Identification and Characterization of Aldehyde Oxidase 5 in the Pheromone Gland of the Silkworm (Lepidoptera: Bombycidae).

Aldehyde oxidases (AOXs) are a subfamily of cytosolic molybdo-flavoenzymes that play critical roles in the detoxification and degradation of chemicals. Active AOXs, such as AOX1 and AOX2, have been identified and functionally analyzed in insect antennae but are rarely reported in other tissues. This is the first study to isolate and characterize the cDNA that encodes aldehyde oxidase 5 (BmAOX5) in the pheromone gland (PG) of the silkworm, Bombyx mori. The size of BmAOX5 cDNA is 3,741 nucleotides and includes an open reading frame, which encodes a protein of 1,246 amino acid residues. The theoretical molecular weight and isoelectric point of BmAOX5 are approximately 138 kDa and 5.58, respectively. BmAOX5 shares a similar primary structure with BmAOX1 and BmAOX2, containing two [2Fe-2S] redox centers, a FAD-binding domain, and a molybdenum cofactor (MoCo)-binding domain. RT-PCR revealed BmAOX5 to be particularly highly expressed in the PG (including ovipositor) of the female silkworm moth, and the expression was further confirmed by in situ hybridization, AOX activity staining, and anti-BmAOX5 western blotting. Further, BmAOX5 was shown to metabolize aromatic aldehydes, such as benzaldehyde, salicylaldehyde, and vanillic aldehyde, and fatty aldehydes, such as heptaldehyde and propionaldehyde. The maximum reaction rate (Vmax) of benzaldehyde as substrate was 21 mU and Km was 1.745 mmol/liter. These results suggested that BmAOX5 in the PG could metabolize aldehydes in the cytoplasm for detoxification or participate in the degradation of aldehyde pheromone substances and odorant compounds to identify mating partners and locate suitable spawning sites.

Identification of an aldehyde oxidase involved in indole-3-acetic acid synthesis in Bombyx mori silk gland.

Auxin is thought to be an important factor in the induction of galls by galling insects. We have previously shown that both galling and nongalling insects synthesize indole-3-acetic acid (IAA) from tryptophan (Trp) via two intermediates, indole-3-acetaldoxime (IAOx) and indole-3-acetaldehyde (IAAld). In this study, we isolated an enzyme that catalyzes the last step "IAAld → IAA" from a silk-gland extract of Bombyx mori. The enzyme, designated "BmIAO1", contains two 2Fe-2S iron-sulfur-cluster-binding domains, an FAD-binding domain, and a molybdopterin-binding domain, which are conserved in aldehyde oxidases. BmIAO1 causes the nonenzymatic conversion of Trp to IAAld and the enzymatic conversion of IAOx to IAA, suggesting that BmIAO1 alone is responsible for IAA production in B. mori. However, a detailed comparison of pure BmIAO1 and the crude silk-gland extract suggested the presence of other enzymes involved in IAA production from Trp. Abbreviations: BA: benzoic acid; CE: collision energy; CXP: collision cell exit potential; DP: declustering potential; IAA: indole-3-acetic acid; IBI1: IAA biosynthetic inhibitor-1; IAAld: indole-3-acetaldehyde; ICA: indole-3-carboxylic acid; IAOx: indole-3-acetaldoxime; IEtOH: indole-3-ethanol; LC-MS/MS: liquid chromatography-tandem mass spectrometry; Trp: tryptophan.

Crystal structure of an aldehyde oxidase from Methylobacillus sp. KY4400.

Hetero-trimeric aldehyde oxidases of bacterial origin, which use O2 to catalyse the oxidation of various aldehydes but not those of aromatic N-heterocycles, belong to the xanthine oxidase family. In the present study, the crystal structure of a recombinant aldehyde oxidase from Methylobacillus sp. KY4400 (Mb-AOX) was determined at 2.5 Å resolution. The structures of its subunits resemble those of the corresponding subunits or domains of other structurally characterised enzymes belonging to the family, and include a [4Fe-4 S] cluster in the medium subunit like that found in Escherichia coli periplasmic aldehyde oxidoreductase (EP-AOR). A funnel leading to the si-face of the isoalloxazine ring of FAD, which is narrower than those in mouse liver AOX3 and human AOX1, is also present and it is even narrower than that in EP-AOR. The environment surrounding the ring in Mb-AOX and EP-AOR is subtly different, which might account for their different abilities to use O2. A remarkable characteristic of the Mo catalytic centre in Mb-AOX is a tryptophan situated near the centre instead of the alanine present in other xanthine oxidase family members. The tryptophan residue together with other residue differences might play an important role in binding to aldehydes such as n-heptylaldehyde in Mb-AOX.

A single nucleotide polymorphism causes enhanced radical oxygen species production by human aldehyde oxidase.

