Regulation of the chorismic acid branch-point in aromatic acid synthesis in blue-green and green algae

Summary In extension of previous studies on the regulation of the aromatic amino acid pathway in blue-green and green algae the control of two branch-point enzymes, namely chorismate mutase and anthranilate synthetase has been studied. The activity of chorismate mutase in these organisms is effectively inhibited by l-tyrosine or l-phenylalanine. l-tryptophan, in contrast, proved to be a positive effector of the enzyme: in the absence of phenylalanine or tyrosine tryptophan slightly stimulated chorismate mutase activity; this stimulation was even brought about in the presence of excess phenylalanine or tyrosine, irrespective if the enzyme had been preincubated with these inhibitors or not. Tryptophan thus proved to completely revert the feedback inhibition of this enzyme by phenylalanine or tyrosine. Substrate saturation curves of chorismate mutase activity are hyperbolic in the presence of tryptophan and sigmoid in the presence of phenylalanine or tyrosine. In contrast to the enzymes of the green algae investigated, chorismate mutase activity of Anacystis nidulans, a member of the class of the blue-green algae was not affected by any of the aromatic amino acids.The activity of anthranilate synthetase, the second enzyme of the chorismic acid branch-point of the pathway was consistently inhibited by l-tryptophan in all the organisms tested. The results described here bear significance on the regulation of a multi-branched pathway the first enzyme of which is inhibited just by one endproduct.     

Resistance of active monovalent cation transport to pronase digestion of intact human erythrocytes

Chorismate mutase CM-1, an isozyme that is inhibited by phenylalanine and tyrosine and activated by tryptophan was purified 1200-fold from etiolated mung bean seedlings with a final yield of 18–20%. Loss of activity was rapid in highly purified preparations but was reduced by the addition of bovine serum albumin. Enzyme activity was unaffected by thiol-alkylating agents, reducing agents, EDTA, or divalent cations.The enzyme displayed pH-sensitive, positive homotrophic cooperativity toward chorismate with greatest cooperativity at the pH optimum of the tryptophan-free enzyme (pH 7.2–7.4) and least cooperativity at the pH optimum of the enzyme fully activated with tryptophan (pH 7.0). Activation by tryptophan reduced the K_m for the enzyme, and modified the sigmoid substrate saturation kinetics to a rectangular hyperbola. Feedback inhibition by the end product amino acids phenylalanine and tyrosine was not additive but revealed heterotrophic cooperativity with chorismate. Tyrosine (K_i = 31 μM) was a slightly more effective inhibitor than phenylalanine (K_i = 37 μM) at 1 mm chorismate. Tryptophan at equimolar concentration antagonized the feedback inhibition by phenylalanine and tyrosine. The latter two, however, at higher concentrations reversed the tryptophan activation in a noncompetitive fashion with respect to either tryptophan or chorismate. The enzyme was responsive only to the l-isomers of the amino acids. The results indicate a primary role for chorismate mutase CM-1 from mung bean in the regulation of the synthesis of phenylalanine and tyrosine for protein synthesis.     

Regulatory Properties of Chorismate Mutase from Corynebacterium glutamicum

Regulatory properties of chorismate mutase from Corynebacterium glutamicum were studied using the dialyzed cell-free extract. The enzyme activity was strongly feedback inhibited by l-phenylalanine (90% inhibition at 0.1~1 mm) and almost completely by a pair of l-tyrosine and l-phenylalanine (each at 0.1~1 mm). The enzyme from phenylalanine auxotrophs was scarcely inhibited by l-tyrosine alone but the enzyme from a wild-type strain or a tyrosine auxotroph was weakly inhibited by l-tyrosine alone (40~50% inhibition, l-tyrosine at 1 mm). The enzyme activity was stimulated by l-tryptophan and the inhibition by l-phenylalanine alone or in the simultaneous presence of l-tyrosine was reversed by l-tryptophan. The Km value of the reaction for chorismate was 2.9 } 10^?3 m. Formation of chorismate mutase was repressed by l-phenylalanine. A phenylalanine auxotrophic l-tyrosine producer, C. glutamicum 98–Tx–71, which is resistant to 3-amino-tyrosine, p-aminophenylanaine, p-fluorophenylalanine and tyrosine hydroxamate had chorismate mutase derepressed to two-fold level of the parent KY 10233. The enzyme in C. glutamicum seems to have two physiological roles; one is the control of the metabolic flow to l-phenylalanine and l-tyrosine biosynthesis and the other is the balanced partition of chorismate between l-phenylalanine-l-tyrosine biosynthesis and l-tryptophan biosynthesis.     

