Indole-3-pyruvic Acid Research Articles

Role of Pseudomonas putida indoleacetic acid in development of the host plant root system.

Many plant-associated bacteria synthesize the phytohormone indoleacetic acid (IAA). While IAA produced by phytopathogenic bacteria, mainly by the indoleacetamide pathway, has been implicated in the induction of plant tumors, it is not clear whether IAA synthesized by beneficial bacteria, usually via the indolepyruvic acid pathway, is involved in plant growth promotion. To determine whether bacterial IAA enhances root development in host plants, the ipdc gene that encodes indolepyruvate decarboxylase, a key enzyme in the indolepyruvic acid pathway, was isolated from the plant growth-promoting bacterium Pseudomonas putida GR12-2 and an IAA-deficient mutant constructed by insertional mutagenesis. The canola seedling primary roots from seeds treated with wild-type P. putida GR12-2 were on average 35 to 50% longer than the roots from seeds treated with the IAA-deficient mutant and the roots from uninoculated seeds. In addition, exposing mung bean cuttings to high levels of IAA by soaking them in a suspension of the wild-type strain stimulated the formation of many, very small, adventitious roots. Formation of fewer roots was stimulated by treatment with the IAA-deficient mutant. These results suggest that bacterial IAA plays a major role in the development of the host plant root system.

A Novel Enzyme,L-Tryptophan Oxidase, from a Basidiomycete,Coprinussp. SF-1: Purification and Characterization

A basidiomycete, Coprinus sp. SF-1, was found to produce an L-Trp-oxidizing enzyme by screening from the culture collection of our laboratory. After solubilization by 1 M NaSCN from the particulate fraction of disrupted cells of the strain, the enzyme was purified about 76-fold to essential homogeneity. The enzyme had a molecular mass of about 420 kDa and the subunit molecular mass was 68 kDa. The enzyme contained 1 mol of non-covalently bound FAD per mol of the subunit. It catalyzed the simultaneous reactions of oxidative deamination and oxygenative decarboxylation of L-Trp to form indolepyruvic acid and indole-3-acetamide, the former of which was further oxidized to indole-3-acetic acid. The molar ratio of the respective reaction products was about 9:1. The enzyme specifically oxidized L-Trp, and slightly acted on L-Phe and L-Tyr. The Km for L-Trp was about 0.5 mM in both oxidase and oxygenase reactions. Thus, the enzyme is a novel one and was tentatively designated "L-Trp oxidase (deaminating and decarboxylating)". The optimum pHs of oxidase and oxygenase activities were 7.0 and 9.0, respectively. The optimum temperatures of both activities were 50 degrees C. The enzyme was stable at pH 6.0-10.5 and below 50 degrees C, and at 4 degrees C for 1 year.

Tryptophan biosynthesis and production of other related compounds from indole and L-serine by mixed ruminal bacteria, protozoa, and their mixture in vitro.

Tryptophan (Trp) biosynthesis and production of other related compounds from 1 mM each of indole (IND), L-serine (Ser), and IND plus Ser by mixed ruminal bacteria (B), protozoa (P), and their mixture (BP) in an in vitro system were quantitatively investigated. Ruminal microorganisms were anaerobically incubated at 39 degrees C for 12 h. Trp and other related compounds produced in both the supernatants and microbial hydrolyzates of the incubation were analyzed by HPLC. B, P, and BP suspensions were not able to produce Trp when incubated with only IND or Ser. Appreciable amounts of Trp (9.8, 3.1, and 6.6% of substrate) were produced from IND plus Ser after 12 h by B, P, and BP suspensions, respectively. Trp produced from IND + Ser in B was found only in the hydrolyzate, whereas it was found in the medium as a free form in P after a 12-h incubation period. Rumen bacteria and protozoa were separately demonstrated for the first time to produce Trp from IND plus Ser, and the ability of P to produce Trp from IND plus Ser was about one-third that of B in 12 h. Trp produced from IND plus Ser by B, P, and BP suspensions was simultaneously degraded into its related compounds, and, among them, indoleacetic acid (IAA) was a major product found in B. Production of IAA was 4.3, 0.3, and 3.2% of IND in 12 h by B, P, and BP suspensions, respectively. A small amount of skatole (SKT) (1.1 and 2.5% in B and BP, respectively) and p-cresol (CRL) (2.4 and 3.4% in B and BP, respectively) were also produced from IND plus Ser during 12-h incubation. P suspension produced no SKT or CRL from IND plus Ser in 12-h incubation. These results suggested for the first time that both rumen bacteria and protozoa have an ability to produce Trp from IND plus Ser, and the ability was higher in B than in P. The ratios of Trp produced from IND plus Ser to that from indolepyruvic acid by B, P, and BP were 1:3.4, 1:14.2, and 1:6.6 during 12-h incubation period. From these results, the degree of importance of producing Trp from IND plus Ser in the rumen was indicated.

