Recent advances in the application of microbial diamine oxidases and other histamine-oxidizing enzymes.

The consumption of foods fraught with histamine can lead to various allergy-like symptoms if the histamine is not sufficiently degraded in the human body. The degradation occurs primarily in the small intestine, naturally catalyzed by the human diamine oxidase (DAO). An inherent or acquired deficiency in human DAO function causes the accumulation of histamine and subsequent intrusion of histamine into the bloodstream. The histamine exerts its effects acting on different histamine receptors all over the body but also directly in the intestinal lumen. The inability to degrade sufficient amounts of dietary histamine is known as the 'histamine intolerance'. It would be preferable to solve this problem initially by the production of histamine-free or -reduced foods and by the oral supplementation of exogenous DAO supporting the human DAO in the small intestine. For the latter, DAOs from mammalian, herbal and microbial sources may be applicable. Microbial DAOs seem to be the most promising choice due to their possibility of an efficient biotechnological production in suitable microbial hosts. However, their biochemical properties, such as activity and stability under process conditions and substrate selectivity, play important roles for their successful application. This review deals with the advances and challenges of DAOs and other histamine-oxidizing enzymes for their potential application as processing aids for the production of histamine-reduced foods or as orally administered adjuvants to humans who have been eating food fraught with histamine.

Autophagy induction by exogenous polyamines is an artifact of bovine serum amine oxidase activity in culture serum.

Polyamines are small polycationic alkylamines involved in many fundamental cellular processes, including proliferation, nucleic acid synthesis, apoptosis, and protection from oxidative damage. It has been proposed that in addition to these functions, elevated levels of polyamines promote longevity in various biological systems, including yeast, Drosophila, and murine models. A series of in vitro mechanistic studies by multiple investigators has led to the conclusion that addition of exogenous spermidine promotes longevity through autophagy induction; however, these experiments were confounded by the use of mammalian cell culture systems supplemented with fetal bovine serum. Using cell viability assays, LC3B immunoblots, and live-cell fluorescence microscopy, we report here that in the presence of ruminant serum, exogenously added polyamines are quickly oxidized by the copper-containing bovine serum amine oxidase. This polyamine oxidation resulted in the production of harmful byproducts including hydrogen peroxide, ammonia, and reactive aldehydes. Our data demonstrate that it is critically important to prevent confounding bovine serum amine oxidase-induced cytotoxicity in mechanistic studies of the roles of polyamines in autophagy.

Evaluation of porcine diamine oxidase for the conversion of histamine in food-relevant amounts.

Histamine exists in a multitude of foods and displays an emerging role within food intolerances. Our aim was to identify the activity of porcine diamine oxidase (DAO) required for the in vitro degradation of histamine amounts that are found in typical meals containing histamine (75 mg, equaled 150 mg/L). Furthermore, we investigated an actual dietary supplement that is commercially available for histamine intolerant individuals for its histamine reduction capability. Kinetic investigations of porcine DAO showed a substrate inhibition by histamine concentrations greater than 56 mg/L (0.5 mM). The stability of free porcine DAO was tested in a fed state simulated intestinal fluid and exhibited a half-life period of around 19 min. A total of 50 nanokatal (nkat) free porcine DAO, which equaled the amount of enzyme isolated from around 100 g pig kidney, were necessary for the in vitro reduction of around 90% of the histamine. The dietary supplement that contains a pig kidney extract did not show DAO activity. Instead, the used histamine (0.75 mg) was apparently reduced due to the adsorption of histamine onto a capsule component by 18.9 ± 2.3% within 5 hr. Although the capsule preparation retained its overall structure and shape for at least 90 min in simulated gastric fluid, the apparent histamine reduction was significantly reduced to 12.1 ± 2.3% (P ≤ 0.05). In conclusion, an alternative to the pig kidney DAO or an improved capsule preparation is needed to ensure an adequate supplementation for histamine-intolerant humans. PRACTICAL APPLICATION: Histamine intolerance is an emerging issue in our society and the intolerance-related physiological symptoms are currently not reliably treatable due to a lack of scientific investigation. A commercially available dietary supplement for histamine intolerance does not fulfil the requirements for a satisfactory histamine reduction in intolerant humans. The activity of the histamine degrading enzyme diamine oxidase, required for a satisfactory histamine degradation, is by far higher than the theoretical amount apparently given in the dietary supplement. With this knowledge, it is obvious that improved food supplements must be developed to help histamine intolerant humans.

