Euphorbia characias Latex Amine Oxidase and Peroxidase: Interacting Enzymes?

This minireview deals the enzymatic transformation of some amino acids as arginine and ornithine, amines as tyramine, putrescine, spermine and spermidine, and other substances as nitric oxide and thiocyanate. These reactions, catalyzed by two proteins purified from the latex of Euphorbia characias, a copper/quinone containing amine oxidase and a cationic peroxidase, show enzymatic activity interactions probably occurring between these proteins in Euphorbia latex.     

Enzymatic production of 4-hydroxyphenylacetaldehyde by oxidation of the amino group of tyramine with a recombinant primary amine oxidase

In this study, an efficient enzymatic process for the synthesis of 4-hydroxyphenylacetaldehyde (4-HPAA) from tyramine was developed using whole cells of recombinant Escherichia coli co-expressing primary amine oxidase (PrAO) from E. coli and catalase (CAT) from Bacillus pumilus. The reaction conditions for the synthesis of 4-HPAA were systematically optimized starting from a monophasic aqueous buffer. The optimum reaction temperature, pH, and biocatalyst loading were 33 °C, 7.5, and 20 g/L wet cells, respectively. Substrate feeding strategies were employed to alleviate substrate inhibition, providing a 14.8 % increase in yield. A biphasic catalytic system was explored to avoid product inhibition and thus further improve the 4-HPAA yield. Ethyl acetate was found to be the best organic solvent, and the optimum volume ratio of the organic phase to the aqueous phase was 40 % (v/v). Under the optimized conditions on a 1 L scale, a yield of 76.5 % was obtained with a substrate concentration of 120 mM. Thus, the bioconversion was more efficient in the ethyl acetate/buffer biphasic system than in the monophasic aqueous system, and the yield of 4-HPAA was improved 1.89-fold.     

Involvement of Cytochrome P-450 in the Biosynthesis of Dhurrin in Sorghum bicolor (L.) Moench

The biosynthesis of the tyrosine-derived cyanogenic glucoside dhurrin involves N-hydroxytyrosine, (E)- and (Z)-p-hydroxyphenylacetaldehyde oxime, p-hydroxyphenylacetonitrile, and p-hydroxymandelonitrile as intermediates and has been studied in vitro using a microsomal enzyme system obtained from etiolated sorghum (Sorghum bicolor [L.] Moench) seedlings. The biosynthesis is inhibited by carbon monoxide and the inhibition is reversed by 450 nm light demonstrating the involvement of cytochrome P-450. The combined use of two differently prepared microsomal enzyme systems and of tyrosine, p-hydroxyphenylacetaldehyde oxime, and p-hydroxyphenylacetonitrile as substrates identify two cytochrome P-450-dependent monooxygenases: the N-hydroxylase which converts tyrosine into N-hydroxytyrosine and the C-hydroxylase converting p-hydroxyphenylacetonitrile into p-hydroxymandelonitrile. The inhibitory effect of a number of putative cytochrome P-450 inhibitors confirms the involvement of cytochrome P-450. Monospecific polyclonal antibodies raised toward NADPH-cytochrome P-450-reductase isolated from sorghum inhibits the same metabolic conversions as carbon monoxide. No cytochrome P-450-dependent monooxygenase catalyzing an N-hydroxylation reaction has previously been reported in plants. The metabolism of p-hydroxyphenylacetaldehyde oxime is completely dependent on the presence of NADPH and oxygen and results in the production of p-hydroxymandelonitrile with no accumulation of the intermediate p-hydroxyphenylacetonitrile in the reaction mixture. The apparent NADPH and oxygen requirements of the oxime-metabolizing enzyme are identical to those of the succeeding C-hydroxylase converting p-hydroxyphenylacetonitrile to p-hydroxymandelonitrile. Due to the complex kinetics of the microsomal enzyme system, these requirements may not appertain to the oxime-metabolizing enzyme, which may convert p-hydroxyphenylacetaldehyde oxime to p-hydroxyacetonitrile by a simple dehydration.     