Biosynthesis of indole-3-acetic acid in Azospirillum brasilense. Insights from quantum chemistry.

Quantum chemical methods AM1 and PM3 and chromatographic methods were used to qualitatively characterize pathways of bacterial production of indole-3-acetic acid (IAA). The standard free energy changes (delta G(o)'sum) for the synthesis of tryptophan (Trp) from chorismic acid via anthranilic acid and indole were calculated, as were those for several possible pathways for the synthesis of IAA from Trp, namely via indole-3-acetamide (IAM), indole-3-pyruvic acid (IPyA), and indole-3-acetonitrile (IAN). The delta G(o)'sum for Trp synthesis from chorismic acid was -402 (-434) kJ.mol-1 (values in parentheses were calculated by PM3). The delta G(o)'sum for IAA synthesis from Trp were -565 (-548) kJ.mol-1 for the IAN pathway, -481 (-506) kJ.mol-1 for the IAM pathway, and -289 (-306) kJ.mol-1 for the IPyA pathway. By HPLC analysis, the possibility was assessed that indole, anthranilic acid, and Trp might be utilized as precursors for IAA synthesis by Azospirillum brasilense strain Sp 245. The results indicate that there is a high motive force for Trp synthesis from chorismic acid and for IAA synthesis from Trp, and make it unlikely that anthranilic acid and indole act as the precursors to IAA in a Trp-independent pathway.

Tryptophan biosynthesis and production of other related compounds from indolepyruvic acid by mixed ruminal bacteria, protozoa, and their mixture in vitro.

Tryptophan (Trp) biosynthesis and the production of other related compounds by mixed ruminal bacteria (B), protozoa (P), and their mixture (BP) in an in vitro system were quantitatively investigated by using 1 mM of indole-3-pyruvic acid (IPA) as substrate. Ruminal microorganisms were anaerobically incubated at 39 degrees C for 12 h. Trp and other related compounds in both the supernatants and the microbial hydrolyzates of the incubation were analyzed by HPLC. As a whole, about 334, 440, and 436 &mgr;M of Trp were produced from IPA in 12 h by B, P, and BP suspensions, respectively. In the B suspension, a greater portion of synthesized Trp (242 &mgr;M) from IPA was accumulated as free form in the medium, whereas a large amount of Trp (92 &mgr;M) was incorporated into cell protein in a 12-h incubation. On the other hand, in the P suspension, a large amount of Trp (475 &mgr;M) from IPA was also found as free form in the supernatant in a 12-h incubation. Protozoa did not incorporate Trp into cell protein, but they liberated endogenous Trp (34 &mgr;M) into the medium. The net productions of Trp from IPA were 344.3 and 447.7 &mgr;mol/g of microbial nitrogen in 12 h by B and P suspensions, respectively. The ability of P to synthesize Trp from IPA was about 30% higher than that of B in 12 h. Trp produced from IPA by B, P, and BP suspensions were simultaneously degraded into its related compounds, and among them, indoleacetic acid (IAA) was a major product found in all microbial suspensions. Productions of IAA were 124, 25, and 99 &mgr;M from IPA in 12 h by B, P, and BP suspensions, respectively. The formation of indolelactic acid (ILA) from IPA was observed for the first time in all microbial suspensions, and it was about 84, 24, and 54 &mgr;M in 12 h by B, P, and BP, respectively. Higher IAA and ILA productions were observed in B when compared with P. A small amount of skatole (SKT) (26 &mgr;M) was produced from IPA in B, whereas a sizable amount of SKT (38 &mgr;M) was found in BP after a 12-h incubation. p-Cresol (CRL) was also produced from IPA by both B (43 &mgr;M) and BP (65 &mgr;M) suspensions in 12 h, and this is also the first discovery to show the formation of CRL from IPA by B and BP suspensions. BP suspension was more active to produce both SKT and CRL from IPA, though P suspension has no ability to produce either SKT or CRL from IPA during a 12-h incubation.