Stability of Vegetal Diamine Oxidase in Simulated Intestinal Media: Protective Role of Cholic Acids.

Food biogenic amines, in particular, histamine, are often responsible for various enteric and vascular dysfunctions. Several years ago, the oral administration of copper-containing diamine oxidase (DAO), also called histaminase, able to oxidatively deaminate biogenic amines, had been suggested as a food supplement to control food allergy and enteric dysfunctions. This report is aimed to generate a global image on the behavior of orally administrated DAO dosage forms in the intestinal tract. The catalytic stability of DAO from Lathyrus sativus seedlings in various simulated intestinal media with different pH and containing different association of cholic acids, pancreatic proteases, bicarbonate, lipids, or alcohol was investigated. Cholic acids and lipids protected the enzyme in the simulated intestinal fluids. However, they were not able to protect against the inhibitory effect of 24-36% (v/v) ethanol. These observations may be relevant for oral administration of enzymes as food supplements or therapeutic bioactive agents.

Syncephalastrum racemosum amine oxidase with high catalytic efficiency toward ethanolamine and its application in ethanolamine determination.

Our screening study yielded a copper amine oxidase (SrAOX) from Syncephalastrum racemosum, which showed much higher affinity and catalytic efficiency toward ethanolamine (EA) than any other amine oxidase (AOX). Following purification of the enzyme to electrophoretic homogeneity from a cell-free extract, the maximum activity toward EA was detected at pH 7.2-7.5 and 45 °C. The SrAOX complementary DNA (cDNA) was composed of a 2052-bp open reading frame encoding a 683-amino acid protein with a molecular mass of 77,162 Da. The enzyme functions as a homodimer. The deduced amino acid sequence of SrAOX showed 55.3 % identity to Rhizopus delemar AOX and contains two consensus sequences of Cu-AOX, NYDY, and HHQH, suggesting SrAOX is a type 1 Cu-AOX (i.e., a topaquinone enzyme). Structural homology modeling showed that residues (112)ML(113), (141)FADTWG(146) M158, and N318 are unique, and T144 possibly characterizes the substrate specificity of SrAOX. The recombinant enzyme (rSrAOX) was produced using Escherichia coli. Steady-state kinetic analysis of rSrAOX activity toward EA (pH 7.5 and 45 °C) gave K m and k cat values of 0.848 ± 0.009 mM and 9.11 ± 0.13 s(-1), respectively. The standard curves were linear between 0.1 and 2 mM EA, and 10 µg mL(-1)-2.5 mg mL(-1) (15 µM-3.6 mM) phosphatidylethanolamine using Streptomyces chromofuscus phospholipase D, respectively, was sufficiently sensitive for clinical use.

Quinone-Catalyzed Selective Oxidation of Organic Molecules.

Quinones are common stoichiometric reagents in organic chemistry. Para-quinones with high reduction potentials, such as DDQ and chloranil, are widely used and typically promote hydride abstraction. In recent years, many catalytic applications of these methods have been achieved by using transition metals, electrochemistry, or O2 to regenerate the oxidized quinone in situ. Complementary studies have led to the development of a different class of quinones that resemble the ortho-quinone cofactors in copper amine oxidases and mediate the efficient and selective aerobic and/or electrochemical dehydrogenation of amines. The latter reactions typically proceed by electrophilic transamination and/or addition-elimination reaction mechanisms, rather than hydride abstraction pathways. The collective observations show that the quinone structure has a significant influence on the reaction mechanism and has important implications for the development of new quinone reagents and quinone-catalyzed transformations.

Experimental and computational evidence of metal-O2 activation and rate-limiting proton-coupled electron transfer in a copper amine oxidase.

The mechanism of O(2) reduction by copper amine oxidase from Arthrobacter globiformus (AGAO) is analyzed in relation to the cobalt-substituted protein. The enzyme utilizes a tyrosine-derived topaquinone cofactor to oxidize primary amines and reduce O(2) to H(2)O(2). Steady-state kinetics indicate that amine-reduced CuAGAO is reoxidized by O(2) >10(3) times faster than the CoAGAO analogue. Complementary spectroscopic studies reveal that the difference in the second order rate constant, k(cat)/K(M)(O(2)), arises from the more negative redox potential of Co(III/II) in relation to Cu(II/I). Indistinguishable competitive oxygen-18 kinetic isotope effects are observed for the two enzymes and modeled computationally using a calibrated density functional theory method. The results are consistent with a mechanism where an end-on (η(1))-metal bound superoxide is reduced to an η(1)-hydroperoxide in the rate-limiting step.