Control of Aromatic Amino Acid Biosynthesis in Cultured Plant Tissues: Effect of Intermediates and Aromatic Amino Acids on Free Levels

Shikimate, anthranilate, indole, l -tryptophan, phenylpyruvate, l -p henylalanine, p-hydroxyphenylpyruvate or l -tyrosine were added to suspension-cultured Nicotiana tabacum (tabacco) and Daucus carota (carrot) tissues and incubated for 24 hours. Uptake of each compound was substantial as measured by its decrease in the medium. The levels of free tryptophan, phenylalanine and tyrosine were determined in the tissues after the 24 hours incubation. Shikimate did not change the aromatic animo acid levels in carrot tissue, but did increase all three in tobacco (3-fold or more), indicating a less stringent feedback control in tobacco. Anthranilate and indole increased the tissue tryptophan levels in both species by at least 17-fold, showing that the flow from anthranilate and indole to tryptophan was apparently unhindered by enzymatic control mechanisms. When tryptophan levels were elevated in both carrot and tobaccotissues by anthranilate, indole or tryptophan addition, there was also an increase in free phyenylalanine and tyrosine. This might be due to the reversal of phenylalanine and tyrosine feedback inhibition of chorismate mutase by the high tryptophan in the tissue. Chorismate mutase activity in tobacco crude extracts could be inhibited by 66–90% by 1 mM phenylalanine and /or tyrosine. Tryptophan at 1 mM stimulated the enzyme activity by 1/3 and completely reversed the phenylalanine and/or tyrosine inhibition of enzyme activity. Chorsimate mutase activity amino acids under a variety of conditions. Phenylpyruvate increased the phenylalanine levels and p-hydroxyphenylpyruvate increased the tyrosine levels in carrot and tobacco tissues indicating that there was no feedback control of the last step in phenylalanine and tyrosine biosynthesis.     

Regulation of phenylalanine biosynthesis in Rhodotorula glutinis.

The phenylalanine biosynthetic pathway in the yeast Rhodotorula glutinis was examined, and the following results were obtained. (i) 3-Deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase in crude extracts was partially inhibited by tyrosine, tryptophan, or phenylalanine. In the presence of all three aromatic amino acids an additive pattern of enzyme inhibition was observed, suggesting the existence of three differentially regulated species of DAHP synthase. Two distinctly regulated isozymes inhibited by tyrosine or tryptophan and designated DAHP synthase-Tyr and DAHP synthase-Trp, respectively, were resolved by DEAE-Sephacel chromatography, along with a third labile activity inhibited by phenylalanine tentatively identified as DAHP synthase-Phe. The tyrosine and tryptophan isozymes were relatively stable and were inhibited 80 and 90% by 50 microM of the respective amino acids. DAHP synthase-Phe, however, proved to be an extremely labile activity, thereby preventing any detailed regulatory studies on the partially purified enzyme. (ii) Two species of chorismate mutase, designated CMI and CMII, were resolved in the same chromatographic step. The activity of CMI was inhibited by tyrosine and stimulated by tryptophan, whereas CMII appeared to be unregulated. (iii) Single species of prephenate dehydratase and phenylpyruvate aminotransferase were observed. Interestingly, the branch-point enzyme prephenate dehydratase was not inhibited by phenylalanine or affected by tyrosine, tryptophan, or both. (iv) The only site for control of phenylalanine biosynthesis appeared to be DAHP synthase-Phe. This is apparently sufficient since a spontaneous mutant, designated FP9, resistant to the growth-inhibitory phenylalanine analog p-fluorophenylalanine contained a feedback-resistant DAHP synthase-Phe and cross-fed a phenylalanine auxotroph of Bacillus subtilis.     

