Arabidopsis indole synthase, a homolog of tryptophan synthase alpha, is an enzyme involved in the Trp-independent indole-containing metabolite biosynthesis

The plant tryptophan (Trp) biosynthetic pathway produces many secondary metabolites with diverse functions.Indole-3-acetic acid (IAA),proposed as a derivative from Trp or its precursors,plays an essential role in plant growth and development.Although the Trp-dependant and Trp-independent IAA biosynthetic pathways have been proposed,the enzymes,reactions and regulatory mechanisms are largely unknown.In Arabidopsis,indole-3-glycerol phosphate (IGP) is suggested to serve as a branchpoint component in the Trp-independent IAA biosynthesis.To address whether other enzymes in addition to Trp synthase α(TSA1) catalyze IGP cleavage,we identified and characterized an indole synthase (INS) gene,a homolog of TSA1 in Arabidopsis.INS exhibits different subcellular localization from TSA1 owing to the lack of chloroplast transit peptide (cTP).In silico data show that the expression levels of INS and TSA1 in all examined organs are quite different.Histochemical staining of INS promoter-GUS transgenic lines indicates that INS is expressed in vascular tissue of cotyledons,hypocotyls,roots and rosette leaves as well as in flowers and siliques.INS is capable of complementing the Trp auxotrophy of Escherichia coil △trpA strain,which is defective in Trp synthesis due to the deletion of TSA.This implies that INS catalyzes the conversion of IGP to indole and may be involved in the biosynthesis of Trp-independent IAA or other secondary metabolites in Arabidopsis.     

Physiological Effects of a Constitutive Tryptophanase in Bacillus alvei.

Hoch, J. A. (University of Illinois, Urbana), and R. D. DeMoss. Physiological effects of a constitutive tryptophanase in Bacillus alvei. J. Bacteriol. 90:604-610. 1965.-Tryptophanase synthesis in B. alvei is not under the control of tryptophan and is not subject to catabolite repression. Exogenously supplied tryptophan was converted to indole by tryptophanase, and was excreted into the culture medium. The amount of indole excreted was dependent upon the concentration of tryptophan supplied. At intermediate levels of tryptophan (5 to 15 mug/ml), the excreted indole was completely reutilized by the cell, in contrast to the result with higher levels. Indole reutil zation was shown to be dependent upon a functional tryptophan synthetase. In the absience of exogenous tryptophan, indole was excreted into the culture medium at an earlier physiological age. The early indole was shown not to be a consequence of tryptophanase action. The early indole accompanied uniformly the normal process of tryptophan biosynthesis, and the fission of indole-3-glycerol phosphate was suggested as the origin of the excreted indole.     

Secondary metabolite biosynthesis in cultured cells of Catharanthus roseus (L.) G. Don immobilized by adhesion to glass fibres

Summary Suspension-cultured cells of Catharanthus roseus (L.) G. Don were immobilized on glass fibre mats and cultivated in shake flasks. The highly-aggregated immobilized cells exhibited a slower growth rate and accumulated reduced levels of tryptamine and indole alkaloids, represented by catharanthine and ajmalicine, in comparison to cells in suspension. The increased total protein synthesis in immobilized cells suggests a diversion of the primary metabolic flux toward protein biosynthetic pathways and away from other growth processes. In vitro assays for the specific activity of tryptophan decarboxylase (TDC) and tryptophan synthase (TS) suggest that the decreased accumulation of tryptamine in immobilized cells was due to reduced tryptophan biosynthesis. The specific activity of TDC was similar in immobilized and suspension-cultured cells. However, the expression of TS activity in immobilized cells was reduced to less than 25% of the maximum level in suspension-cultured cells. The reduced availability of a free tryptophan pool in immobilized cells is consistent with the reduced TS activity. Reduced tryptamine accumulation, however, was not responsible for the decreased accumulation of indole alkaloids in immobilized cells. Indole alkaloid accumulation increased to a similar level in immobilized and suspension-cultured cells only after the addition of exogenous secolaganin to the culture medium. The addition of tryptophan resulted in increased accumulation of tryptamine, but had no effect on indole alkaloid levels. Reduced biosynthesis of secologanin, the monoterpenoid precursor to indole alkaloids, in immobilized cells is suggested. Immobilization does not appear to alter the activity of indole alkaloid biosynthetic enzymes in our system beyond, and including, strictosidine synthase. Offprint requests to: P. J. Facchini     

A colorimetric method for the determination of indole, and its application to assay of tryptophanase.

