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.     

Comparative investigations of IAA metabolism in suspension cultures and plant organs fromBeta vulgaris (sugar beet) andChenopodium album L. (common lamb’s quarters)

Exogenoualy applicated indol-3-ylaeetic acid (IAA) is metabolized mainly to IAA aspartate in intact plants and plant segments and to IAA glucose in suspension cultures fromBeta vulgaris andChenopodium album. Main metabolic product of D-tryptophan is N-malonyltryptophan in both suspension cultures and hypocotyl segments of both species. The turnover rate of L-tryptophan to IAA is comparatively low (0.1 %); inBeta the turnover rate is higher than inChenopodium. In sugar beets phenmedipham leads to a decrease in the IAA. biosynthesis rate in suspension cultures of both plant species. There is, however, an increase in the IAA content in all intact plants. The metabolic activity is substantially higher in suspension cultures than in intact plants and plant segments.     

Lazy rice gotten up with tryptophan

In culture of seedlings of the lazy strain of rice “Lazy Kamenoo”, the lazy character, i.e., a-geotropism, begins to appear when the coleoptile reaches about 3 cm in length. This character is diminished and the lazy coleoptile gets up upon exogenously applied IAA or tryptophan. The effect is much more remarkable with L-tryptophan than with D-tryptophan. It seems that the lazy character is due to deficiency of auxin, and it is caused by deficiency of tryptophan.     

Conversion of D- and L-tryptophan to brain serotonin and 5-hydroxyindoleacetic acid and to blood serotonin

Abstract— Intraperitoneal administration of both D- or L-tryptophan elevated the levels of serotonin and 5-hydroxyindoleacetic acid in the brains of hypophysectomized and intact rats. In intact rats, the increase in brain 5-hydroxyindoles was slower after D-tryptophan than after L-tryptophan. Similarly, brain tryptophan rose more slowly after administration of D-tryptophan. The uptake of L-tryptophan from blood into brain was at a rate about one-third that of ³H₂O. D-tryptophan uptake was at 1/25 that of ³H₂O. Brain and liver tryptophan aminotransferase activities were stereospecific for the L-isomer and no evidence could be found for a tryptophan racemase in brain. Evisceration prevented the increase in brain 5-hydroxyindoles following peripheral administration of D-tryptophan administration but not that after L-tryptophan. The serotonin ratios between the two brain regions examined remained constant following administration of either D- or L-tryptophan. On the basis of these results we concluded that the increase in brain 5-hydroxyindoles following administration of L-tryptophan was not dependent upon stress-induced changes in pituitary hormones and that the elevations after D-tryptophan were dependent upon its prior conversion to L-tryptophan via peripheral deamination and subsequent transamination.     

Evolutionary significance of the system utilizing D-amino acid enantiomers.

The evolutionary significance of the system utilizing D-amino acid enantiomers in living organisms is discussed, based on an experiment in which the mutant from Escherichia coli K--12 4627 grown on D-tryptophan was used. The mutant shows the ability of D-tryptophan is degraded to indol which can be utilized for the synthesis of L-tryptophan in the presence of serine.     

Tryptophanase-catalysed degradation of D-tryptophan in highly concentrated diammonium hydrogen phosphate solution

Summary Tryptophanase is and is perfectly inert to D-tryptophan under ordinary conditions. However, activity that can degrade D-tryptophan into indole is observed when tryptophanase is in highly concentrated diammoniumhydrogen phosphate solution. The reaction has been so far unknown in tryptophanase metabolic pathways. Here we report the characteristic of the reaction. We also discuss its significance in relation to selection of an amino acid optical isomer from a racemic mixture.Abbreviations AP diammoniumhydrogen phosphate - TPase tryptophanase - L-Trp L-tryptophan - D-Trp D-tryptophan - PLP pyridoxal 5-phosphate     

Kidney clearance of D- and L-tryptophan by rats

Renal clearances of D-tryptophan and of L-tryptophan by rats were compared. Both D- and L-tryptophan were reabsorbed by the rat kidney tubules very efficiently. Compared to the rapid excretion of D-tryptophan by the chick, the good retention of this isomer by the rat kidney might be responsible for its efficient utilization by rats.     

Efficiency of D-tryptophan as Niacin in rats

L-tryptophan is a very important precursor of niacin in mammals. Food preparation in which proteins are exposed to an alkali and/or high temperature for a long period generate appreciable amounts of D-amino acids from racemization. The efficiency of D-tryptophan as niacin was thus investigated by using weanling rats. The availability of D-tryptophan was almost the same as that in L-tryptophan as the precursor of niacin and was 1/6 as active as niacin.     