Determination of indole-3-pyruvic acid levels in Arabidopsis thaliana by gas chromatography–selected ion monitoring-mass spectrometry

A rapid and simple method is described for the determination of indole-3-pyruvic acid (IPA) levels in Arabidopsis thaliana by gas chromatography–selected ion monitoring-mass spectrometry (GC–SIM-MS). The method includes derivatization of IPA with hydroxylamine in the crude extract, followed by ethyl acetate partitioning, solid-phase extraction with C 18 resin, reversed-phase high-performance liquid chromatography (HPLC), and GC–SIM-MS. Three derivatizing reagents were tested; these were pentafluorobenzylhydroxylamine, pentafluorophenylhydrazine, and hydroxylamine. Hydroxylamine proved to be the most useful, as the IPA-oxime was easiest to purify from plant extracts and was the most stable. IPA was quantified in Arabidopsis seedlings ranging in age from 5 to 12 days; levels varied from 4 to 13 ng/g, peaking at 7–9 days.

インドールピルビン酸から生成される未同定化合物(X1)とトリプトファンの高速液体クロマトグラフィーによる分離

Indole-3-pyruvic acid (IPA) is an unstable compound and some unidentified compounds (X1-X4) were naturally produced from IPA immediately after dissolving in 25% ethanol or after incubation in MB9 buffer solution at 39°C. Unfortunately, one of the unidentified compounds (X1) overlapped with the peak of tryptophan (Trp), when analyzed by the previously reported method for analysis of Trp and its related compounds by high-performance liquid chromatography (HPLC). In the present study, a rapid method for the separation of Trp from X1 was established using HPLC. LiChrospher 100 RP-18 column and methanol-50mM sodium acetate buffer (pH 4.2) (20:80, v/v) as a mobile phase for isocratic elution were used for the analysis. The compounds were monitored at 280nm with a UV absorbance detect. When a 5μl portion of deproteinized and filtered mixed sample of Trp and IPA was injected onto the HPLC system, the peaks of Trp and X1 were separated within 7min. The recovery of Trp was more than 96% and the minimum detectable limit was 40pmol.

Aromatic amino acid catabolism by lactococci

Summary - While catabolism of amino acids is believed to play an important role in cheese f1avor development, the pathways present in cheese microflora are poorly understood. To determine the pathways of aromatic amino acid catabolism in lactococci and effects of Cheddar cheese ripening conditions on catabolic enzymes and products, eight starter lactococcal strains were screened. Cell-free extracts prepared from these strains were found to contain an œ-ketoglutarate-dependent aminotransferase activity with tryptophan, tyrosine and phenylalanine. Tryptophan, tyrosine and phenylalanine aminotransferase specifie activities (Ilmol product formed/mg proteinlmin) ranged from 0.30 to 2.8 10-3, 0.93 to 7.3 10-3 and 1.5 to 7.2 10-3, respectively. Metabolites produced from tryptophan by a cellfree extract of Lactococcus lactis S3 were indolepyruvic acid, indoleacetic acid and indole-3-aldehyde. Indoleacetic acid and indole-3-aldehyde can form spontaneously from indolepyruvic acid under the conditions employed. A defined medium was used to determine whether the aminotransferase(s) was expressed and which metabolite(s) accumulate under conditions that simulated those of ripening Cheddar cheese in terms of pH, salt, temperature and carbohydrate starvation. The results indicated that the aminotransferase(s) was expressed and stable under these conditions. The tryptophan metabolites that accumulated were determined to be strain-specific. lactococcus / aminotransferase / aromatic amino acid / catabolism / cheese flavor

Indolepyruvate Pathway for Indole-3-Acetic Acid Biosynthesis in Bradyrhizobium elkanii

Indole-3-acetaldehyde (IAAId) was detected in the culture supernatant of Bradyrhizobium elkanii. Deuteriumlabelled L-tryptophan (Trp) was incorporated into IAAId and indole-3-acetic acid (IAA), suggesting that B. elkanii produces IAA via IAAId from Trp. In B. elkanii cell suspension, indole-3-pyruvic acid (IPyA) was converted to IAAId, and exogenously added IAAId was rapidly converted to IAA. Furthermore, the activity of indolepyruvat e decarboxylase (IPDC), which catalyzes the decarboxylation of IPyA to produce IAAId and is a key enzyme for IPyA pathway, was detected in B. elkanii cell-free extract. The IPDC activity depended on Mg 2+ and thiamine pyrophosphate, cofactors of decarboxylati on. This mounting evidence strongly suggests that IAA synthesis occurs via IPyA pathway (Trp -»• IPyA -*• IAAId -*• IAA) in B. elkanii.