Fluorometric assay for intestinal peptidases

A fluorometric assay for intestinal peptidases has been developed. Amino acids liberated by hydrolysis are estimated by use of l-amino-acid oxidase, peroxidase, and the fluorogenic reagent p-hydroxyphenylacetic acid, which yields a highly fluorescent compound on oxidation. During development of fluorescence, continued hydrolysis of peptides by peptidases which contaminate available preparations of l-amino-acid oxidase is prevented by the use of two inhibitors, 1,10-phenanthroline and p-hydroxymercuribenzoate. The assay is at least 10 times more sensitive than comparable spectrophotometric methods which employ the potentially carcinogenic chromogen o-dianisidine for detection of amino acids.     

Nucleotide sequence of the gene for monoamine oxidase (maoA) from Escherichia coli

We found that the structural gene for monoamine oxidase was located at 30.9 min on the Escherichia coli chromosome. Deletion analysis showed that two amine oxidase genes are located in this region. The nucleotide sequence of one of the two genes was determined. The peptide sequence of the first 40 amino acids from the N terminus of monoamine oxidase purified from E. coli agrees with that deduced from the nucleotide sequence of the gene. The leader peptide extends over 30 amino acids. The nucleotide sequence of the gene and amino acid sequence of the predicted mature enzyme (M.W. 81,295) were highly homologous to those of the maoA_K gene and monoamine oxidase from Klebsiella aerogenes, respectively. From these results and analysis of the enzyme activity, we concluded that the gene encodes for monoamine oxidase (maoA_E). The tyrosyl residue, which may be converted to topa quinone in the E. coli enzyme, was located by comparison with amino acid sequences at the cofactor sites in other copper/topa quinone-containing amine oxidases.     

Changes in Amines and Biosynthetic Enzyme Activities in p-Fluorophenylalanine Resistant and Wild Type Tobacco Cell Cultures

The levels of free amines and the activities of their biosynthetic enzymes were measured in a p-fluorophenylalanine resistant Nicotiana tabacum L. cv Xanthi cell line (TX4) which accumulates high levels of cinnamoylamides, and a wild type cell line (TX1). Putrescine in TX1 and spermidine in TX1 and TX4 increased 4-fold by day 4 but declined by day 8 of the culture period. Spermine levels were consistently low, while tyramine was not found in TX1 until day 9 when a gradual rise was noted. Ornithine decarboxylase activity in TX1 and TX4 increased slightly through day 2 but declined gradually thereafter. S-Adenosylmethionine decarboxylase activity remained low throughout the culture period, and tyrosine and arginine decarboxylases in TX1 were very low in activity. In contrast, the activities of tyrosine and arginine decarboxylases were elevated in TX4, but a 3-fold increase in tyramine after a subculture was not accompanied by a rise in tyrosine decarboxylase. However, tyrosine decarboxylase activity did increase during a second rise in tyramine levels in aging cells, late in the culture period. Although significant differences exist in amine levels, between TX4 and TX1, it is unclear how altered amine metabolism relates to p-fluorophenylalanine resistance.     

Monophenolase activity of avocado polyphenol oxidase

Polyphenol oxidase of avocado mesocarp catalyses (a) the orthohydroxylation of monophenols like l-tyrosine, d-tyrosine, tyramine and p-cresol, and (b) the oxidation of the corresponding o-dihydroxyphenols to quinones. The rate of step b is much greater than that of step a. The hydroxylation of monophenols occurs after a lag period. DOPA or ascorbate effectively eliminate the lag but not dl-6-methyltetrahydropteridine or tetrahydrofolic acid. At 1.66 × 10^?4 M, α,α-dipyridyl has no effect, while diethyldithiocarbamate at this concentration inhibits the hydroxylation reaction by 90%. The tyrosinase activity of avocado polyphenol oxidase is inactivated in the course of the reaction; this inactivation occurs faster and is more pronounced in the presence of exogenously added DOPA. This inactivation is partially prevented by a large excess of ascorbate. The K_m values indicate that tyramine, dopamine, p-cresol and 4-methyl catechol are better substrates for avocado polyphenol oxidase than tyrosine or DOPA.     