Isolation of cyanobacterial auxotrophs by direct selection of regulatory-mutant phenotypes

We have isolated a chorismate mutase bradytroph (leaky auxotroph) ofAnabaena sp. PCC 7119 (ATCC 29151) as a spontaneous 6-fluorotryptophan-resistant mutant. The decreased chorismate mutase activity resulted in the production of quantities of the phenylalanine and tyrosine that limited rate of growth. 3-Deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase activity in the mutant was elevated more than twofold over the wild-type activity, suggesting derepression of this enzyme. The physiological deregulation of DAHP synthase and the genetic-based deficiency of chorismate mutase promoted an elevated level of intracellular chorismate, which then overwhelmed the competitive inhibition of anthranilate synthase by tryptophan, resulting in the overproduction of tryptophan and indoleglycerolphosphate. The presence of exogenous serine increased the production of tryptophan at the expense of indoleglycerolphosphate. This indicated that the endogenous potential for increasing the amount of serine available for increased tryptophan production is limited.     

Amino Acid Composition of Elm Seeds

In the biosynthetic pathway of aromatic amino acids of Brevibacterium flavum, ratios of each biosynthetic flow at the chorismate branch point were calculated from the reaction velocities of anthranilate synthetase for tryptophan and chorismate mutase for phenylalanine and tyrosine at steady state concentrations of chorismate. When these aromatic amino acids were absent, the ratio was 61, showing an extremely preferential synthesis of tryptophan. The presence of tryptophan at 0.01 mM decreased the ratio to 0.07, showing a diversion of the preferential synthesis to phenylalanine and tyrosine. Complete recovery by glutamate of the ability to synthesize the Millon-positive substance in dialyzed cell extracts confirmed that tyrosine was synthesized via pretyrosine in this organism. Partially purified prephenate aminotransferase, the first enzyme in the tyrosine-specific branch, had a pH optimum of 8.0 and Km’s of 0.45 and 22 mM for prephenate and glutamate, respectively, and its activity was increased 15-fold by pyridoxal-5-phosphate. Neither its activity nor its synthesis was affected at all by the presence of the end product tyrosine or other aromatic amino acids. The ratio of each biosynthetic flow for tyrosine and phenylalanine at the prephenate branch point was calculated from the kinetic equations of prephenate aminotransferase and prephenate dehydratase, the first enzyme in the phenylalanine-specific branch. It showed that tyrosine was synthesized in preference to phenylalanine when phenylalanine and tyrosine were absent. Furthermore, this preferential synthesis was diverted to a balanced synthesis of phenylalanine and tyrosine through activation of prephenate dehydratase by the tyrosine thus synthesized. The feedback inhibition of prephenate dehydratase by phenylalanine was proposed to play a role in maintaining a balanced synthesis when supply of prephenate was decreased by feedback inhibition of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP*) synthetase, the common key enzyme. Overproduction of the end products in various regulatory mutants was also explained by these results.     

Cytosolic and plastidic chorismate mutase isozymes from Arabidopsis thaliana: molecular characterization and enzymatic properties

The three aromatic amino acids phenylalanine, tyrosine, and tryptophan are synthesized in the plastids of higher plants. There is, however, biochemical evidence that a cytosolic isoform exists of the enzyme catalysing the first step of that branch of the pathway which is specific for the synthesis of phenylalanine and tyrosine, i.e. chorismate mutase (CM). We now report on the isolation of a cDNA clone encoding a cytosolic CM isozyme from Arabidopsis thaliana that was identified by complementing a CM-deficient Escherichia coli strain. The deduced amino acid sequence of this isozyme was 50% identical to that of a previously isolated plastidic CM, and 41% identical to that of yeast CM. The organ-specific expression patterns of the two CM genes were rather similar, but only the gene encoding the plastidic isozyme was elicitor- and pathogen-inducible. The plastidic CM expressed in E. coli was activated by tryptophan and inhibited by phenylalanine and tyrosine, whereas the cytosolic isozyme was insensitive. The existence of a cytosolic CM isozyme implies that either a cytosolic pathway (partial or complete) for the biosynthesis of phenylalanine and tyrosine exists, or that prephenate, originating from chorismate in the cytosol, is utilized for the synthesis of metabolites other than these two aromatic amino acids.     