Characterization of two new matrices for matrix-assisted laser desorption/ionization mass spectrometry.

Three structurally related compounds, 4-hydroxy-33-methoxyphenylpyruvic acid (HMPPA), indole-3 pyruvic acid (IPA), and indole-3-glyoxylic acid have been evaluated as matrix-assisted laser desorption/ionization (MALDI) matrices. HMPPA and IPA were found to be effective matrices for MALDI-MS analysis of proteins and peptides and have been characterized in this work. HMPPA was found to be a particularly good matrix for the analysis of proteins and peptides because of its tolerance towards impurities and the resulting sensitivity in MALDI-MS experiments. Analysis of model proteins and peptides, including those in biological fluid, are demonstrated.

A critical examination of the specificity of the salkowski reagent for indolic compounds produced by phytopathogenic bacteria

We examined the sensitivity and the specificity of three versions of the Salkowski colorimetric technique. Two of these allowed the detection of indoleacetic acid (IAA) over a low range of concentrations (0.5 to 20 (mu)g/ml), while the third permitted the detection of IAA over a range of higher concentrations (5 to 200 (mu)g/ml). Overall, the three formulations reacted not only with auxin (IAA) but also with indolepyruvic acid and indoleacetamide. Therefore, these techniques appear to be specific for IAA, indolepyruvic acid, and indoleacetamide rather than for IAA alone.

Review: Genetics of theAzospirillum-Plant Root Association

Abstract Recent advances in four aspects of the Azospririllum-plant root association, namely, the biosynthesis of phytohormones, nitrogen fixation, the mechanisms of plant root attachment, and genetic analysis of Azospirillum brasilense megaplasmids, are presented. At least three biosynthetic pathways are involved in indole-3-acetic acid (IAA) production in A. brasilense, that is, two Trp-dependent pathways identified as the indole-3-acetamide (LAM) pathway, the indole-3-pyruvic acid (IPyA) pathway, and a third as-yet unidentified pathway not using Trp as a precursor. So far, Azospirillum is the only bacterium for which a non-Trp-dependent route for IAA biosynthesis has been reported. The gene encoding the key enzyme in the IPyA pathway, indole-3-pyruvate decarboxylase, has been characterized recently. Azospirillum fixes atmospheric dinitrogen only at low oxygen tensions in media devoid of a combined nitrogen source. Both the synthesis and the activity of the nitrogenase enzyme are regulated according to ...

Anxiolytic Effect of Indole-3-Pyruvic Acid (IPA) in Mice

In an elevated plus-maze indole-3-pyruvic acid (IPA, 100-200mg/kg), an endogenous metabolite of tryptophan, possesses in mice an activity typical of anxiolytics. IPA increased the ratio of the number of entries into open arms over the total number of entries into open and closed arms and the time spent in open arms. Similar effect was observed for diazepam, a standard anxiolytic. Pretreatment with IPA attenuated the anxiogenic effect of caffeine (50 mg/kg) and 3-hydroxykynurenine (1.2 μg, i.c.v.) but not that of pentylenetetrazole (10 mg/kg), or phenylethylamine (5 and 10 mg/kg). Pretreatment with IPA (50-200 mg/kg) did not attenuate pentylenetetrazole- or phenylethylamine-induced seizures in contrast to diazepam which prevents both types of seizures. The data suggest that IPA is an endogenous anxiolytic with novel profile.

Indole-pyruvic acid treatment reduces damage in striatum but not in hippocampus after transient forebrain ischemia in the rat.