Indole reacts with sodium nitrite and glycine-HCl buffer, pH 2.6, to form a red color that is stable for more than 1 week. The reaction is reproducible and is linear over a wide range of indole concentrations (0.05–1.00 μmol). Twelve indole derivatives, including tryptophan, and 17 protein amine acids do not interfere. Indole-3-acetic acid, indole-3-acrylic acid, indole-3-pyruvic acid, 5-indole carboxylic acid, and 5-hydroxyindole-3-acetic acid interfere to varying extents (16–27%). Free indole was determined in biological material containing tryptophan by the present method. The method is also applicable to the assay of tryptophanase activity without prior indole extraction.     

Production of indole-3-acetic acid and related indole derivatives from L-tryptophan by <Emphasis Type="Italic">Rubrivivax benzoatilyticus</Emphasis> JA2

Rubrivivax benzoatilyticus JA2 produces indoles with simultaneous utilization of L-tryptophan. Fifteen chromatographically distinct indole derivatives were detected from the L-tryptophan-supplemented cultures of R. benzoatilyticus JA2. Nine of these were identified as, indole 3-acetamide, Methoxyindole-3-aldehyde, indole 3-aldehyde, methoxyindole-3-acetic acid, indole 3-acetic acid, indole-3-carboxylic acid, indole-3-acetonitrile, indole, and trisindoline. Tryptophan stable isotope feeding confirmed the indoles produced are from the supplemented L-tryptophan. Indole 3-acetic acid is one of the major products of L-tryptophan catabolism by R. benzoatilyticus JA2 and its production was influenced by growth conditions. Identification of indole 3-acetamide and tryptophan monooxygenase activity suggests indole 3-acetamide routed IAA biosynthesis in R. benzoatilyticus JA2. The study also indicated the possible multiple pathways of IAA biosynthesis in R. benzoatilyticus JA2.     

Crystallization and X-ray crystallographic studies of wild-type and mutant tryptophan synthase alpha-subunits from Escherichia coli

The alpha-subunit of Escherichia coli tryptophan synthase (aTS), a component of the tryptophan synthase alpha2beta2 complex, is a monomeric 268-residues protein (Mr = 28,600). alphaTS by itself catalyzes the cleavage of indole-3-glycerol phosphate to glyceraldehyde-3-phosphate and indole, which is converted to tryptophan in tryptophan biosynthesis. Wild-type and P28L/Y173F double mutant alpha-subunits were overexpressed in E. coli and crystallized at 298 K by the hanging-drop vapor-diffusion method. X-ray diffraction data were collected to 2.5 angstroms resolution from the wild-type crystals and to 1.8 angstroms from the crystals of the double mutant, since the latter produced better quality diffraction data. The wild-type crystals belonged to the monoclinic space group C2 (a = 155.64 angstroms, b = 44.54 angstroms, c = 71.53 angstroms and beta = 96.39 degrees) and the P28L/Y173F crystals to the monoclinic space group P21 (a = 71.09 angstroms, b = 52.70, c = 71.52 angstroms, and beta = 91.49 degrees). The asymmetric unit of both structures contained two molecules of aTS. Crystal volume per protein mass (V(m)) and solvent content were 2.15 angstroms3 Da(-1) and 42.95% for the wild-type and 2.34 angstroms3 Da(-1) and 47.52% for the double mutant.     