Aldehyde oxidases (AOXs) are molybdo-flavoenzymes characterized by broad substrate specificity, oxidizing aromatic/aliphatic aldehydes into the corresponding carboxylic acids and hydroxylating various heteroaromatic rings. The enzymes use oxygen as the terminal electron acceptor and produce reduced oxygen species during turnover. The physiological function of mammalian AOX isoenzymes is still unclear, however, human AOX (hAOX1) is an emerging enzyme in phase-I drug metabolism. Indeed, the number of xenobiotics acting as hAOX1 substrates is increasing. Further, numerous single-nucleotide polymorphisms (SNPs) have been identified within the hAOX1 gene. SNPs are a major source of inter-individual variability in the human population, and SNP-based amino acid exchanges in hAOX1 reportedly modulate the catalytic function of the enzyme in either a positive or negative fashion. In this report we selected ten novel SNPs resulting in amino acid exchanges in proximity to the FAD site of hAOX1 and characterized the purified enzymes after heterologous expression in Escherichia coli. The hAOX1 variants were characterized carefully by quantitative differences in their ability to produce superoxide radical. ROS represent prominent key molecules in physiological and pathological conditions in the cell. Our data reveal significant alterations in superoxide anion production among the variants. In particular the SNP-based amino acid exchange L438V in proximity to the isoalloxanzine ring of the FAD cofactor resulted in increased rate of superoxide radical production of 75%. Considering the high toxicity of the superoxide in the cell, the hAOX1-L438V SNP variant is an eventual candidate for critical or pathological roles of this natural variant within the human population.

Identification of enzymes responsible for nitrazepam metabolism and toxicity in human.

Nitrazepam (NZP) is a hypnotic agent that rarely causes liver injuries in humans and teratogenicity in rodents. In humans, NZP is primarily metabolized to 7-aminonitrazepam (ANZP) by reduction and subsequently to 7-acetylamino nitrazepam (AANZP) by acetylation. ANZP can be regenerated from AANZP by hydrolysis in rodents, but it is still unclear whether this reaction occurs in humans. In rodents, AANZP may be associated with teratogenicity, while in humans, it is known that drug-induced liver injuries may be caused by NZP reactive metabolite(s). In this study, we attempted to identify the enzymes responsible for NZP metabolism to obtain a basic understanding of this process and the associated metabolite toxicities. We found that the NZP reductase activity in human liver cytosol (HLC) was higher than that in human liver microsomes (HLM). We purified the responsible enzyme(s) from HLC and found that the NZP reductase was aldehyde oxidase 1 (AOX1). The role of AOX1 was confirmed by an observed increase in the NZP reductase activity upon addition of N1-methylnicotinamide, an electron donor of AOX1, as well as inhibition of this activity in HLC in the presence of AOX1 inhibitors. ANZP was acetylated to form AANZP by N-acetyltransferase (NAT) 2. An experiment using recombinant esterases in an inhibition study using HLM revealed that AANZP is hydrolyzed by arylacetamide deacetylase (AADAC) in the human liver. N-Hydroxylamino NZP, which is suspected to be a reactive metabolite, was detected as a conjugate with N-acetyl-l-cysteine through NZP reduction and ANZP hydroxylation reactions. In the latter reaction, the conjugate was readily formed by recombinant CYP3A4 among the various P450 isoforms tested. In sum, we found that AOX1, NAT2, AADAC, and CYP3A4 are the determinants for the pharmacokinetics of NZP and that they confer interindividual variability in sensitivity to NZP side effects.

Purification and mechanism of human aldehyde oxidase expressed in Escherichia coli.

Human aldehyde oxidase 1 (AOX1) has been subcloned into a vector suitable for expression in Escherichia coli, and the protein has been expressed. The resulting protein is active, with sulfur being incorporated in the molybdopterin cofactor. Expression levels are modest, but 1 liter of cells supplies enough protein for both biochemical and kinetic characterization. Partial purification is achieved by nickel affinity chromatography through the addition of six histidines to the amino-terminal end of the protein. Kinetic analysis, including kinetic isotope effects and comparison with xanthine oxidase, reveal similar mechanisms, with some subtle differences. This expression system will allow for the interrogation of human aldehyde oxidase structure/function relationships by site-directed mutagenesis and provide protein for characterizing the role of AOX1 in drug metabolism.

Purification and characterization of a new aldehyde oxidase from pseudomonas sp. AIU 362.

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.

Characterization of superoxide production from aldehyde oxidase: an important source of oxidants in biological tissues.

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.

Stereospecific oxidation of the (S)-enantiomer of RS-8359, a selective and reversible monoamine oxidase A (MAO-A) inhibitor, by aldehyde oxidase.

In a previous paper by the authors on RS-8359, a new selective and reversible monoamine oxidase A (MAO-A) inhibitor, it was reported that the (S)-enantiomer of RS-8359 is rapidly eliminated from rats, monkeys and humans as a result of the formation of a 2-oxidative metabolite. The present study investigates the properties of the enzyme responsible for the 2-oxidation of RS-8359. Subcellular localization, cofactor requirement and the inhibitory effects of typical compounds were studied using rat liver preparations. In addition, the enzyme was purified from rat liver cytosol for further characterization. The enzyme activity was localized in the cytosolic fraction without the need for any cofactor and was extensively inhibited by menadione, chlorpromazine and quinacrine. The purified enzyme was also a homodimer with a monomeric molecular weight of 140 kDa and it had an A280/A450 ratio of 5.1 in the absorption spectrum. The results suggest that the enzyme responsible for the biotransformation of RS-8359 to give the 2-keto derivative is aldehyde oxidase (EC 1.2.3.1). The reaction of aldehyde oxidase is highly stereoselective for the (S)-configuration of RS-8359 and the (9R)-configuration of cinchona alkaloids.