Ralstonia solanacearum induces soluble amine-oxidase activity in Solanum torvum stem calli.

Solanum torvum is reported to carry resistance to bacterial wilt caused by Ralstonia solanacearum. So, this wild species is used as rootskock for eggplants or tomatoes in naturally infected soil. This study aimed to investigate the involvement of the polyamine metabolism pathway in the resistance mechanisms of this species. Calli induced from Solanum torvum stem explants were inoculated with the bacteria under partial vacuum. All calli showed a hypersensitive response after infiltration. Furthermore, amine oxidase activity with aldehyde and H(2)O(2) production was detected in soluble protein extracts of calli infiltrated by the bacteria. Due to its preferential affinity for aliphatic amines, this enzyme was supposed to have amine oxidase-like (AO-like) activity. Moreover, the length of aliphatic chain cycle altered the oxidative deamination kinetics of potential substrates. The AO-like catalytic activity was significantly inhibited by chelator agents such as ethylene-diamine-tretraacetic (EDTA), and also by semi-carbazide as aminoguanidine. These results suggested that (i) the prosthetic group of the AO-like enzyme could be a tyrosine-derived 6-hydroxytopaquinone structure, copper containing; (ii) this enzyme could be a semi-carbazide sensitive amine oxidase (SSAO).

Vascular amine oxidases are needed for leukocyte extravasation into inflamed joints in vivo.

OBJECTIVE: Leukocyte traffic from the blood to the joints is crucial in the pathogenesis of arthritis. A bifunctional endothelial cell-surface glycoprotein, AOC3 (amine oxidase, copper-containing 3; also known as vascular adhesion protein 1), has both adhesive and enzymatic properties. We undertook this study to determine the contribution of AOC3 and its oxidase activity to leukocyte trafficking into inflamed joints in vivo. METHODS: We used gene-modified animals, molecular modeling, an AOC3 enzyme inhibitor, oxidase assays, and arthritis models (adjuvant-induced arthritis [AIA] in rats and anti-type II collagen antibody-induced arthritis in mice) to dissect the importance of AOC3 in vivo. RESULTS: The AOC3 inhibitor fitted well with a covalent binding mode into the active site of the AOC3 crystal structure. It selectively blocked the oxidase activity of AOC3 in enzyme assays. Intraperitoneal and oral administration of the AOC3 inhibitor significantly ameliorated rat AIA. In anti-type II collagen antibody-induced arthritis in mice, the AOC3 inhibitor also improved the outcome of the joint inflammation. The acute semicarbazide-sensitive amine oxidase blockade by the inhibitor had even more pronounced effects than genetic deletion of AOC3. Enzymatic analyses showed that the inhibitor also blocked 2 other structurally very closely related AOCs, but not any of more than 100 other enzymes tested. CONCLUSION: These are the first data to demonstrate that the enzymatic activity of the atypical endothelial adhesion molecule AOC3, and possibly that of other closely related ecto-oxidases, is crucial for leukocyte exit from the vessels in inflamed joints in vivo.

Reactions of plant copper/topaquinone amine oxidases with N6-aminoalkyl derivatives of adenine.

Plant copper/topaquinone-containing amine oxidases (CAOs, EC 1.4.3.6) are enzymes oxidising various amines. Here we report a study on the reactions of CAOs from grass pea (Lathyrus sativus), lentil (Lens esculenta) and Euphorbia characias, a Mediterranean shrub, with N6-aminoalkyl adenines representing combined analogues of cytokinins and polyamines. The following compounds were synthesised: N6-(3-aminopropyl)adenine, N6-(4-aminobutyl)adenine, N6-(4-amino-trans-but-2-enyl) adenine, N6-(4-amino-cis-but-2-enyl) adenine and N6-(4-aminobut-2-ynyl) adenine. From these, N6-(4-aminobutyl) adenine and N6-(4-amino-trans-but-2-enyl)adenine were found to be substrates for all three enzymes (Km approximately 10(-4)M). Absorption spectroscopy demonstrated such an interaction with the cofactor topaquinone, which is typical for common diamine substrates. However, only the former compound provided a regular reaction stoichiometry. Anaerobic absorption spectra of N6-(3-aminopropyl)adenine, N6-(4-amino-cis-but-2-enyl)adenine and N6-(4-aminobut-2-ynyl)adenine reactions revealed a similar kind of initial interaction, although the compounds finally inhibited the enzymes. Kinetic measurements allowed the determination of both inhibition type and strength; N6-(3-aminopropyl)adenine and N6-(4-amino-cis-but-2-enyl)adenine produced reversible inhibition (Ki approximately 10(-5) - 10(-4) M) whereas, N6-(4-aminobut-2-ynyl)adenine could be considered a powerful inactivator.

