI rapidly. In contrast, dog, cat, and chicken liver cytosols do not reduce IMI at appreciable rates. AOX, as a neonicotinoid nitroreductase, may limit the persistence of IMI, and possibly other neonicotinoids, in mammals.

Density functional calculations on geometry-optimized oxidized (Mo(VI)) and reduced (Mo(IV)) analogues of the isolated active site of aldehyde oxidase (MOP), a member of the xanthine oxidase family of pyranopterindithiolate enzymes, show that fold angle changes of the dithiolate ligand modulate the relative metal and dithiolate contributions to the frontier redox orbitals. Proton abstraction from the equatorial aqua ligand of the oxidized Mo(VI) site also flattens the metal dithiolate fold angle. It is proposed that static and/or dynamic changes in the structure of the protein surrounding the active site can induce changes in the dithiolate fold angle and thereby provide a mechanism for electronic buffering of the redox orbital, for fine-tuning the nucleophilicity of the equatorial aqua/hydroxide ligand, and for modulating the electron-transfer regeneration of the active sites of molybdenum and tungsten enzymes via a "dithiolate folding effect".

The selective estrogen receptor modulator, raloxifene, has been demonstrated as a potent uncompetitive inhibitor of human liver aldehyde oxidase-catalyzed oxidation of phthalazine, vanillin, and nicotine-Delta1'(5')-iminium ion, with K(i) values of 0.87 to 1.4 nM. Inhibition was not time-dependent. Raloxifene has also been shown to be a noncompetitive inhibitor of an aldehyde oxidase-catalyzed reduction reaction of a hydroxamic acid-containing compound, with a K(i) of 51 nM. However, raloxifene had only small effects on xanthine oxidase, an enzyme related to aldehyde oxidase. In addition, several other compounds of the same therapeutic class as raloxifene were examined for their potential to inhibit aldehyde oxidase. However, none were as potent as raloxifene, since IC(50) values were orders of magnitude higher and ranged from 0.29 to 57 micro M. In an examination of analogs of raloxifene, it was shown that the bisphenol structure with a hydrophobic group on the 3-position of the benzthiophene ring system was the most important element that imparts inhibitory potency. The relevance of these data to the mechanistic understanding of aldehyde oxidase catalysis, as well as to the potential for raloxifene to cause drug interactions with agents for which aldehyde oxidase-mediated metabolism is important, such as zaleplon or famciclovir, is discussed.

An aldehyde oxidase, which oxidizes various aliphatic and aromatic aldehydes using O(2) as an electron acceptor, was purified from the cell-free extracts of Pseudomonas sp. KY 4690, a soil isolate, to an electrophoretically homogeneous state. The purified enzyme had a molecular mass of 132 kDa and consisted of three non-identical subunits with molecular masses of 88, 39, and 18 kDa. The absorption spectrum of the purified enzyme showed characteristics of an enzyme belonging to the xanthine oxidase family. The enzyme contained 0.89 mol of flavin adenine dinucleotide, 1.0 mol of molybdenum, 3.6 mol of acid-labile sulfur, and 0.90 mol of 5'-CMP per mol of enzyme protein, on the basis of its molecular mass of 145 kDa. Molecular oxygen served as the sole electron acceptor. These results suggest that aldehyde oxidase from Pseudomonas sp. KY 4690 is a new member of the xanthine oxidase family and might contain 1 mol of molybdenum-molybdpterin-cytosine dinucleotide, 1 mol of flavin adenine dinucleotide, and 2 mol of [2Fe-2S] clusters per mol of enzyme protein. The enzyme showed high reaction rates toward various aliphatic and aromatic aldehydes and high thermostability.