Studies on the destruction of indole-3-acetic acid by a species of Arthrobacter IV. Decomposition products

Products of IAA decomposition by IAA destroying enzyme-inducedand non-induced cells of Arthrobacter sp. were examined. Catecholand pyrogallol, and 3-methyldioxindole were respectively identifiedfor non-induced and induced cells. Exogenous catechol was readilyoxidized by induced cells, but was not oxidized by non-inducedcells. Exogenous pyrogallol, 3-methyldioxindole, indole-3-aldehyde,skatole, 2,3-dihydroxindole, 3-methyleneoxindole, o-aminoacetophenone,3-hydroxyanthranilic acid, anthranilic acid and salicylic acidwere oxidized by neither induced nor non-induced cells. (Received June 16, 1969; )     

Studies on the destruction of indole-3-acetic acid by a species of Arthrobacter I. On the induction of the enzyme responsible for the destruction

1. The induction of an IAA-destroying enzyme in Arthrobacter sp.that can utilize IAA as its sole source of carbon and nitrogenwas investigated.
2. 1. The enzyme was most effectively inducedby 10–³ to2x10–³ M IAA, at pH 6.5.
3. 2. All testedIAA analogs were unable to induce the enzyme.Analogs otherthan indole-3-lactic acid were rather inhibitoryon the inductionwith IAA.
4. 3. The induction period was shortened with the ageof culturein both polypeptone and acetate media.
5. 4. Pretreatmentof the bacterium with IAA caused a shorteningof the inductionperiod.
6. 5. The induction was inhibited by various antibiotics,aminoacid analogs and nucleobase analogs.
7. 6. The inductionwas less remarkable in actively proliferatingcells than itwas in slowly proliferating ones.

(Received July 1, 1967; )     

Studies on the oxidation of indole-3-acetic Acid by peroxidase enzymes. I. Colorimetric determination of indole-3-acetic Acid oxidation products

The method described here is based on a brief report by Harley-Mason and Archer. It involves the use of p-dimethylaminocinnamaldehyde (DMACA), a vinylogue of Ehrlich's reagent, as a color reagent for indoles. Colorimetric analyses of indoleacetic acid (IAA) oxidation reaction mixtures were made with the DMACA reagent as a solution rather than a spray. DMACA reagent will yield a wine-red color with IAA oxidation products in solution. Under similar conditions DMACA reacts with authentic IAA to yield only slight coloration at best. In comparison with other indoles, DMACA is more relative with IAA oxidation reaction products than either Salkowski or Ehrlich's reagents. Data discussed support a concept that the color produced with DMACA is due to the presence of tautomeric oxidation product(s) of IAA.     

Horseradish peroxidase-catalyzed aerobic oxidation of indole-3-acetic acid. II. Oxygen uptake and chemiexcitation.

Light emission from the horseradish peroxidase-catalyzed aerobic or anaerobic oxidation of indole-3-acetic acid has been investigated under opposite extreme conditions of enzyme/substrate ratio. The O2-dependent chemiluminescent processes represent a minor part of the total oxygen consumption. Superoxide is involved in chemiexcitation as is evident from the observed inhibitory effect of superoxide dismutase. At high enzyme/substrate ratio, only a part of the emission is dependent on superoxide ion; at low ratio the dependence is extensive. At high ratio, some of the emission is independent of superoxide and O2. The identical quenching effects of D- and L-tryptophan are consistent with the formation of the quenching species only in bulk solution. The similarity of the emission spectra under extreme conditions indicates that the same main emitters are formed. This is also supported by the effect of quenchers. Possibly some of the emitters originate in the oxidative cleavage of the 2,3-double bond of the indole ring.     

Gibberellin-enhanced indole-3-acetic acid biosynthesis: D-Tryptophan as the precursor of indole-3-acetic acid

Stem segments excised from light-grown Pisum sativum L. (cv. Little Marvel) plants elongated in the presence of indole-3-acetic acid and its precursors, except for L-tryptophan, which required the addition of gibberellin A, for induction of growth. Segment elongation was promoted by D-tryptophan without a requirement for gibberellin, and growth in the presence of both D-tryptophan and L-tryptophan with gibberellin A3, was inhibited by the D-aminotransferase inhibitor D-cycloserine. Tryp-tophan racemase activity was detected in apices and promoted conversion of L-tryptophan to the D isomer; this activity was enhanced by gibberellin A3. When applied to apices of intact untreated plants, radiolabeled D-tryptophan was converted to indole-3-acetic acid and indoleacetylaspartic acid much more readily than L-tryptophan. Treatment of plants with gibberellin A3, 3 days prior to application of labeled tryptophan increased conversion of L-tryptophan to the free auxin and its conjugate by more than 3-fold, and led to labeling of N-malonyl-D-tryptophan. It is proposed that gibberellin increases the biosynthesis of indole-3-acetic acid by regulating the conversion of L-tryptophan to D-tryptophan, which is then converted to the auxin.     