Two distinct types of mutations conferring to Escherichia coli K12 capability of D-tryptophan utilization

We showed that the ability of Escherichia coli K12 tryptophan auxotrophs to utilize D-tryptophan as a substitute for L-tryptophan may result from two types of mutations. The first type consisted in changes in the dadR regulatory site of the dad operon increasing the synthesis of D-amino acid dehydrogenase. The mutations of the second type mapped within the dad A structural gene. They changed the apparent substrate specificity of D-amino acid dehydrogenase. We suppose that the change may be due to an altered enzyme structure which make it more accessible to D-tryptophan.     

Reaction pathway of tryptophanase degrading D-tryptophan

Summary Tryptophanase, which has the very strict stereospecificity to L-tryptophan under ordinary condition, becomes active to D-tryptophan in highly concentrated diammoniumhydrogen phosphate solution. The reaction process of D-tryptophan degradation is studied in terms of kinetics. Diammoniumhydrogen phosphate acts on tryptophanase as activator below 3.1 M, and as noncompetitive inhibitor over it. Additionally, the pathway of the reaction is provided on the basis of kinetic parameters.Abbreviations TPase tryptophanase - L-Trp L-tryptophan - D-Trp D-tryptophan - DAP diammoniumhydrogen phosphate - PLP pyridoxal 5-phosphate     

Human indolylamine 2,3-dioxygenase. Its tissue distribution, and characterization of the placental enzyme.

The presence of indolylamine 2,3-dioxygenase was examined in human subjects by determining its activity with L-tryptophan as substrate. Enzyme activity was detected in various tissues, and was relatively high in the lung, small intestine and placenta. Human indolylamine 2,3-dioxygenase, partially purified from the placenta, had an Mr of about 40 000 by gel filtration and exhibited a single pI of 6.9. The human enzyme required a reducing system, ascorbic acid and Methylene Blue, for maximal activity and was able to oxidize D-tryptophan, 5-hydroxy-L-tryptophan as well as L-tryptophan, but kinetic studies indicated that the best substrate of the enzyme was L-tryptophan.     

Role of D-tryptophan oxidase in D-tryptophan utilization by Escherichia coli.

Mutants of Escherichia coli K-12 that require L-tryptophan (trp) are normally unable to utilize D-tryptophan to fulfill their requirement. However, secondary mutations (dadR) that confer this ability can be isolated. In such strains two distinct enzymes are found to be produced at high levels: D-amino acid oxidase (EC 1.4.3.3) and D-tryptophan oxidase. A convenient assay procedure for D-tryptophan oxidase is described. The two enzymes could be distinguished on the basis of their sensitivity to inhibition by L-phenylalanine and L-tyrosine. Strains that were trp dadR could not grow with D-tryptophan in the presence of L-phenylalanine, but further mutations, Fyo, could be isolated that allowed growth under these conditions. Some of them were characterized by further increases in the level of D-tryptophan oxidase activity and a sharp decrease in D-amino acid oxidase. These kinds of Fyo mutations lay in or near the dadR gene. The substrate specificity of the two enzymes toward a large number of compounds was examined. The transamination of aromatic keto acids was investigated. In the wild-type strain only a single enzyme, transaminase A (EC 2.6.1.5), was found, and it was irreversibly activated when subjected to elevated temperatures. The present state of our knowledge on D-amino acid utilization in E. coli is summarized.     

The avoidance of D-tryptophan by the nematode Caenorhabditis elegans.

In chemotactic studies employing countercurrent separation the nematode aenorhabditis elegans was found to avoid D-tryptophan with a threshold in the range 10(-4) to 10(-3) M. There was no response to L-tryptophan up to 10(-2) M although it appeared to partially inhibit the response to D-tryptophan.     

Conversion of D-tryptophan to indole-3-acetic acid in coleoptiles of a normal and a semi-dwarf barley (Hordeum vulgare) strain

Exogenously applied D-tryptophan (D-Trp) was more effective than L-Trp in inducing elongation of coleoptile segments of a normal barley ( Hordeum vulgare L. cv. Akashinriki) strain and a semi-dwarf strain with lower endogenous indole-3-acetic acid (IAA) level. D-cycloserine, an inhibitor of D-aminotransferase, completely inhibited both the D- and L-Trp-induced elongation of both strains. Addition of D-Trp increased IAA levels in both strains 4-fold over endogenous levels. The increase in IAA level was completely inhibited by D-cycloserine. The endogenous L-Trp level of semi-dwarf coleoptiles was similar to that of normal ones. These results suggested that IAA is synthesized by the conversion of L-Trp to indole-3-pyruvic acid via D-Trp in both strains, and that the lower IAA level of the semi-dwarf strain probably is a result of the impeded IAA biosynthesis involved in D-Trp.     