Altered control of chorismate mutase leads to tryptophan auxotrophy of Pichia guilliermondii

We have isolated the tryptophan auxotrophic mutant strain, PK101, of Pichia guilliermondii. This strain is not defective in any of the tryptophan biosynthetic enzymes, but its chrismate mutase, an enzyme of the phenylalanine-tyrosine biosynthesis, is changed. In comparison with the wild type chorismate mutase, the enzyme of PK101 is characterized by a complete loss of sensitivity to l-phenylalanine inhibition and to a considerable loss of sensitivity to l-tryptophan activation. Furthermore, the chorismate mutase activity of the mutant is more than 7-fold higher in the absence of l-tryptophan than in the wild type. The PK101 enzyme is also changed in the pH optimum and in some kinetic constants. We found an increased intracellular pool of both phenylalanine and tyrosine and a reduced contents of tryptophan in the mutant cells. Our genetic data indicate that the mutant phenotype is dominant over the wild type.     

Yarrowia lipolytica chassis strains engineered to produce aromatic amino acids via the shikimate pathway

Yarrowia lipolytica is widely used as a microbial producer of lipids and lipid derivatives. Here, we exploited this yeast’s potential to generate aromatic amino acids by developing chassis strains optimized for the production of phenylalanine, tyrosine and tryptophan. We engineered the shikimate pathway to overexpress a combination of Y. lipolytica and heterologous feedback-insensitive enzyme variants. Our best chassis strain displayed high levels of de novo Ehrlich metabolite production (up to 0.14 g l⁻¹ in minimal growth medium), which represented a 93-fold increase compared to the wild-type strain (0.0015 g l⁻¹). Production was further boosted to 0.48 g l⁻¹ when glycerol, a low-cost carbon source, was used, concomitantly to high secretion of phenylalanine precursor (1 g l⁻¹). Among these metabolites, 2-phenylethanol is of particular interest due to its rose-like flavour. We also established a production pathway for generating protodeoxyviolaceinic acid, a dye derived from tryptophan, in a chassis strain optimized for chorismate, the precursor of tryptophan. We have thus demonstrated that Y. lipolytica can serve as a platform for the sustainable de novo bio-production of high-value aromatic compounds, and we have greatly improved our understanding of the potential feedback-based regulation of the shikimate pathway in this yeast.     

Altered prephenate dehydratase in phenylalanine-excreting mutants of Brevibacterium flavum.

The regulatory properties of three key enzymes in the phenylalanine biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase (DAHP synthetase) [EC 4.1.2.15], chorismate mutase [EC 5.4.99.5], and prephenate dehydratase [prephenate hydro-lyase (decarboxylating), EC 4.2.1.51] were compared in three phenylalanine-excreting mutants and the wild strain of Brevibacterium flavum. Regulation of DAHP synthetase by phenylalanine and tyrosine in these mutants did not change at all, but the specific activities of the mutant cell extracts increased 1.3- to 2.8-fold, as reported previously (1). Chorismate mutase activities in both the wild and the mutant strains were cumulatively inhibited by phenylalanine and tyrosine and recovered with tryptophan, while the specific activities of the mutants increased 1.3- to 2.8-fold, like those of DAHP synthetase. On the other hand, the specific activities of prephenate dehydratase in the mutant and wild strains were similar, when tyrosine was present. While prephenate dehydratase of the wild strain was inhibited by phenylalanine, tryptophan, and several phenylalanine analogues, the mutant enzymes were not inhibited at all but were activated by these effectors. Tyrosine activated the mutant enzymes much more strongly than the wild-type enzyme: in mutant 221-43, 1 mM tyrosine caused 28-fold activation. Km and the activation constant for tyrosine were slightly altered to a half and 6-fold compared with the wild-type enzyme, respectively, while the activation constants for phenylalanine and tryptophan were 500-fold higher than the respective inhibition constants of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 x 10(5), a half of that of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 X 10(5) a half of that of the wild type enzyme, while in the presence of tyrosine, phenylalanine, or tryptophan, it increased to that of the wild-type enzyme. Immediately after the mutant enzyme had been activated by tyrosine and then the tyrosine removed, it still showed about 10-fold higher specific activity than before the activation by tyrosine. However, on standing in ice the activity gradually fell to the initial level before the activation by tyrosine. Ammonium sulfate promoted the decrease of the activity. On the basis of these results, regulatory mechanisms for phenylalanine biosynthesis in vivo as well as mechanisms for the phenylalanine overproduction in the mutants are discussed.     