Isolation of auxotrophs and analysis of regulation of tryptophan biosynthesis in Zymomonas mobilis

Tryptophan auxotrophs were isolated and used to analyze the regulation of tryptophan biosynthesis in Zymomonas mobilis. Twelve tryptophan auxotrophs were cassified as trp E, B or A based on accumulation of, or growth on, indole and anthranilic acid. Trp B mutants were found to accumulate indole when grown on limiting, but not on excess tryptophan, suggesting that tryptophan plays a role in regulating its biosynthesis. Tryptophan synthase and indoleglycerol phosphate synthase specific activities were measured in the wild-type strain and two trp mutants grown in limiting or excess tryptophan. Neither activity was repressed by exogenous tryptophan.Abbreviations CDRP O-(carboxyphenol amino)-1 deoxyribulose 5-phosphate - IGPS indoleglycerol phosphate synthase - TS tryptophan synthase Dedicated in memory of Dr. O. H. Smith     

Physiological and enzymatic aspects of histidine-mediated control of the tryptophan pathway

Summary It would thus appear that in Saccharomyces cerevisiae there are two forms of histidine-mediated control on the tryptophan pathway. In some strains histidine increases anthranilate synthetase and indole glycerol phosphate synthetase activities, while tryptophan synthetase decreases. In other strains histidine affects coordinately all enzymatic activities involved in tryptophan biosynthesis. The two groups of strains also differ in the formation, during the growth of the enzymatic activities involved in tryptophan biosynthesis. This difference in the relative rates at which the two enzymes are formed may explain the accumulation of intermediates in the cultural media of some strains. The derepression of anthranilate synthetase and indole glycerol phosphate synthetase activities by histidine is particularly manifest in the auxotrophic his₃ strains that show these activities very depressed in histidine starvation; large amounts of this amino acid stimulate them to a considerably greater extent than in prototrophic strains.Abbreviations IGP imidazole glycerol phosphate - InGP indole glycerol phosphate - ASase anthranilate synthetase - InGPase indole-3-glycerol phosphate synthetase - TSase tryptophan synthetase - Tris tris (hydroxymethyl)-aminomethane This investigation was supported by a research grant of C.N.R. (Consiglio Nazionale delle Ricerche, Roma).     

Identification of an indigo precursor from leaves of Isatis tinctoria (Woad).

Indole is presumably a product of indole-3-glycerol phosphate catabolism in Isatis tinctoria. It is oxidized into indoxyl and stored in young leaves as indigo precursor. Further oxidation and dimerization of indoxyl produces indigoid pigments. In this work, we describe an HPLC method dedicated to the identification and quantification of indigoid pigments (indigo, indirubin, isoindigo and isoindirubin) and indigo precursors produced in I. tinctoria (Woad). This work, carried out with two cultivars of I. tinctoria, has confirmed that the quantity of indigo precursors is dependent on the species and the harvest period. In addition we have shown for the first time that young leaves of I. tinctoria, harvested in June contained a new indigo precursor in addition to isatan B (indoxyl-5-ketogluconate) and indican (indoxyl-beta-D-glucoside). We suggest the name "isatan C" for this new indigo precursor in I. tinctoria. Its chemical characteristics point to an dioxindole ester with PM of 395. We have shown that isatan C reacts with isatan B increasing the red pigment production.     

Tryptophan Synthase and Production of l-Tryptophan in Regulatory Mutants

A 5-fluorotryptophan-resistant mutant of Brevibacterium flavum, No. 187, accumulated 2.6 g of indole 3-glycerol (InG) in addition to 8.0 g of l-tryptophan per liter in the culture medium. The addition of l-serine to the medium decreased the accumulation of InG and increased that of l-tryptophan up to a concentration of 10.3 g/liter, while the addition of l-tryptophan increased the InG accumulation, suggesting that InG was formed by hydrolysis of indole 3-glycerol phosphate (InGP), the substrate of tryptophan synthase (TS) which catalyzed the final step reaction of tryptophan biosynthesis. Then, in order to examine the mechanism of the InG accumulation, TS was purified from tryptophan auxotroph, TA-60. The reaction mechanism of TS was Ordered Bi Bi with Km’s of 0.63 and 0.038 mm for serine and InGP, respectively. Tryptophan, a product of the TS reaction, inhibited TS competitively with respect to serine and the Ki for tryptophan was estimated to be 2.0 mm. On the other hand, anthranilate synthase (AS), the first enzyme in the tryptophan biosynthetic pathway, was much less sensitive to the feedback inhibition by tryptophan in strain No. 187 than in the wild strain. The tryptophan concentration giving 50% inhibition of AS in strain No. 187 was estimated to be 2.4 mm, almost comparable to that of TS, 7.7 mm. From these results, it was concluded that the accumulation of InG in strain No. 187 would result from the product inhibition of TS by the tryptophan accumulated.     