Is glycine "sweet" to mice? Mouse strain differences in perception of glycine taste

Glycine is an amino acid tasting sweet to humans. In 2-bottle tests, C57BL/6ByJ (B6) mice strongly prefer glycine solutions, whereas 129P3/J (129) mice do not, suggesting that they differ in perception of glycine taste. We examined this question using the conditioned taste aversion (CTA) generalization technique. CTA was achieved by injecting LiCl after drinking glycine, and next its generalization to 10 taste solutions (glycine, sucrose, saccharin, D-tryptophan, L-tryptophan, L-alanine, L-proline, L-glutamine, NaCl, and HCl) was examined by video recording licking behavior. Both B6 and 129 mice generalized the aversion to sucrose, saccharin, L-alanine, and L-proline and did not generalize it to NaCl, HCl, and L-tryptophan. This indicates that both B6 and 129 mice perceive the sweetness (i.e., a sucrose-like taste) of glycine. Thus, the lack of a glycine preference by 129 mice cannot be explained by their inability to perceive its sweetness. Strain differences were observed for CTA generalization to 2 amino acids: 129 mice generalized aversion to L-glutamine but not D-tryptophan, whereas B6 mice generalized it to D-tryptophan but not L-glutamine. 129.B6-Tas1r3 congenic mice with 2 genotypes of the Tas1r3 locus (B6/129 heterozygotes and 129/129 homozygotes) did not differ in aversion generalization, suggesting that the differences between 129 and B6 strains are not attributed to the Tas1r3 allelic variants and that other, yet unknown, genes are involved in taste perception of amino acids.     

Growth behaviour and bioproduction of indole acetic acid by a Rhizobium sp. isolated from root nodules of a leguminous tree Dalbergia lanceolaria

The Rhizobium sp. isolated from healthy and mature root nodules of a leguminous tree, Dalbergia lanceolaria Linn. f., preferred mannitol and KNO3 for growth as carbon and nitrogen sources, respectively. The bacterium produced a high amount (22.3 microg/ml) of indole acetic acid (IAA) from L-tryptophan supplemented basal medium. Growth and IAA production started simultaneously. IAA production was maximum at 20 hr when the bacteria reached the stationary phase of growth. Cultural requirements were optimized for maximum growth and IAA production. The IAA production by the Rhizobium sp. was increased by 270.8% over control when the medium was supplemented with mannitol (1%,w/v), SDS (1 microg/ml), L-asparagine (0.02%,w/v) and biotin (1 microg/ml) in addition to L-tryptophan (2.5 mg/ml). The possible role of IAA production in the symbiosis is discussed.     

Stereospecificity of hepatic L-tryptophan 2,3-dioxygenase.

Tryptophan 2,3-dioxygenase [L-tryptophan--oxygen 2,3-oxidoreductase (decyclizing), EC 1.13.11.11] has been reported to act solely on the L-isomer of tryptophan. However, by using a sensitive assay method with D- and L-[ring-2-14C]tryptophan and improved assay conditions, we were able to demonstrate that both the D- and L-stereoisomers of tryptophan were cleaved by the supernatant fraction (30000 g, 30 min) of liver homogenates of several species of mammals, including rat, mouse, rabbit and human. The ratio of activities toward D- and L-tryptophan was species variable, the highest (0.67) in ox liver and the lowest (0.07) in rat liver, the latter being hitherto exclusively used for the study of hepatic tryptophan 2,3-dioxygenase. In the supernatant fraction from mouse liver, the ratio was 0.23 but the specific activity with D-tryptophan was by far the highest of all the species tested. To identify the D-tryptophan cleaving enzyme activity, the enzyme was purified from mouse liver to apparent homogeneity. The specific activities toward D- and L-tryptophan showed a parallel rise with each purification step. The electrophoretically homogeneous protein had specific activities of 0.55 and 2.13 mumol/min per mg of protein at 25 degrees C toward D- and L-tryptophan, respectively. Additional evidence from heat treatment, inhibition and kinetic studies indicated that the same active site of a single enzyme was responsible for both activities. The molecular weight (150000), subunit structure (alpha 2 beta 2) and haem content (1.95 mol/mol) of the purified enzyme from mouse liver were similar to those of rat liver tryptophan 2,3-dioxygenase. The assay conditions employed in the previous studies on the stereospecificity of hepatic tryptophan 2,3-dioxygenase were apparently inadequate for determination of the D-tryptophan cleaving activity. Under the assay conditions in the present study, the purified enzyme from rat liver also acted on D-tryptophan, whereas the pseudomonad enzyme was strictly specific for the L-isomer.     