A single point mutation results in a constitutively activated and feedback-resistant chorismate mutase of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae ARO7 gene product chorismate mutase, a single-branch-point enzyme in the aromatic amino acid biosynthetic pathway, is activated by tryptophan and subject to feedback inhibition by tyrosine. The ARO7 gene was cloned on a 2.05-kilobase EcoRI fragment. Northern (RNA) analysis revealed a 0.95-kilobase poly(A)+ RNA, and DNA sequencing determined a 771-base-pair open reading frame capable of encoding a protein 256 amino acids. In addition, three mutant alleles of ARO7 were cloned and sequenced. These encoded chorismate mutases which were unresponsive to tyrosine and tryptophan and were locked in the on state, exhibiting a 10-fold-increased basal enzyme activity. A single base pair exchange resulting in a threonine-to-isoleucine amino acid substitution in the C-terminal part of the chorismate mutase was found in all mutant strains. In contrast to other enzymes in this pathway, no significant homology between the monofunctional yeast chorismate mutase and the corresponding domains of the two bifunctional Escherichia coli enzymes was found.     

Regulation of aromatic amino Acid biosynthesis in higher plants: I. Evidence for a regulatory form of chorismate mutase in etiolated mung bean seedlings

Etiolated mung bean seedlings were examined for chorismate mutase activity. Evidence for the occurrence of two forms of the enzyme (designated CM-1 and CM-2) was obtained by ammonium sulfate fractionation, anion exchange cellulose chromatography, and isoelectric focusing. The two forms showed distinctly different properties, as CM-1 was inhibited by phenylalanine and tyrosine and activated by tryptophan, but inhibition by phenylalanine and tyrosine was reversed by tryptophan. The other form, CM-2, was unaffected by any of the three aromatic amino acids. Isoelectric points of the two forms were CM-1, pH 4.6, and CM-2, pH 5.6. The molecular weights estimated by molecular sieving on Sephadex G-200 were CM-1, 50,000, and CM-2, 36,000.     

Elicitor induced secondary metabolism in Ruta graveolens L.

