Singlet oxygen production from the peroxidase-catalyzed oxidation of indole-3-acetic acid

The aerobic oxidation of indole-3-acetic acid catalyzed by horseradish peroxidase produces 1268 nm emission characteristic of singlet oxygen. Lactoperoxidase also oxidizes indole-3-acetic acid to produce singlet oxygen, but in contrast to horseradish peroxidase, this enzyme system requires hydrogen peroxide. In both of these systems, the intensity of the 1268 nm emission is small due to quenching of the singlet oxygen by indole-3-acetic acid and by reaction products derived from indole-3-acetic acid. The biomolecular reaction of peroxyl radicals via a Russell mechanism is a plausible mechanism for the singlet oxygen generation in these systems. Under typical conditions of p2H 4.0, 1 microM horseradish peroxidase, 1 mM indole-3-acetic acid, and 240 microM oxygen, the singlet oxygen yield was 15 +/- 1 microM or 13% of the amount predicted by the Russell mechanism.     

The Mechanism of the Scopoletin-induced Inhibition of the Peroxidase Catalyzed Degradation of Indole-3-acetate

The naturally occurring coumarin, scopoletin, has been found to modify horseradish peroxidase rapidly to give a stable, spectroscopically distinguishable form of the enzyme. Peroxidase treated with scopoletin is less active in reactions with molecular oxygen and indole-3-acetic acid. Kinetic data for the degradation of this growth regulator were obtained with a continuously monitored fluorometric procedure. Lineweaver-Burk plots of the reciprocal rate of degradation against the reciprocal substrate concentration were markedly curved in the presence of the inhibitor, scopoletin. Excess indole-3-acetate restored the scopoletin-treated enzyme to a reactive state. In the presence of molecular oxygen, concentrations of indole-3-acetic acid which were at least 10-fold greater than the inhibitor concentration led to the rapid oxidation of the coumarin and converted peroxidase to compound III as expected from previous studies. This form of the enzyme is the catalytically active species in the oxidative degradation of the growth regulator. The kinetically preferential reaction of scopoletin or related coumarins with peroxidase and the suppression of indole-3-acetic acid degradation may provide a possible control mechanism over the oxidative degradation of indole-3-acetate by this plant enzyme.     

Indole-3-methanol as an intermediate in the oxidation of indole-3-acetic acid by peroxidase

During oxidation of indole-3-acetic acid catalyzed by horseradish peroxidase, indole-3-aldehyde and 3-hydroxymethayloxindole cease to be produced a few minutes after initiation of the reaction even though IAA is still being consumed. At the same time an increased accumulation of indole-3-methanol is observed and the ratio of oxygen to indole-3-acetic acid consumed becomes less than unity. Indole-3-niethanol can be a substrate for horseradish peroxidase provided that H₂O₂ is present. In this reaction, indole-3-aldehyde but not 3-hydroxymethyloxindole is formed. H₂O₂ is not merely an activating agent for the enzyme but also a true oxidant because it is consumed stoichiometrically (1 mol of H₂O₂ per mol of indole-3-methanol) and the reaction is independent of the presence of oxygen. Indole-3-methanol is proposed as an intermediate in the process of oxidation of indole-3-acetic acid into indole-3-al-denyde, the second step of which requires peroxide as an oxidant.     

The inhibition of peroxidase and indole-3-acetic acid oxidase activity by British Anti-Lewisite

British Anti-Lewisite (BAL) binds to horseradish peroxidase in a manner which results in inhibition of both peroxidatic and oxidative functions of the enzyme. BAL competes with hydrogen peroxide for binding on peroxidase, and the inhibition of peroxidatic activity is irreversible. Solutions of purified horseradish peroxidase and individually resolved peroxidase isozymes show a gradual loss of peroxidatic activity with time when incubated with BAL. In these same treatments, however, the inhibition of indole-3-acetic acid (IAA) oxidase activity is immediate. With increasing amounts of enzyme in the incubation mixture, IAA oxidase activity is not completely inhibited and is observed following a lag period in the assay which shortens with longer incubation times. Peroxidase activity during this same time interval shows a lag period which increases with longer incubation times. Lowering the pH removed the lag period for oxidase activity, but did not change the pattern of peroxidase activity. These results suggest that the sites for the oxidation of indole-3-acetic acid and for peroxidatic activity may not be identical in horseradish peroxidase isozymes.     