Purification, cloning, and three-dimensional structure prediction of Micrococcus luteus FAD-containing tyramine oxidase

The FAD-containing tyramine oxidase enzyme and gene from the Gram (+) bacterium Micrococcus luteus were isolated, and computer prediction was used to propose a preliminary 3D model of the protein. A 2.8-kb Sau3AI fragment containing the structural gene of tyramine oxidase was cloned from a M. luteus genomic DNA library. The 1332 bp gene encodes a protein of 443 amino acids, with a calculated molecular mass of 49.1 kDa. The enzyme was found to be a homodimer with a molecular weight of 49,000. It oxidizes tyramine, adrenaline, 3-hydroxytyramine, dopamine, and noradrenaline, and was reversibly inhibited by FAD-containing monoamine oxidase A and B specific inhibitors. Sequence comparison show that tyramine oxidase is smaller than other FAD-amine oxidases but that it contains well-conserved amino acid residues reported in all other FAD-amine oxidases. A hypothetical three-dimensional structure of tyramine oxidase has also been proposed based on secondary structure predictions, threading, and comparative modeling.     

The function of free carboxyl groups in the action of peroxidase and indole-3-acetic acid oxidase

The extracellular peroxidase from cultures of Inonotus radiatus and of peanut (Arachis hypogeaL.) cells as well as the mycelial peroxidase from Trametes versicolor were used for studies of immobilizing this protein either by its free amino or its carboxyl groups. The immobilization process was carried out either on keratin proteins derived from feathers or on polyamide coated over silica gel. Coupling was established either through the free amino or carboxyl groups. In general the indolyl-3-acetic acid oxidase activity of fungal peroxidases exceeds that of peanut peroxidase. When the peroxidase of the three sources was immobilized on the matrices by the free amino groups, little if any effect on the IAA oxidase activity could be measured. However, immobilization through the carboxyl groups resulted in a drastic reduction of indole-3-acetic acid oxidase activity. Since identical amounts of peroxidase were linked in all cases, the loss of indolyl-3-acetic acid oxidase activity implies that the carboxyl group is essential for the active site.     

Bacterial Monoamine Oxidases

A procedure for obtaining crystalline preparations of tyramine oxidase of Sarcina lutea has been developed. The procedure included fractionation with ammonium sulfate, treatment with protamine sulfate and separation by column chromatographies on DEAE-cellulose, hydroxylapatite and sephadex G-150. The specific activity of enzyme was increased 5,700~ 6,000-fold through the procedure, over the crude cell extract. Crystals were prepared from solutions of the purified enzyme by adding solid ammonium sulfate. The crystals appeared as minute, highly refractive needles, with a bright yellow color.

With the use of crystalline preparations of tyramine oxidase of Sarcina lutea, substrate and inhibitor specificities of the enzyme were investigated. The enzyme oxidized tyramine and dopamine at almost the same rates. Other monoamines, diamines, polyamines and amino acids were not oxidized at all. The oxidation of tyramine proceeded as follows: Tyramine+O₂+H₂O→p-Hydroxyphenylacetaldehyde +NH₃+H₂O₂. Ammonia and hydrogen peroxide were formed in stoichiometric amounts.

The enzyme was not inhibited by carbonyl reagents, such as hydroxylamine, hydrazine, semicarbazide and isoniazid, but was inhibited by p-CMB and iproniazid.     