In vitro cultures of Ruta graveolens L. respond with rapid accumulation of acridone epoxides, furoquinolines and furanocoumarins, when challenged with autoclaved homogenate of the yeast Rhodotorula rubra. A transient increase of several enzymes of the respective biosynthetic pathways was measured but we still look for the key regulatory enzymes. We investigated whether the branch point enzymes of the shikimic acid pathway anthranilate synthase (AS) and chorismate mutase (CM) possibly play such a role. The two enzymes compete for chorismate. AS forms anthranilate, the precursor amino acid of acridone and furoquinoline alkaloids. CM channels chorismate into phenylalanine, tyrosine and phenylpropanoid biosynthesis. Elicitation resulted in a transient increase of the activity of both enzymes. Relative induction rates were 2–4 fold for AS and about 1.5 fold for CM. Constitutive CM activity, however, is about 1000 fold higher than AS activity. As in other plants 2 isoforms of CM are expected to be present in R. graveolens. A differential determination of the activity of the isoforms via the tryptophan activation rate proved to be ambiguous. Some evidence for the specific induction of a plastidic form of CM was obtained by inhibition of translation. The time courses of CM induction show CM not to be a key enzyme in elicitor induction of furanocoumarin accumulation. In comparison to other enzyme activities induction of anthranilate synthase activity corresponds closest to inducible acridone epoxide accumulation indicating a key role in its regulation. Induction of AS and CM was inhibited by actinomycin D and chloramphenicol while cycloheximid inhibited AS induction only.Abbreviations ACT actinomycin D - AS anthranilate synthase - CAP chloramphenicol - CHX cycloheximid - 4-CL 4-coumarate CoA ligase - CM chorismate mutase - DTT dithiothreitol - NMT S-adenosyl-L-methionine:anthranilic acid N-methyltransferase - PAL phenylalanine ammonia lyase - XOMT S-adenosylmethionine: xanthotoxol-O-methyltransferase     

Regulation of aromatic amino acid biosynthesis in higher plants. Properties of an aromatic amino acid-insensitive chorismate mutase (CM-2) from mung bean.

Chorismate mutase activity in etiolated mung bean seedlings is comprised of two isozyme forms designated CM-1 and CM-2. The chorismate mutase CM-2 form representing 50% of the extractable activity was purified 420-fold with a final yield of 30%. The final preparation contained three electrophoretically distinct species, one of which exhibited chorismate mutase activity. The highly purified CM-2 form possessed an estimated molecular weight of 36,000 and displayed a pH optimum of 6.9. Enzyme activity was unaffected by thiol-alkylating agents, reducing agents, EDTA, or divalent cations.In contrast to the CM-1 form, which was inhibited by phenylalanine and tyrosine and activated by tryptophan, the CM-2 form reported here was insensitive to all three aromatic amino acids and displayed normal Michaelis-Menten substrate saturation kinetics. The apparent K_0.5S of the CM-2 form was sensitive to temperature with values of 0.28, 0.20, and 0.094 mm at 20, 25, and 40 °C, respectively. Although a biphasic Arrhenius plot was observed with a break at 25 °C, further studies failed to reveal any temperature-, pH-, or substrate concentration-influenced cooperative interactions with either the aromatic amino acids or a number of secondary metabolites derived from the shikimic acid pathway. In addition, mixtures of potential metabolic effectors failed to reveal cooperative or synergistic regulatory patterns with the metabolites tested. Thus, although a primary role for the CM-1 form was proposed in regulation of the synthesis of phenylalanine and tyrosine for protein synthesis, no similar role can be proposed for the CM-2 form of the enzyme.     

Structure and function of a complex between chorismate mutase and DAHP synthase: efficiency boost for the junior partner

Chorismate mutase catalyzes a key step in the shikimate biosynthetic pathway towards phenylalanine and tyrosine. Curiously, the intracellular chorismate mutase of Mycobacterium tuberculosis (MtCM; Rv0948c) has poor activity and lacks prominent active‐site residues. However, its catalytic efficiency increases >100‐fold on addition of DAHP synthase (MtDS; Rv2178c), another shikimate‐pathway enzyme. The 2.35 Å crystal structure of the MtCM–MtDS complex bound to a transition‐state analogue shows a central core formed by four MtDS subunits sandwiched between two MtCM dimers. Structural comparisons imply catalytic activation to be a consequence of the repositioning of MtCM active‐site residues on binding to MtDS. The mutagenesis of the C‐terminal extrusion of MtCM establishes conserved residues as part of the activation machinery. The chorismate‐mutase activity of the complex, but not of MtCM alone, is inhibited synergistically by phenylalanine and tyrosine. The complex formation thus endows the shikimate pathway of M. tuberculosis with an important regulatory feature. Experimental evidence suggests that such non‐covalent enzyme complexes comprising an AroQ_δ subclass chorismate mutase like MtCM are abundant in the bacterial order Actinomycetales.     