Structure-function studies of N-acyl derivatives of tryptophan that function as specific cholecystokinin receptor antagonists

Benzotript, N-p-chlorobenzoyl-L-tryptophan, is a specific cholecystokinin receptor antagonist. In the present study we used dispersed pancreatic acini to examine tryptophan as well as several different N-acyl derivatives of tryptophan for their abilities to function as cholecystokinin receptor antagonists. L-Tryptophan, D-tryptophan as well as each acyl derivative tested inhibited cholecystokinin-stimulated amylase secretion and outflux of ⁴⁵Ca and there was a good correlation between the ability of a particular agent to inhibit the action of cholecystokinin on acinar function and its ability to inhibit binding of ¹²⁵I-labeled cholecystokinin to pancreatic acini. Results with butyloxycarbonyl-L-tryptophan indicated that the inhibition of the action of cholecystokinin caused by L-tryptophan and various acyl derivatives is specific, competitive and fully reversible. In functioning as a cholecystokinin receptor antagonist the relative potencies of the agents tested were: carbobenzoxyl-L-tryptophan >benzotript >benzoyl-L-tryptophan = butyloxycarbonyl-L-tryptophan >acetyl-L-tryptophan >L-tryptophan. In inhibiting the actions of cholecystokinin, native as well as N-acyl derivatives of D-tryptophan were equipotent with the corresponding compound containing L-tryptophan. Although L-tryptophan inhibited the actions of cholecystokinin, L-phenylalanine, L-methionine or L-aspartic acid, even when tested at concentrations as high as 3 mM, did not alter the action of cholecystokinin on pancreatic acini. The antagonism of the actions of cholecystokinin was not restricted to N-acyl derivatives of L-tryptophan because butyloxycarbonyl-L-methionine and butyloxycarbonyl-L-phenylalanine but not butyloxycarbonyl-L-aspartic acid also antagonized the actions of cholecystokinin. These results demonstrate that both the nature of the N-Prmacyl group and the amino acid residue are important determinants of the affinity of the antagonist for the cholecystokinin receptor. For derivatives of L-tryptophan, the more hydrophobic the N-acyl moiety, the greater the affinity of the derivative for the cholecystokinin receptor.     

NMR studies of the activation of the Escherichia coli trp repressor

The Escherichia coli trp repressor binds to the trp operator in the presence of tryptophan, thereby inhibiting tryptophan biosynthesis. Tryptophan analogues lacking the alpha-amino group act as inducers of trp operon expression. We have used one- and two-dimensional 1H-NMR spectroscopy to compare the binding to the repressor of the corepressors L-tryptophan, D-tryptophan and 5-methyl-DL-tryptophan with that of the inducer indole-3-propionic acid. We have determined the chemical shifts of the indole ring protons of the ligands when bound to the protein, principally by magnetization-transfer experiments. The chemical shifts of the indole NH and C4 protons differ between corepressors and inducer. At the same time, the pattern of intermolecular NOE between protons of the protein and those of the ligand also differ between the two classes of ligand. These two lines of evidence indicate that corepressors and inducers bind differently in the binding site, and the evidence suggests that the orientation of the indole ring in the binding site differs by approximately 180 degrees between the two kinds of ligand. This is in contrast to a previous solution study [Lane, A.N. (1986) Eur. J. Biochem. 157, 405-413], but consistent with recent X-ray crystallographic work [Lawson, C.L. & Sigler, P.B. (1988) Nature 333, 869-871]. D-Tryptophan and 5-methyltryptophan, which are more effective corepressors than L-tryptophan, bind similarly to L-tryptophan. The indole ring of D-tryptophan appears to bind in essentially the same orientation as that of the L isomer. There are, however, some differences in chemical shifts and NOE for 5-methyltryptophan, which indicate that there are significant differences between the two corepressors L-tryptophan and 5-methyltryptophan in the orientation of the indole ring within the binding site.     

Possible role of N-malonyl-D-tryptophan as an auxin precursor

N-malonyl-D-tryptophan (MT) and D-tryptophan added to the medium instead of auxin stimulated growth of soybean and tomato cell and tissue cultures. Effects of 50–100 μmol 1^-1 MT and 100 –300 μmol 1^-1 D-tryptophan were equal to the effect of 3–10 μmol 1^-1 IAA. Soybean cells grown in the presence of 100 μmol 1^-1 MT contained 125–170 ng IAA per 1 g fresh mass (as determined by spectrofluorimetric indole-α-pyrone method), whereas the cells grown in the presence of NAA 10. 7 μmol 1^-1 contained 50 –60 ng IAA and the cells grown in the absence of auxin failed to show endogenous IAA. MT as proposed can be hydrolyzed by plant cells with liberation of D-tryptophan, which in turn can be used in IAA synthesis. It is proposed that MT is a possible source of endogenous auxin in plants.