Characterization of Anionic Peroxidases in Tomato Isolines Infected by Meloidogyne incognita

Changes in peroxidase activity during nematode infection were studied using root extracts of tomato near-isogenic lines differing in resistance to Meloidogyne incognita. Total peroxidase activity increased slightly in crude extracts of four susceptible isolines but doubled in two resistant lines, Monita and Motaci. Nematode infection enhanced levels of both p-phenylenediamine-pyrocatechol oxidase and syringaldazine oxidase 7 days after inoculation, especially in resistant lines. This elevated peroxidase activity in resistant isolines was caused by an increase in anionic peroxidase activity. These enzymes, which likely are involved in lignification, were isolated and purified from tomato isolines by ammonium sulfate precipitation, high performance ion-exchange chromatography, and gel electrophoresis. The purified anionic peroxidase extracts contained an electrophoretic band with Rf 0.51 that was present in extracts of infected but not uninfected roots.     

An amine oxidase in seedlings of <Emphasis Type="Italic">Papaver somniferum</Emphasis> L.

Amine oxidase (AO) from 4-d-old seedlings of Papaver somniferum L. (Papaveraceae) was purified (58-fold) by using ammonium sulphate precipitation and chromatography on Sephadex G-150 and HA-Ultrogel columns. The most readily oxidized substrate was tyramine and other aromatic amines, while aliphatic amines cadaverine and putrescine were oxidized more slowly. Cu chelating and carbonyl reagents are the most effective inhibitors of poppy amine oxidase. Immunoblotting analysis showed cross reactivity of AO protein from poppy seedlings with polyclonal antisera against AO from pea. Obtained Mr value for AO from poppy (83 kDa) corresponds to that of copper AOs (75 – 90 kDa). These results suggest that the amine oxidase from poppy seedlings is a copper containing and tyramine specific AO.This work was supported by the grant of Slovak Grant Agency (VEGA 1/1197/04) and by the Comenius University, Faculty of Pharmacy Grant (FaF UK/1191/2002).     

Monoamine oxidase electrode in freshness testing of meat

Monoamine oxidase (monoamine: oxygen oxidoreductase, EC 1.4.3.4 from Aspergillus niger and beef plasma) was immobilized in a collagen membrane. An enzyme electrode consisting of a monoamine oxidase - collagen membrane (10 units) and an oxygen electrode was prepared for the determination of monoamines. Monoamines were oxidized to aldehydes by the immobilized enzyme and oxygen consumption was monitored amperometrically by the oxygen electrode. The response time of the electrode was 4 min. The optimum conditions for the enzyme electrode were pH 7.4 and 30°C. A linear relationship was observed between the amine (tyramine) concentration in the range 50–200 μm and the difference in current. No decrease in the output current was observed over an observation period of one week. The difference in current was reproducible with an average relative error of 8%. Monoamines in meat extracts were determined by the enzyme electrode.     

Brain tyramine and reproductive states of workers in honeybees

To explore the role of tyramine in the transformation of reproductive states of honeybee workers, brain levels of tyramine and N-acetyltyramine were measured in both normal and queenless workers. Queenless workers had higher tyramine levels and lower N-acetyltyramine levels than normal workers did. Intermediate reproductive workers that were transferred into a normal colony from a queenless colony had intermediate levels of tyramine and N-acetyltyramine. Elevation of tyramine in the queenless workers occurred at an earlier adult stage than elevation of dopamine. Tyramine levels in intermediate reproductive workers returned to the levels seen in normal workers, but dopamine levels in intermediate reproductive workers remained elevated at the same level as in queenless workers. Thus, brain tyramine may be regulated by the colony condition with or without a queen. Injection of an amine uptake inhibitor, reserpine, depleted tyramine and elevated N-acetyltyramine. Distributions of tyramine and dopamine within the brain were distinctively different, whereas distributions of N-acetyltyramine and N-acetyldopamine were similar, suggesting that each functional amine is stored in specific neurosecretory cells and released to the relevant receptor sites but that metabolism into each N-acetylmetabolite is determined by diffusion.     