Exploring the binding mode of semicarbazide-sensitive amine oxidase/VAP-1: identification of novel substrates with insulin-like activity.

We previously reported that substrates of semicarbazide-sensitive amine oxidase in combination with low concentrations of vanadate exert potent insulin-like effects. Here we performed homology modeling of the catalytic domain of mouse SSAO/VAP-1 and searched through chemical databases to identify novel SSAO substrates. The modeling of the catalytic domain revealed that aromatic residues Tyr384, Phe389, and Tyr394 define a pocket of stable size that may participate in the binding of apolar substrates. We identified a number of amines as substrates of human, rat, and mouse SSAO. The compounds PD0119035, 2,3-dimethoxy-benzylamine, and C-naphthalen-1-yl-methylamine showed high affinity as substrates of rat SSAO. C-Naphthalen-1-yl-methylamine was the only substrate that showed high affinity for human SSAO. C-Naphthalen-1-yl-methylamine and 4-aminomethyl-benzenesulfonamide showed the highest capacity to stimulate glucose transport in isolated rat adipocytes. The impact of these findings on the development of new treatments for diabetes is discussed.

Inhibition of lentil copper/TPQ amine oxidase by the mechanism-based inhibitor derived from tyramine.

Copper amine oxidase from lentil (Lens esculenta) seedlings was shown to catalyze the oxidative deamination of tyramine and three similar aromatic monoamines, benzylamine, phenylethylamine and 4-methoxyphenylethylamine. Tyramine, an important plant intermediate, was found to be both a substrate and an irreversible inhibitor of the enzyme whereas the other amines were not inhibitory. In the course of tyramine oxidation the enzyme gradually became inactivated with the concomitant appearance of a new absorption at 560 nm due to the formation of a stable adduct. Inactivation took place only in the presence of oxygen and was probably due to the reaction of the enzyme with the oxidation product of tyramine, p-hydroxyphenylacetaldehyde. The kinetic data obtained in this study indicate that tyramine represents a new interesting type of physiological mechanism-based inhibitor for plant copper amine oxidases.

Gene organization and molecular modeling of copper amine oxidase from Aspergillus niger: re-evaluation of the cofactor structure.

Amine oxidase AO-I from Aspergillus niger AKU 3302 has been reported to contain topa quinone (TPQ) as a cofactor; however, analysis of the p-nitrophenylhydrazine-derivatized enzyme and purified active site peptides showed the presence of a carboxylate ester linkage of TPQ to a glutamate. The catalytic functionality of such a cross-linked cofactor has recently been shown unlikely by spectroscopic and voltammetric studies on synthesized model compounds. We have obtained resonance Raman spectra of native and substrate-reduced AO-I demonstrating that the catalytically active cofactor is unmodified TPQ. The primary structure of the enzyme (GenBank acc. no. U31869) has been reviewed and updated by repeated isolation and sequencing of AO-I cDNA. This allowed rectification of several errors that account for previously reported low homology to other amine oxidases in the regions around copper binding histididyl residues. The results were confirmed by cloning the ao-1 structural gene (GenBank acc. no. AF362473). Analysis of the gene 5'-upstream region of the gene revealed potential binding sites for an analog of NIT2, the nitrogen metabolism regulatory protein found in Neurospora crassa and other fungi. The molecular structure of AO-I was modeled by a comparative method using published crystal structures of amine oxidases as templates.

Structure and nucleotide sequence of Euphorbia characias copper/TPQ-containing amine oxidase gene.

A cDNA encoding for a copper containing amine oxidase has been isolated and sequenced from young leaves of Euphorbia characias, a perennial mediterranean shrub. A single long open reading frame of 2068 pb encodes a protein composed of 653 amino acids with a molecular mass of about 74 kDa. A putative 24-aminoacid signal peptide precedes the sequence of the mature protein, with characteristics of a secretion signal peptide. Alignments of Euphorbia amine oxidase cDNA nucleotide sequence with that of amine oxidase from the seedlings of the pulses lentil, pea, and chickpea reveal several conserved regions, especially in the C-terminus, with a homology 90%-97%. The near 5' region shows several insertions, deletions, and different nucleotide sequence with ca. 60% homology. The enzyme contains 1%-2% carbohydrate deduced by deglycosylation experiments. Five cysteine residues are present in the deduced aminoacid sequence with a single disulfide bridge as judged by titration with cysteine reagents.