Glucuronidation of oxygenated benzo(a)pyrene derivatives by UDP-glucuronyl transferase of nuclear envelope

The variation of the spectra and its reactivity towards 2-methylpropanal, indole-3-acetic acid and malonaldehyde of solutions of horseradish peroxidase in dimethyl sulfoxide-water mixtures has been studied. A broad pattern of changes was observed in the CD spectra of peroxidase, especially in the 400 nm region. These variations influenced strongly the excited triplet acetone emission from the 2-methylpropanal system which is generated in the active site of the enzyme protected from external quenching. This means that presumably the active site is more uncovered in the presence of dimethyl sulfoxide than the native form. Energy transfer parameters indicate that in fact there is a conformational effect produced by dimethyl sulfoxide in the horseradish peroxide active site. Dimethyl sulfoxide appears to be an important conformational probe in biochemistry.     

Primary or secondary? Versatile nitrilases in plant metabolism

The potential of plant nitrilases to convert indole-3-acetonitrile into the plant growth hormone indole-3-acetic acid has earned them the interim title of "key enzyme in auxin biosynthesis". Although not widely recognized, this view has changed considerably in the last few years. Recent work on plant nitrilases has shown them to be involved in the process of cyanide detoxification, in the catabolism of cyanogenic glycosides and presumably in the catabolism of glucosinolates. All plants possess at least one nitrilase that is homologous to the nitrilase 4 isoform of Arabidopsis thaliana. The general function of these nitrilases lies in the process of cyanide detoxification, in which they convert the intermediate detoxification product beta-cyanoalanine into asparagine, aspartic acid and ammonia. Cyanide is a metabolic by-product in biosynthesis of the plant hormone ethylene, but it may also be released from cyanogenic glycosides, which are present in a large number of plants. In Sorghum bicolor, an additional nitrilase isoform has been identified, which can directly use a catabolic intermediate of the cyanogenic glycoside dhurrin, thus enabling the plant to metabolize its cyanogenic glycoside without releasing cyanide. In the Brassicaceae, a family of nitrilases has evolved, the members of which are able to hydrolyze catabolic products of glucosinolates, the predominant secondary metabolites of these plants. Thus, the general theme of nitrilase function in plants is detoxification and nitrogen recycling, since the valuable nitrogen of the nitrile group is recovered in the useful metabolites asparagine or ammonia. Taken together, a picture emerges in which plant nitrilases have versatile functions in plant metabolism, whereas their importance for auxin biosynthesis seems to be minor.     

Proposed Model for the Peroxidase-Catalyzed Oxidation of Indole-3-acetic Acid in the Presence of the Inhibitor Ferulic Acid

Linear increments in ferulic acid concentration produce logarithmic increases in the ferulic acid-induced lag periods prior to the peroxidase-catalyzed oxidation of indole-3-acetic acid in a system containing 2,4-dichlorophenol and MnCl₂ in acetate buffer at pH 5.6. Maintaining the ratio of indole-3-acetic acid to ferulic acid constant at 100 while linearly raising the ferulic acid concentration results in linear increases in the lag period. Both indole-3-acetic acid and ferulic acid are substrates of horseradish peroxidase in the presence of H₂O₂, and indole-3-acetic acid competitively inhibits the oxidation of ferulic acid. A model for the enzymatic oxidation of indole-3-acetic acid catalyzed by peroxidase is proposed.     