Brown Pigments Produced by Yarrowia lipolytica Result from Extracellular Accumulation of Homogentisic Acid

Yarrowia lipolytica produces brown extracellular pigments that correlate with tyrosine catabolism. During tyrosine depletion, the yeast accumulated homogentisic acid, p-hydroxyphenylethanol, and p-hydroxyphenylacetic acid in the medium. Homogentisic acid accumulated under all aeration conditions tested, but its concentration decreased as aeration decreased. With moderate aeration, equimolar concentrations of alcohol and p-hydroxyphenylacetic acid (1:1) were detected, but with lower aeration the alcohol concentration was twice that of the acid (2:1). p-Hydroxyphenylethanol and p-hydroxyphenylacetic acid may result from the spontaneous disproportionation of the corresponding aldehyde, p-hydroxyphenylacetaldehyde. The catabolic pathway of tyrosine in Y. lipolytica involves the formation of p-hydroxyphenylacetaldehyde, which is oxidized to p-hydroxyphenylacetic acid and then further oxidized to homogentisic acid. Brown pigments are produced when homogentisic acid accumulates in the medium. This acid can spontaneously oxidize and polymerize, leading to the formation of pyomelanins. Mn²⁺ accelerated and intensified the oxidative polymerization of homogentisic acid, and lactic acid enhanced the stimulating role of Mn²⁺. Alkaline conditions also accelerated pigment formation. The proposed tyrosine catabolism pathway appears to be unique for yeast, and this is the first report of a yeast producing pigments involving homogentisic acid.     

The Effect of Pseudomonas putida Colonization on Root Surface Peroxidase

Increased activities of peroxidase and indole 3-acetic acid (IAA) oxidase were detected on root surfaces of bean (Phaseolus vulgaris) seedlings colonized with a soil saprophytic bacterium, Pseudomonas putida. IAA oxidase activity increased over 250-fold and peroxidase 8-fold. Enhancement was greater for 6-day-old than for 4- or 8-day-old inoculated plants No IAA oxidase or peroxidase activities were associated with the bacterial cells. Native polyacrylamide gel electrophoresis demonstrated that washes of P. putida-inoculated roots contained two zones of peroxidase activity. Only the more anodic bands were detected in washes from noninoculated roots. Ion exchange and molecular sizing gel chromatography of washes from P. putida-colonized roots separated two fractions of peroxidase activity. One fraction corresponded to the anodic bands detected in washes of P. putida inoculated and in noninoculated roots. A second fraction corresponded to the less anodic zone of peroxidase, which was characteristic of P. putida-inoculated plants. This peroxidase had a higher IAA oxidase to peroxidase ratio than the more anodic, common enzyme. The changes in root surface peroxidases caused by colonization by a saprophytic bacterium are discussed with reference to plant-pathogen interactions.     

The role of octopamine in locusts and other arthropods

The biogenic amine octopamine and its biological precursor tyramine are thought to be the invertebrate functional homologues of the vertebrate adrenergic transmitters. Octopamine functions as a neuromodulator, neurotransmitter and neurohormone in insect nervous systems and prompts the whole organism to “dynamic action”. A growing number of studies suggest a prominent role for octopamine in modulating multiple physiological and behavioural processes in invertebrates, as for example the phase transition in Schistocerca gregaria. Both octopamine and tyramine exert their effects by binding to specific receptor proteins that belong to the superfamily of G protein-coupled receptors. Since these receptors do not appear to be present in vertebrates, they may present very suitable and specific insecticide and acaricide targets.     