5-Azidoindole binding to the Escherichia coli tryptophan synthase alpha 2 beta 2 complex

The tryptophan synthase alpha 2 beta 2 complex catalyzes tryptophan (Trp) biosynthesis from serine plus either indole (IN) or indole-3-glycerol phosphate (InGP). The photoreactive 5-azido analog in IN (AzIN), itself a substrate in the dark, was utilized to examine the substrate binding sites on this enzyme. When irradiated with AzIN at concentrations approaching IN saturation for the IN----Trp activity (0.1 mM), in the absence of serine, the enzyme was increasingly inactivated (up to 70-80%) concomitant with the progressive binding of a net of 2 mol AzIN per alpha beta equivalent. Little or no cooperativity in the binding of the 2 mol AzIN was observed. In contrast, there was minimal effect on the IN----InGP activity. Under these conditions AzIN appeared to be incorporated equally into each subunit. No significant inactivation nor binding occurred in the presence of serine. A quantitatively similar inactivation of InGP----Trp activity was observed over the same AzIN concentration range, suggesting common IN sites for Trp biosynthesis from either indole substrate. At higher concentrations (0.1-0.7 mM), no further inactivation occurred, although there was extensive additional binding (up to 10 mol/alpha beta equivalent). These data are consistent, although more clear-cut quantitatively, with the high- and low-affinity sites proposed from equilibrium dialysis studies. AzIN binding studies utilizing the isolated beta 2 subunit confirmed earlier reports suggesting the existence of many nonspecific IN binding sites on this subunit.     

Indigo biosynthesis by Comamonas sp. MQ

An indigo-producing strain was isolated from activated sludge and identified as Comamonas sp. based on 16S rRNA analysis. It produced indigo at 26.5 mg/l with a conversion of indole to indigo of 47%. Indole at 50 mg/l plus 200 mg naphthalene/l gave 32.2 mg indigo/l with a 58% conversion. A pathway for indigo formation is proposed. This is the first study of indigo biosynthesis by Comamonas sp.     

Auxin biosynthesis in maize kernels

Auxin biosynthesis was analyzed in a maize (Zea mays) kernel culture system in which the seeds develop under physiological conditions similar to the in vivo situation. This system was modified for precursor feeding experiments. Tryptophan (Trp) is efficiently incorporated into indole-3-acetic acid (IAA) with retention of the 3, 3' bond. Conversion of Trp to IAA is not competed by indole. Labeling with the general precursors [U-(13)C(6)]glucose and [1, 2-(13)C(2)]acetate followed by retrobiosynthetic analysis strongly suggest that Trp-dependent IAA synthesis is the predominant route for auxin biosynthesis in the maize kernel. The synthesis of IAA from indole glycerol phosphate and IAA formation via condensation of indole with an acetyl-coenzyme A or phosphoenolpyruvate derived metabolite can be excluded.     

Photoaffinity labeling of the indole sites on the Escherichia coli tryptophan synthase alpha-subunit