Molecular cloning and characterization of an amidase from Arabidopsis thaliana capable of converting indole-3-acetamide into the plant growth hormone,indole-3-acetic acid

Acylamidohydrolases from higher plants have not been characterized or cloned so far. AtAMI1 is the first member of this enzyme family from a higher plant and was identified in the genome of Arabidopsis thaliana based on sequence homology with the catalytic-domain sequence of bacterial acylamidohydrolases, particularly those that exhibit indole-3-acetamide amidohydrolase activity. AtAMI1 polypeptide and mRNA are present in leaf tissues, as shown by immunoblotting and RT-PCR, respectively. AtAMI1 was expressed from its cDNA in enzymatically active form and exhibits substrate specificity for indole-3-acetamide, but also some activity against L-asparagine. The recombinant enzyme was characterized further. The results show that higher plants have acylamidohydrolases with properties similar to the enzymes of certain plant-associated bacteria such as Agrobacterium-, Pseudomonas- and Rhodococcus-species, in which these enzymes serve to synthesize the plant growth hormone, indole-3-acetic acid, utilized by the bacteria to colonize their host plants. As indole-3-acetamide is a native metabolite in Arabidopsis thaliana, it can no longer be ruled out that one pathway for the biosynthesis of indole-3-acetic acid involves indole-3-acetamide-hydrolysis by AtAMI1.     

The Role of Indole-3-Acetic Acid in Peroxidase Oxidation of Ascorbic Acid Catalyzed by Horseradish Peroxidase

We studied stationary kinetics of ascorbic acid oxidation in the presence of indole-3-acetic acid catalyzed by horseradish peroxidase. The catalytic (k_cat and K_m) and inhibition (K_i) constants were determined for pH from 4.5 to 7.0. The auxin proved to competitively inhibit the enzyme when a single ascorbic acid molecule is bound, while a non-competitive inhibition by IAA is observed for peroxidase oxidation of two or more substrate molecules. A mechanism of ascorbic acid oxidation in the presence of indole-3-acetic acid is proposed.     

Exogenous auxin affects the oxidative burst in barley roots colonized by Piriformospora indica

Beside a cardinal role in coordination of many developmental processes in the plant, the phytohormone auxin has been recognized as a regulator of plant defense. The molecular mechanisms involved are still largely unknown. Using a sensitive chemiluminescence assay, which measures the oxidation of luminol in the presence of H₂O₂ by horseradish peroxidase (HRP), we report here on the ability of exogenously added indole-3-acetic acid (IAA) to enhance the suppressive effect of the root endophyte Piriformospora indica on the chitin-elicited oxidative burst in barley roots. Thus, the potential of P. indica to produce free IAA during the early colonization phase in barley might provide the symbiont with a means to interfere with the microbe-associated molecular patterns (MAMP)-triggered immunity.     

Tomato peroxidase: purification, characterization, and catalytic properties

A major peroxidase has been found in the tomato pericarp (Lycopersicon esculentum var. Tropic) of the ripe and green fruit. A purification scheme yielding this enzyme approximately 85% pure has been developed. The tomato enzyme resembles horseradish peroxidase (HRP) in a standard peroxidase assay and in its ability to be reduced to ferroperoxidase, to be converted to oxyferroperoxidase (compound III), and to form peroxidase complexes with hydrogen peroxide (compounds I and II). In contrast to the HRP, the tomato peroxidase fails to catalyze the aerobic oxidation of indole-3-acetic acid in the presence of 2,4-dichlorophenol and manganese. The tomato peroxidase can be resolved into two nonidentical subunits in the presence of dithiothreitol while HRP remains as a single polypeptide chain after such treatment. Dithiothreitol is oxidized in the presence of tomato or horseradish peroxidase with the enzymes accumulating in their oxyferroperoxidase forms during the oxidation reaction. Whereas HRP returns to its free ferric form at the end of the reaction, the tomato enzyme is converted into a form that absorbs at 442 nanometers.     