Destruction of indole-3-acetic acid during the aerobic oxidation of sulfite

Indole-3-acetic acid (IAA) was rapidly destroyed in the presence of Mn²⁺, oxygen and sulfite ion. The optimal pH for the reaction was between 5 and 6. The destruction was dependent on the aerobic oxidation of sulfite, but was not inhibited by superoxide dismutase. Tracer studies indicate that IAA was converted into at least 3 compounds. Decarboxylation of IAA was not involved in the destruction.     

New phenolic inhibitors of the peroxidse-catalysed oxidation of indole-3-acetic acid

Previously we reported two metabolites of the insecticide carbofuran as persistent inhibitors of the peroxidase-catalysed oxidtion ofindole-3-acetic acid. In searching for more active inhibitors of this type, we have found that 5-hydroxy-2,2-dimethylchromene (β-tubanol), 2′,6′-dihydroxycetophenone oxime, 5-hydroxy-2,2-dimethylchroman, 2′,6′-dihydroxyacetophenone and 2,6-dihydroxybenzoic acid methyl ester were more active than the carbofuran metabolite 7-hydroxy-2,2-dimethyl-3-oxo-2,3-dihydrobenzofuran. Resorcinol, 5-hydroxy-2,2-dimethylchroman-4-one, 3-hydroxy-5-methoxy-2,2-dimethylchroman-4-one and 5-hydroxy-2-methylchrom-4-one were also inhibitory but with less activity. The new inhibitors differed from the well-known phenolic inhibitors such as caffeic acid in inhibition kinetics as demonstrated by the rate of disappearance of indole-3-acetic acid, the rate of formation of the oxidation products, and the transient spectral change in the enzyme.     

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.     

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.     

Indole-3-acetic acid (IAA) production by Arthrobacter species isolated from Azolla.

Arthrobacter species, isolated from the leaf cavities and the microsporocarps of the aquatic fern species Azolla pinnata and Azolla filiculoides, produced indole-3-acetic acid (IAA) in culture when the precursor tryptophan was added to the medium. No IAA production was detected in the absence of tryptophan. Maximum IAA formation was obtained in the first 2 d of incubation. Part of the tryptophan was transformed to N alpha-acetyl-L-tryptophan.     

Inhibition of enzymic oxidation of indole-3-acetic acid by metabolites of the insecticide carbofuran

Metabolites of carbofuran, a carbamate insecticide, inhibit the enzymic oxidation of indole-3-acetic acid. The metabolites differ in stability and effectiveness. 2,2-Dimethyl-7-hydroxy-2,3-dihydrobenzofuran represents one type which is broken down in the IAA oxidation reaction; thus the induced inhibition is limited by depletion of the the inhibitor. 2,2-Dimethyl-3-keto-7-hydroxy-2,3-dihydrobenzofuran represents the other type which is stable in the reaction; thus the inhibition is persistent. With both types of inhibitors the inhibition is reversible by higher substrate concentrations, but the Lineweaver-Burk plot is curvilinear suggesting the complex nature of competitive inhibition.     

Regulation of enzymic oxidation of indole-3-acetic acid by phenols: Structure-activity relationships

Mono- and diphenols were tested for their effects on the decarboxylation of [1-¹⁴C]IAA catalysed by purified horseradish peroxidase (EC 1.11.1.7) in the presence or absence of 2,4-dichlorophenol (DCP). The number of hydroxyl groups and their position relative to each other and the nature and position of other substituents on the aromatic ring were found to affect the activity. Although the effects were complex, the following generalizations may be made. (1) Monophenols produce activation when no other cofactor is present. p-Substituted monophenols are more active than o- or m-compounds. In the presence of DCP, the activity varies from slight activation to strong inhibition. (2) m-Diphenols also produce activation in the absence of other cofactors while o- and p-diphenols, with the exception of 3,4-dihydroxyacetophenone and 3,4-dihydroxypropiophenone, produce strong inhibition in the presence or absence of DCP. The o-diphenolsare degraded in the IAA-oxidizing enzyme system and thus produce only a temporary inhibition. (3) m-Diphenols and 3,4-dihydroxyacetophenone produce a sustained inhibition in the presence of DCP. (4) Substitution at position 2 significantly alters the activity of m-diphenols. (5) O-Methylation alters the activity of most o-diphenols. In the absence of DCP, o-methoxyphenols and certain other phenols such as 3,4-dihydroxyacetophenone and 2,6-dihydroxyacetophenone either promote or inhibit IAA oxidation depending on concentration.     