Characterization and potential application of purified aldehyde oxidase from Pseudomonas stutzeri IFO12695.

The molecular weight of purified aldehyde oxidase from Pseudomonas stutzeri IFO12695 was estimated to be 160 kDa by a gel filtration method. SDS-PAGE showed that the enzyme consisted of three non-identical subunits with molecular weights of 18, 38, and 83 kDa. The enzyme exhibited an absorption spectrum with maxima at 277, 325, 365, 415, 450, 480, and 550 nm and possessed molybdenum, CMP, iron, sulfur, and FAD as its cofactors, indicating that it belonged to the xanthine oxidase family. A variety of aliphatic and aromatic aldehydes were oxidized; and among them n-hexylaldehyde gave the most rapidly action. When 10 mM formaldehyde was treated with the aldehyde oxidase in the presence of catalase for 240 min, the formaldehyde concentration was reduced to 0.8 mM, suggesting this enzyme might be effective for the removal of formaldehyde contained in wastewater.

Enzymatic oxidation of phthalazine with guinea pig liver aldehyde oxidase and liver slices: inhibition by isovanillin.

The enzymes aldehyde oxidase and xanthine oxidase catalyze the oxidation of a wide range of N-heterocycles and aldehydes. These enzymes are widely known for their role in the metabolism of N-heterocyclic xenobiotics where they provide a protective barrier by aiding in the detoxification of ingested nitrogen-containing heterocycles. Isovanillin has been shown to inhibit the metabolism of aromatic aldehydes by aldehyde oxidase, but its inhibition towards the heterocyclic compounds has not been studied. The present investigation examines the oxidation of phthalazine in the absence and in the presence of the inhibitor isovanillin by partially purified aldehyde oxidase from guinea pig liver. In addition, the interaction of phthalazine with freshly prepared guinea pig liver slices, both in the absence and presence of specific inhibitors of several liver oxidizing enzymes, was investigated. ldehyde oxidase rapidly converted phthalazine into 1-phthalazinone, which was completely inhibited in the presence of isovanillin (a specific inhibitor of aldehyde oxidase). In freshly prepared liver slices, phthalazine was also rapidly converted to 1-phthalazinone. The formation of 1-phthalazinone was completely inhibited by isovanillin, whereas disulfiram (a specific inhibitor of aldehyde dehydrogenase) only inhibited 1-phthalazinone formation by 24% and allopurinol (a specific inhibitor of xanthine oxidase) had little effect. Therefore, isovanillin has been proved as an inhibitor of the metabolism of heterocyclic substrates, such as phthalazine, by guinea pig liver aldehyde oxidase, since it had not been tested before. Thus it would appear from the inhibitor results that aldehyde oxidase is the predominant enzyme in the oxidation of phthalazine to 1-phthalazinone in freshly prepared guinea pig liver slices, whereas xanthine oxidase only contributes to a small extent and aldehyde dehydrogenase does not take any part.

The aldehyde oxidase gene cluster in mice and rats. Aldehyde oxidase homologue 3, a novel member of the molybdo-flavoenzyme family with selective expression in the olfactory mucosa.

Mammalian molybdo-flavoenzymes are oxidases requiring FAD and molybdopterin (molybdenum cofactor) for their catalytic activity. This family of proteins was thought to consist of four members, xanthine oxidoreductase, aldehyde oxidase 1 (AOX1), and the aldehyde oxidase homologues 1 and 2 (AOH1 and AOH2, respectively). Whereas the first two enzymes are present in humans and various other mammalian species, the last two proteins have been described only in mice. Here, we report on the identification, in both mice and rats, of a novel molybdo-flavoenzyme, AOH3. In addition, we have cloned the cDNAs coding for rat AOH1 and AOH2, demonstrating that this animal species has the same complement of molybdo-flavoproteins as the mouse. The AOH3 cDNA is characterized by remarkable similarity to AOX1, AOH1, AOH2, and xanthine oxidoreductase cDNAs. Mouse AOH3 is selectively expressed in Bowman's glands of the olfactory mucosa, although small amounts of the corresponding mRNA are present also in the skin. In the former location, two alternatively spliced forms of the AOH3 transcript with different 3'-untranslated regions were identified. The general properties of AOH3 were determined by purification of mouse AOH3 from the olfactory mucosa. The enzyme possesses aldehyde oxidase activity and oxidizes, albeit with low efficiency, exogenous substrates that are recognized by AOH1 and AOX1. The Aoh3 gene maps to mouse chromosome 1 band c1 and rat chromosome 7 in close proximity to the Aox1, Aoh1, and Aoh2 loci and has an exon/intron structure almost identical to that of the other molybdo-flavoenzyme genes in the two species.

Purification and characterization of an aldehyde oxidase from Pseudomonas sp. KY 4690.

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.