Interaction of pig kidney and lentil seedling copper-containing amine oxidases with guanidinium compounds.

The effect of guanidinium compounds on the catalytic mechanism of pig kidney and lentil seedling amine oxidases has been investigated by polarographic techniques and spectroscopy. Guanidine does not inhibit the lentil enzyme and is a weak inhibitor for pig kidney amine oxidase (Ki=1 mM), whereas aminoguanidine is an irreversible inhibitor of both enzymes, with a Ki value of 10(-6) M. 1,4-Diguanidino butane (arcaine) is a competitive inhibitor for both pig and lentil amine oxidases. Amiloride is a competitive inhibitor for pig enzyme, but upon prolonged incubation with this drug the enzyme gradually loses its activity in an irreversible manner.

A study on the reactions of plant copper amine oxidase with C3 and C4 aliphatic diamines.

The paper reports a study on the reactions of grass pea (Lathyrus sativus) amine oxidase (GPAO) with several aliphatic diamines. The influence of the chain length and of unsaturations in the molecules was examined. Kinetic measurements confirmed that trans-, i.e., (E)-2-butene-1,4-diamine (TDABE) and cis-, i.e., (Z)-2-butene-1,4-diamine (CDABE) could be classified as good substrates. Propane-1,3-diamine (DAP) and propene-1,3-diamine (DAPE) were only weakly oxidized, whereas 1,3-diamino-2-propanol (DAPL) was not utilized as a substrate. Contrary to the inactivator 2-butyne-1,4-diamine (DABI), DAPE was shown to be only a competitive inhibitor. DAP itself did not inhibit the catalytic activity. Irreversible inhibition of the activity occurred only after the incubation of GPAO with DABI; other diamines were without this effect. Differential pulse polarography and chromatofocusing confirmed that the aminoaldehyde product of DABI oxidation binds to the enzyme. Activity assay of pea aminoaldehyde dehydrogenase enabled us to detect the products of the oxidation of TDABE, CDABE, and DAP by GPAO. As the product of DAP oxidation, 3-amino-propanal (APAL) was detected by mass spectrometry and confirmed to be a potent noncompetitive inhibitor of GPAO. The absorption changes that occurred in the course of the reaction of GPAO with the diamines were investigated using rapid-scanning spectrophotometry. DABI, TDABE, CDABE, DAP, and DAPE reacted with GPAO providing characteristic maxima of the Cu(I)-semiquinolamine species that is formed in the catalytic cycle. The results presented here confirm that with the exception of DAPL, all the studied diamines could be classified as GPAO substrates, but only DABI can be considered as a mechanism-based inhibitor.

Effect of metal substitution in copper amine oxidase from lentil seedlings.

The reaction with substrates and carbonyl reagents of native lentil Cu-amine oxidase and its modified forms, i.e. Cu-fully-depleted, Cu-half-reconstituted, Cu-fully-reconstituted, Co-substituted, Ni-substituted and Zn-substituted, has been studied. Upon removal of only one of the two Cu ions, the enzyme loses 50% of its enzymatic activity. Using several substrates, Co-substituted lentil amine oxidase is shown to be active but the k(c) value is different from that of native or Cu-fully-reconstituted enzyme, while K(m) is similar. On the other hand, the Ni- and Zn-substituted forms are catalytically inactive. Enzymatic activity measurements and optical spectroscopy show that only in the Co-substituted enzyme is the organic cofactor 6-hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Co-substituted amine oxidase does not form the semiquinone radical as an intermediate of the catalytic reaction. While devoid or reduced of catalytic activity, all the enzyme preparations are still able to oxidise two moles of substrate and to release two moles of aldehyde per mole of dimeric enzyme. The results obtained show that although Co-substituted amine oxidase is catalytically competent, copper is essential for the catalytic mechanism.

CuI-semiquinone radical species in plant copper-amine oxidases.

The intermediate CuI-semiquinone radical species in the catalytic mechanism of copper-amine oxidase from Lens esculenta and Pisum sativum seedlings has been studied by optical, Raman resonance and ESR spectroscopies and by stopped-flow and temperature-jump measurements. Treatment of highly purified enzyme preparations with good, poor or suicide substrates, under anaerobic and aerobic conditions, at different pH values and temperatures, makes it possible to generate, detect and characterize this free radical intermediate.