Cloning and sequencing of a copper-containing,topa quinone-containing monoamine oxidase from human placenta

A 4040-bp cDNA was cloned from a human placenta library by screening with a polymerase chain reaction-amplified fragment. The fragment was generated from the library using primers corresponding to conserved sequences encompassing the topa quinone (TPQ) cofactor sites of the copper-containing proteins, bovine serum amine oxidase (BSAO) and human kidney diamine oxidase (DAO). The cloned cDNA contains a coding sequence from positions 161 to 2449. Between bases 2901 and 2974, in a very long 1591-bp 3′-untranslated region, there is a G/A-rich region in the minus strand, which contains a (AGG)₅ tandem repeat. The human placenta cDNA sequence and its translated amino acid sequence are 84% and 81% identical to the corresponding BSAO sequences, while the identities for the placenta sequences and those for human kidney DAO are 60% and 41%, respectively. The TPQ consensus nucleotide and protein sequences are identical for the placenta enzyme and BSAO, but the corresponding sequences for human kidney DAO are nonidentical. Three His residues that have been identified as Cu(II) ligands in other amine oxidases are conserved in the human placenta amine oxidase protein sequence. It was concluded that the placenta cDNA open-reading frame codes for a copper-containing, TPQ-containing monoamine oxidase. A putative 19-amino acid signal peptide was identified for human placenta amine oxidase. The resulting mature protein would be composed of 744 amino acids, and would have a M_r of 82 525. Comparison of the human placenta amine oxidase with DNA sequences found in GenBank suggests that the gene for this enzyme is located in the q21 region of human chromosome 17, near the BRCA1 gene.     

The relative participation of liver microsomal amine oxidase and cytochrome P-450 in N-demethylation reactions.

N-Demethylation of benzphetamine and p-chloro-N-methylaniline measured in the presence and absence of specific antibodies to NADPH-cytochrome c (P-450) reductase demonstrates that part of the formaldehyde formed from the sec-N-methylamine arises from non-cytochrome P-450-dependent oxidation catalyzed by pig, hamster, and rat liver microsomes. The additional formaldehyde formed can be inhibited by adding methimazole, a non-formaldehyde-producing substrate specific for the microsomal mixed-function amine oxidase, to the reaction media. Purified amine oxidase catalyzes the oxidation of sec-N-methylamines to sec-N-methylhydroxylamines that, upon oxidation and hydrolysis, yield formaldehyde. Approximately 65, 40, and 15% of total formaldehyde is formed by this route during oxidation of p-chloro-N-methylaniline catalyzed by pig, hamster, and rat liver microsomes, respectively.     

Characterization of amine oxidases from Arthrobacter aurescens and application for determination of biogenic amines

Biogenic amines (BAs) that are produced through naturally occurring decarboxylation of amino acids have toxicological effects on humans. Bacterial amine oxidases are useful tools for the rapid quantification of BAs in foods. To develop amine oxidases for the rapid detection of BAs, the genes for amine oxidases from Arthobacter aurescens TC-1, designated AMAO1, AMAO2, and AMAO3, respectively, were cloned and expressed in Escherichia coli. AMAO1 was catalytically inactive to BAs, and AMAO3 showed a narrow substrate spectrum. In contrast, AMAO2 exhibited activity with relative k _cat/K _M values of 100:49.6:7.6 for 2-phenylethylamine, tyramine, and histamine, respectively. AMAO2 also utilized putrescine and spermidine as substrates, with four or five orders of magnitude lower k _cat/K _M values than that of 2-phenylethylamine. AMAO2 and AMAO3 were seriously affected by substrate inhibition. Using BA mixtures (consisting of 2-phenylethylamine, tyramine, and histamine) as samples, the detection range of the enzymatic analysis of BA using AMAO2 was determined to be 2.5–120 μM, and its detection limit was 2.3 μM. Analysis of five commercial cheese products revealed that the BA contents determined by the enzymatic methods showed a good agreement with the sum of three monoamines and histamine by HPLC. Therefore, the enzymatic assay using AMAO2 can be used in quality control of food products through rapid, sensitive, and preliminary estimation of major BAs including the most important TyrN and HisN in foods.