Anaerobic stopped-flow studies of indole-3-acetic acid oxidation by dioxygen catalysed by horseradish C and anionic tobacco peroxidase at neutral pH: catalase effect

The effect of order of reagent mixing in the absence and in the presence of catalase on the transient kinetics of indole-3-acetic acid (IAA) oxidation by dioxygen catalysed by horseradish peroxidase C and anionic tobacco peroxidase at neutral pH has been studied. The data suggest that haem-containing plant peroxidases are able to catalyse the reaction in the absence of exogenous hydroperoxide. The initiation proceeds via the formation of the ternary complex enzyme-->IAA-->oxygen responsible for IAA primary radical generation. The horseradish peroxidase-catalysed reaction is independent of catalase indicating a significant contribution of free radical processes into the overall mechanism. This is in contrast to the tobacco peroxidase-catalysed reaction where the peroxidase cycle plays an important role. The transient kinetics of IAA oxidation catalysed by tobacco peroxidase exhibits a biphasic character with the first phase affected by catalase. The first phase is therefore associated with the common peroxidase cycle while the second is ascribed to native enzyme interaction with skatole peroxy radicals yielding directly Compound II.     

解淀粉芽胞杆菌HZ-12中ysnE参与吲哚-3-乙酸合成的代谢途径研究

【目的】吲哚-3-乙酸是调控植物生长发育和生理活动的重要激素,吲哚-3-乙酸N-乙酰转移酶YsnE在吲哚-3-乙酸合成中发挥重要作用,本研究拟解析解淀粉芽胞杆菌中YsnE参与吲哚-3-乙酸合成的代谢途径。【方法】通过基因ysnE缺失和强化表达,分析ysnE对吲哚-3-乙酸合成影响,结合吲哚-3-乙酸合成中间物(吲哚丙酮酸、吲哚乙酰胺、色胺和吲哚乙腈)添加和体外酶转化实验,解析ysnE参与吲哚-3-乙酸合成的代谢途径。【结果】明确了YsnE在解淀粉芽胞杆菌HZ-12吲哚-3-乙酸合成中发挥重要作用。发现ysnE缺失菌株中的吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈利用显著降低,揭示了YsnE主要发挥吲哚丙酮酸脱羧酶YclB和吲哚乙酰胺水解酶/腈水解酶/腈水合酶YhcX的功能,并通过参与吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈途径来影响吲哚-3-乙酸合成。【结论】初步揭示了YsnE通过影响吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈途径参与吲哚-3-乙酸合成的代谢机理,为吲哚-3-乙酸合成途径解析和代谢工程育种构建吲哚-3-乙酸高产菌株奠定了基础。     

Oxidation of Indole-3-Acetic Acid to Oxindole-3-Acetic Acid by an Enzyme Preparation from Zea mays

Indole-3-acetic acid is oxidized to oxindole-3-acetic acid by Zea mays tissue extracts. Shoot, root, and endosperm tissues have enzyme activities of 1 to 10 picomoles per hour per milligram protein. The enzyme is heat labile, is soluble, and requires oxygen for activity. Cofactors of mixed function oxygenase, peroxidase, and intermolecular dioxygenase are not stimulatory to enzymic activity. A heat-stable, detergent-extractable component from corn enhances enzyme activity 6- to 10-fold. This is the first demonstration of the in vitro enzymic oxidation of indole-3-acetic acid to oxindole-3-acetic acid in higher plants.     

Promotion of peroxidase activity in the cell wall of Nicotiana

Peroxidase catalyzes the oxidation of indole-3-acetic acid. The primary products of this reaction stimulate growth in plants. Therefore, our concept is that an increase in peroxidase activity will increase the effect of indole-3-acetic acid as a growth hormone. Our objective was to study the effect of 2,3,5-triiodobenzoic acid, a growth regulator, on isoperoxidases in the cell wall and cytoplasm of Nicotiana. Isoperoxidases from the cell wall and cytoplasmic fractions were separated by acrylamide gel electrophoresis. We found that 2,3,5-triiodobenzoic acid and indole-3-acetic acid increase peroxidase activity in the cell wall. Since both 2,3,5-triiodobenzoic acid and indole-3-acetic acid increase the activity of the same isoperoxidase, we conclude that 2,3,5-triiodobenzoic acid synergizes rather than antagonizes auxin action, and we suggest that this increase in indole-3-acetic acid oxidase activity sensitizes plant tissues to auxin.     