The effects of treatment with indole-pyruvic acid, an endogenous metabolite of tryptophan converted into kynurenic acid in the brain, were studied in rats after transient forebrain ischemia induced by the 4-vessel occlusion procedure. The histological analysis showed a significant protective effect of indole-pyruvic acid treatment on striatal ischemic lesions assessed by the extent of regional atrophy and the area of neuronal disappearance 14 days after ischemia. Striatal neurons were labelled by dopamine and adenosine 3':5' monophosphate regulated phosphoprotein-32 immunoreactivity. Conversely, increased neuronal loss, regional atrophy and glial fibrillary acidic protein immunoreactivity, an index of post-injury astroglial activation, were observed in the hippocampal formation, especially the CA3 field, of indole-pyruvic acid-treated rats when compared with vehicle-treated ischemic rats. The treatment with indole-pyruvic acid did not produce any improving effects in a test assessing short-term impairments after transient ischemia (motor test score at 24 h and 48 h post-ischemia). Furthermore, no significant effects of indole-pyruvic acid treatment were found on performance in water T-maze studied at 7 and 14 days post-ischemia. The opposite effects of indole-pyruvic acid on ischemic lesion in different brain regions may be related to its multiple neurochemical actions in the brain. The protective effect of indole-pyruvic acid on ischemic damage in striatum may be due to its conversion into kynurenic acid, a broad spectrum glutamate receptor antagonist. At hippocampal level, where glutamate receptor antagonists have been proved ineffective in the present lesion model, indole-pyruvic acid-induced changes in monamine availability may lead to a worsening of neuronal damage.

Indole-pyruvic acid, a tryptophan ketoanalogue, antagonizes the endocrine but not the behavioral effects of repeated stress in a model of depression

Increased glucocorticoid secretion is frequent in mood disorders and is normalized by long-term antidepressant therapy. Many antidepressants act by increasing central serotonin transmission. We investigated the effects of a serotonin precursor, indole-pyruvic acid (IPA), in an animal model of depression based on repeated exposure to unpredictable stress. Rats were divided in groups, and IPA (20 mg/kg), the tricyclic antidepressant imipramine (IMI) (5 mg/kg), or vehicle was administered daily during 3 weeks of repeated exposure to various stressors according to the procedure described by Katz et al [Katz RJ, Roth KA, Carroll BJ (1981): Neurosci Biobehav Rev 5:247–251]. After treatment, rats were evaluated for stress-induced exploratory behavior and killed 24 hr later. Serum corticosterone levels and glucocorticoid receptor (GR) immunoreactivity (IR) in the nuclei of neurons located in the hippocampal subregion CA1 were also measured. Rats exposed to repeated stress showed a lower exploratory behavior score ( p < 0.01), higher basal corticosterone levels ( p < 0.01), and stronger GR IR in the hippocampus ( p < 0.05) than control rats. All of these effects were antagonized by IMI treatment. IPA administration did not affect the behavioral response induced by repeated stress ( p < 0.01) but normalized serum corticosterone levels. In addition, IPA treatment produced a decrease in GR IR ( p < 0.05 versus control group) that was not modified by exposure to repeated stress. Differences in the drug effects suggest that (1) the dose of IPA used for treatment was not sufficient to produce behavior effects; (2) a side product of IPA in vivo transformation, kynurenic acid, may have affected glutamate transmission, interacting with the behavioral outcomes; (3) the serotonin precursor IPA and the uptake blocker IMI may have produced different adaptive changes in hippocampal serotonin transmission, as indicated by nuclear GR IR measurements.

Estimation of Indole Derivatives Effects in Pea Mesophyll Protoplast Suspensions

ABSTRACTThe effects of 3—Indoleacetic acid (IAA), 3—Indolepyruvic acid (IPA), 5—Hydroxytryptamine (5—HT), 5,6—Dihydroxytryptamine (5,6—DHT) added at concentrations 10−7M to pea mesophyll protoplast suspensions were studied. It was demonstrated that IAA and 5,6-DHT decreased the surface potential of Pisum sativum cv. Ran 1 protoplasts. The ability of protoplasts to aggregate was not influenced significantly by treatment with the indole derivatives. Aggregation was not changed even if Ca2+ ions were added to the washing solutions. Pyrene test measurements of membrane fluidity of treated and untreated protoplasts did not show significant changes evoked by the indole derivatives. The experiments carried out with (14C) labelled IAA indicated that the great amount of IAA does not penetrate into the protoplasts. Studying protoplast viability after fusion procedure we found out that the number of dead protoplasts was increased by IPA and 5—HT treatment. Our results indicate that although indole derivatives tested...