The alpha subunit of the Escherichia coli tryptophan synthase catalyzes the reversible aldolytic reaction: Indole-3-glycerol phosphate in equilibrium indole + glyceraldehyde 3-phosphate. The use of 5-azidoindole as a photoaffinity label has made the generation of a number of enzyme-substrate complexes possible, each with a given degree of saturation of the two postulated indole sites. When assayed in the reverse reaction (indole-3-glycerol phosphate synthesis), samples of alpha subunit treated at concentrations of 5-azidoindole less than or equal to 2 mM show a progressive 30-40% activation. A gradual inactivation occurs only in samples irradiated at concentrations in excess of 2 mM 5-azidoindole, and this inactivation is complete at 8-10 mM. A quantitatively similar activation occurs in the forward reaction (indole synthesis), however inactivation in this case is incomplete, with complexes treated at 8-12 mM 5-azidoindole retaining 30-40% relative activity in this reaction. When treated alpha subunits were assayed for their abilities to complement the beta 2-subunit in the reactions indole + L-serine leads to L-tryptophan + H2O and indole-3-glycerol phosphate + L-serine leads to L-tryptophan + glyceraldehyde 3-phosphate, quantitatively lesser amounts of activation followed by total inactivation are observed over a similar range of 5-azidoindole concentrations.     

Two Mechanisms of Allelic Complementation Among Tryptophan Synthetase Mutants of Saccharomyces cerevisiae

Two different types of allelic complementation were observed in tryptophan synthetase mutants of the yeast Saccharomyces cerevisiae. Each type is associated with a different mechanism for the enzymatic conversion of indole-3-glycerol phosphate (InGP) to tryptophan. Mechanism I is utilized by a hybrid tryptophan synthetase that resembles, but is not identical with, the wild-type enzyme. Mechanism II is due to a sequential conversion of InGP to free indole, and indole to tryptophan. Two partially active mutant enzymes rather than a single hybrid enzyme catalyze the sequential reaction steps. This is an example of intracellular cross-feeding. The quantitative evaluation of mechanism II leads to the conclusion that tryptophan synthetase in yeast is most likely a dimer of two identical subunits.     

The role of tryptophan as a biosynthetic precursor of indole-diterpenoid fungal metabolites: Continuing a debate

Studies in the 1980s and 1990s on the origin of the indole moiety in fungal indole-diterpenoids using ¹⁴C-labelled tryptophan consistently showed autoradiographic evidence but gave low % incorporation of the probe. Recent studies on a member of the group (nodulisporic acid A), using more specific ¹³C methodology, demonstrated a role of the tryptophan biosynthetic pathway but, in failing to show involvement of end-product, concluded that the indole was derived from indole-3-glycerol phosphate and suggested that the previous ¹⁴C data arose via metabolic scrambling of label. In considering the protocol for the ¹³C studies, there is concern that the fungal material was starved of an exogenous nitrogen source and thus could have degraded added labelled tryptophan. Consequently, synthesis of the serine necessary for anabolic formation of tryptophan may have been constrained. It is suggested that ¹³C studies on appropriate fungi early in the idiophase of submerged or surface fermentation should be made before the biosynthesis of indole-diterpenoids can become clearer.     

In vivo regulation of intermediate reactions in the pathway of tryptophan biosynthesis in Neurospora crassa

The in vivo regulation of intermediate reactions in the pathway of tryptophan synthesis in Neurospora crassa was examined in a double mutant (tr-2, tr-3) which lacks the functions of the first and last enzymes in the pathway from chorismic acid to tryptophan. The double mutant can convert anthranilic acid to indole and indole-3-glycerol, and the production of these indolyl compounds by germinated conidia was used to estimate the activity of the intermediate enzymes in the pathway. Indole-synthesizing activity was maximal in germinated conidia obtained from cultures in which the levels of l-tryptophan were growth-limiting; the formation of this activity was markedly repressed when the levels of l-tryptophan exceeded those required for maximal growth. d-, 5-methyl-dl-, and 6-methyl-dl-tryptophan were less effective than l-tryptophan, and 4-methyl-dl-tryptophan, tryptamine, and indole-3-acetic acid were ineffective in repressing the formation of indole-synthesizing activity; anthranilic acid stimulated the formation of indole-synthesizing activity. Preformed indole-synthesizing activity was strongly and specifically inhibited by low levels of l-tryptophan; several related compounds were ineffective as inhibitors. These results suggest that, in addition to repression, an end product feedback inhibition mechanism is operative on an intermediate enzyme(s) in tryptophan biosynthesis. The relation of these results to other in vivo and in vitro studies and to general aspects of the regulation of tryptophan biosynthesis in N. crassa are discussed.