Measurement of the rates of oxindole-3-acetic acid turnover, and indole-3-acetic acid oxidation in Zea mays seedlings

Oxindole-3-acetic acid is the principal catabolite of indole-3-acetic acid in Zea mays seedlings. In this paper measurements of the turnover of oxindole-3-acetic acid are presented and used to calculate the rate of indole-3-acetic acid oxidation. [3H]Oxindole-3-acetic acid was applied to the endosperm of Zea mays seedlings and allowed to equilibrate for 24 h before the start of the experiment. The subsequent decrease in its specific activity was used to calculate the turnover rate. The average half-life of oxindole-3-acetic acid in the shoots was found to be 30 h while that in the kernels had an average half-life of 35h. Using previously published values of the pool sizes of oxindole-3-acetic acid in shoots and kernels from seedlings of the same age and variety, and grown under the same conditions, the rate of indole-3-acetic acid oxidation was calculated to be 1.1 pmol plant-1 h-1 in the shoots and 7.1 pmol plant-1 h-1 in the kernels.     

Excitation of indole-3-acetic acid (an auxin) in a linoleate-lipoxygenase system.

The weak luminescence that accompanies the linoleate-lipoxygenase reaction was greatly enhanced by the addition of indole analogues, and especially indole acetic acid. The main emitting species in the indole acetic acid-linoleate-lipoxygenase system was analysed spectrophotometrically in the visible region and ascribed to the transition of excited indole acetate in triplet state to its ground state. Such an excited indole acetate could be generated by transfer of energy from the excited CO2 and excited carbonyl (generated by the linoleate-lipoxygenase reaction) to indole acetate in the ground state, but not by cleavage of the dioxetane analog (positions 2 and 3 on the indole ring).     

Studies on the Destruction of Indole-3-acetic Acid by a Species of Arthrobacter. V. Indole-3-acetic Acid Production from Tryptophan

Tryptophan (Try) metabolism of Arthrobacter sp. was examined. The inducibility of the Try oxidizing enzyme system seems to be correlated with that of the indole-3-acetic acid (IAA) oxidizing enzyme system. Try is metabolized to IAA via indole-3-pyruvic acid (Ip) and indole-3-acetaldehyde (IAAId). Indole-3-acetamide (IAm) is formed as a product of Try oxidation. Exogenous IAm, indole-3-acetonitrile (IAN) and tryptamine are not oxidized by Try-induced cells.     

Computer simulation of damped oscillations during peroxidase-catalyzed oxidation of indole-3-acetic acid

Oscillation patterns in horseradish peroxidase (HRP)-catalyzed oxidation of indole-3-acetic acid (IAA) at neutral pH were studied using computer simulation. Under certain conditions, such as the presence of a reaction promoter and continuous intake of oxygen from the gaseous phase, the simulated system exhibits damped oscillations of the concentrations of oxygen in the aqueous phase, [O(2)](aq), and of all the reaction intermediates. The critical concentration of oxygen in aqueous phase, [O(2)](cr)(aq), was used to describe the nature of the oscillations. The critical concentration is the concentration at which the system abruptly changes its properties. If [O(2)](aq) is higher than [O(2)](cr)(aq) then the reaction develops as an avalanche, otherwise, the reaction stops. The nature of oscillations is accounted for by the interaction of two processes: the consumption/accumulation of oxygen and the accumulation/consumption of reaction intermediates. Oscillations are always damped. Neither HRP or umbelliferone (Umb) deactivation nor IAA consumption can account for the damping. The nature of the damping is determined by the termination reactions of free radical intermediates and ROOH. The three major parameters of oscillations: period of oscillations, initial amplitude of oscillations and the rate of damping were studied as functions of: (i) oxygen concentration in the gaseous phase, (ii) initial oxygen concentration in aqueous phase, (iii) the concentration of IAA and (iv) the initial concentration of HRP.