P-392 - Effects of indolepyruvic acid, a keto-analogue of tryptophan, on indole metabolism in the pineal gland and brain of the rat

P-392 - Effects of indolepyruvic acid, a keto-analogue of tryptophan, on indole metabolism in the pineal gland and brain of the rat

Indole-3-Pyruvic Acid as a Possible Hypnotic Agent in Insomniac Subjects

In a single-blind study six male patients (mean age 39.5 years) with moderate insomnia were treated with placebo for three nights, 100 mg indole-3-pyruvic acid (IPA) for three nights, 200 mg IPA for three nights, 100 mg IPA for two nights and placebo for two nights. Polygraphic recordings were made and total sleep time, sleep efficiency, sleep latency, slow wave sleep latency, rapid eye movement (REM) sleep latency, number of arousals (greater than 1 min), percentage and duration of wakefulness after sleep onset, percentage and duration of wakefulness after sleep onset, percentage and duration of sleep stages 1, 2, 3, 4 and REM were recorded. At the end of 13 days, total sleep time, duration of stage 2 sleep and total non-REM were significantly increased when compared with baseline. Total sleep time and duration of stage 2 and total non-REM sleep on completion were significantly decreased when compared with after 200 mg IPA (night 9). Results suggest an action of IPA on human sleep similar to that of exogenous melatonin and L-tryptophan, thus confirming that IPA could be used to increase serotonin and melatonin turnover.

Proposed Enzymes of Auxin Biosynthesis and Their Regulation II. Tryptophan Dehydrogenase Activity in Plants.

In pea, maize and tomato plants a hitherto undescribed L-tryptophan dehydrogenase activity (TDH) has been detected. This enzyme catalyzes the reversible formation of indolepyruvic acid (IPyA) from L-tryptophan (L-trp). TDH and L-glutamate dehydrogenase (GDH), related enzymes in their mode of action, could be separated by gel chromatography. Enzymatic activity of TDH was sustained by both pyridine coenzymes NAD/NADP. With pea TDH the coenzyme NAD displays, at optimum pH 8.5 and at room temperature, only about 40-70 % of the activity of NADP. The amination of IPyA is catalysed more actively than the deamination of L-trp. L-trp/IPyA, L-glu/ketoglutarate, L-ala/pyruvate reacted as dehydrogenase substrates; L-phe/ phenylpyruvate, D-trp and D-phe did not react with pea enzyme extracts. A considerable similarity between the active centres of TDH and GDH has been found using inhibitors: absence of heavy metals, presence of a carbonyl group, indispensibility of bivalent ions for the enzyme activity. Pea TDH and GDH were distinctly inhibited by sodium azide. For the activity of TDH the presence of SH groups is less important than for GDH. The TDH activity in the investigated plants was lower than the GDH activity. The possible role of TDH in the regulation of the IPyA pool is discussed.Doc. RNDr. PhMr. M. Kutacek died on 28 November, 1989. The final form for print was prepared by dr. Ivana Machdckovd of the same Institute, who will also answer the reprint requests. Received June 6, 1990; accepted October 10, 1990

Molecular cloning of the gene for indolepyruvate decarboxylase from Enterobacter cloacae

Although indole-3-acetic acid (IAA) is a well-known plant hormone, the main IAA biosynthetic pathway from L-tryptophan (Trp) via indole-3-pyruvic acid (IPyA) has yet to be elucidated. Previous studies have suggested that IAA is produced by Enterobacter cloacae isolated from the rhizosphere of cucumbers and its biosynthetic pathway may possibly be the same as that in plants. To elucidate this pathway, the IAA biosynthetic gene was isolated from a genomic library of E. cloacae by assaying for the ability to convert Trp to IAA. DNA sequence analysis showed that this gene codes for only one enzyme and its predicted protein sequence has extensive homology with pyruvate decarboxylase in yeast and Zymomonas mobilis. Cell-free extracts prepared from Escherichia coli harboring this gene could convert IPyA to indole-3-acetaldehyde (IAAld). These results clearly show that this pathway is mediated only by indolepyruvate decarboxylase, which catalyzes the conversion of IPyA to IAAld.