Role of Auxin in Induction of Polarity in Fucus vesiculosus Zygotes

We studied the effects of auxin (indole-3-acetic acid) on formation of the primary polarity axis in zygotes of the brown algae Fucus vesiculosusL. Within the first 2.5 h after fertilization, the zygotes release this phytohormone in the ambient medium. The treatment of developing zygotes with the inhibitor of indole-3-acetic acid transport from the cell 2,3,5-triiodobenzoic acid at 5 mg/l arrests the auxin secretion and leads to its accumulation in the cells. This causes a significant delay in zygote polarization. The treatment of zygotes with the exogenous indole-3-acetic acid at 1 mg/l stimulates cell polarization and formation of a rhizoid protuberance. When auxin was added to the medium with triiodobenzoic acid, the inhibitory effect of the latter was eliminated. It has been proposed that the content of indole-3-acetic acid in the ambient medium is a key factor in the induction of polarity of the F. vesiculosus zygotes.     

An electron spin resonance study of free radical intermediates in the oxidation of indole acetic acid by horseradish peroxidase

The oxidation of indole-3-acetic acid by horseradish peroxidase was studied using the spin traps t-nitrosobutane and 5,5-dimethyl-1-pyrroline N-oxide to trap free radical intermediates. The major free radical metabolite of indole acetic acid was unambiguously determined by the use of indole-3-[2,2-2H2]acetic acid to be the skatole carbon-centered free radical. In the presence of oxygen, superoxide was also trapped.     

Activation of indole-3-acetic acid oxidase from horseradish and Prunus by phenols and H2O2

The enzyme-catalysed oxidation of indole-3-acetic acid (IAA) was sytematically investigated with respect to enzyme source and cofactor influence using differential spectrophotometry and oxygen uptake measurement. Commercially-available horseradish peroxidase (HRP) and a peroxidase preparation from Prunus phloem showed identical catalytic properties in degrading IAA. There was no lag phase of IAA oxidation with any of the reaction mixtures tested. Monophenols exhibited a much stronger stimulatory effect than inorganic cofactors, but during the incubation of IAA the phenols were also gradually oxidised. Hydrogen peroxide (H₂O₂) in combination with monophenols accelerated peroxidation of the monophenol and IAA oxidation simutaneously. Since photometric determination of IAA was affected by oxidation products of dichlorophenol or phenol contamination of the enzyme preparation used, the standard IAA absorption measurements appear to be susceptible to methodological errors. Under certain incubation conditions a catalase-like activity of HRP during the course of IAA oxidation was noted and substrate inhibition was observed above 1.5 × 10^\s-4 M IAA. Some concepts concerning the mode of activation of the enzyme-catalysed IAA oxidation are deduced from the experimental results.     

Oxidation of indole-3-acetic acid by peroxidase: involvement of reduced peroxidase and compound III with superoxide as a product

Kinetic and spectral data establish that peroxidase may oxidize indole-3-acetic acid by either of two pathways depending on the enzyme/substrate ratio. When relatively low enzyme/substrate ratios are employed, the oxidation proceeds through a reduced peroxidase in equilibrium compound III shuttle. Conversely, peroxidase operates through the conventionally accepted pathway involving native enzyme and compounds I and II only when high enzyme/substrate ratios are used. Compound III, a specific oxidase, constitutes the dominant steady-state form of peroxidase when the reduced peroxidase in equilibrium compound III shuttle is operational. Activation of this shuttle also produces a flux of superoxide anion radical at the expense of molecular oxygen. Thus, important biological consequences may follow activation of this shuttle under physiological conditions.