Regulation of aromatic amino acid biosynthesis in the ribulose monophosphate cycle methylotroph Nocardia sp. 239

The regulation of aromatic amino acid biosynthesis in Nocardia sp. 239 was studied. In cell-free extracts 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase activity was inhibited in a cumulative manner by tryptophan, phenylalanine and tyrosine. Chorismate mutase was inhibited by both phenylalanine and tyrosine, whereas prephenate dehydratase was very sensitive to inhibition by phenylalanine. Tyrosine was a strong activator of the latter enzyme, whereas anthranilate synthase was inhibited effectively by tryptophan. No clear repression of the synthesis of these enzymes was observed during growth of the organism in the presence of the aromatic amino acids. It is therefore concluded that in Nocardia sp. 239 synthesis of these amino acids is mainly regulated by feedback inhibition. The molecular organization and kinetic properties of DAHP synthase were studied in more detail following its purification. The molecular weight of the native enzyme and its single subunit species were estimated to be 168,000 and 41,000, respectively, suggesting that the enzyme is a tetramer. Apparent K _m values for phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) were 45 and 370 M, respectively. Tryptophan, phenylalanine and tyrosine inhibited DAHP synthase in a competitive manner with respect to E4P, with apparent K _i values of 3, 160 and 180 M, respectively. In addition, tryptophan and E4P (apparent K _i values of 11 and 530 M, respectively) were found to exert an uncompetitive and competitive inhibition, respectively, towards PEP.Abbreviations DAHP 3-deoxy-D-arabino-heptulosonate 7-phosphate - E4P erythrose-4-phosphate - PEP phosphoenolpyruvate - RuMP ribulose monophosphate - HPLC high performance liquid chromatography - FPLC fast protein liquid chromatography - SDS sodium dodecyl sulphate     

The Biosynthetic Control in Aromatic Amino Acid Producing Mutants of Corynebacterium glutamicum

Regulatory properties of the enzymes involved in aromatic amino acid biosynthesis in the mutant of Corynebacterium glutamicum which produces a large amount of aromatic amino acids were examined. A phenylalanine auxotrophic l-tyrosine producer, pr-20, had a 3-deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthetase released from the feedback inhibition by l-phenylalanine, l-tyrosine and l-tryptophan and had a two-fold derepressed chorismate mutase. A pair of l-phenylalanine and l-tyrosine still strongly inhibited the chorismate mutase activity, though the enzyme was partially released from the inhibition by l-phenylalanine alone. A tyrosine auxotrophic l-phenylalanine producer, PFP-19-31, had a DAHP synthetase sensitive to the feedback inhibition by l-phenylalanine, l-tyrosine and l-tryptophan and had a prephenate dehydratase and a chorismate mutase both partially released from the feedback inhibition by l-phenylalanine. The mutant produced a large amount of prephenate as well as l-phenylalanine. A phenylalanine and tyrosine double auxotrophic l-tryptophan producer, Px-115-97, had an anthranilate synthetase partially released from the feedback inhibition by l-tryptophan and had a DAHP synthetase sensitive to the feedback inhibition. These data explained the mechanism of the production of aromatic amino acids by these mutants and supported the in vivo functioning of the control mechanisms of aromatic amino acid biosynthesis in C. glutamicum previously elucidated in vitro experiments.     

Absolute dependence of phenylalanine and tyrosine biosynthetic enzyme on tryptophan in Candida maltosa

Candida maltosa synthesizes phenylalanine and tyrosine only via phenylpyruvate and p-hydroxyphenylpyruvate. Tryptophan is absolutely necessary for the enzymatic reaction of chorismate mutase and prephenate dehydrogenase; activity of prephenate dehydratase can be increased 2.5-fold in the presence of tryptophan. Activation of the chorismate mutase, prephenate dehydratase and prephenate dehydrogenase by tryptophan is competitive with respect to chorismate and prephenate with Ka 0.06mM, 0.56mM and 1.7mM. In addition tyrosine is a competitive inhibitor of chorismate mutase (Ki = 0.55mM) and prephenate dehydrogenase (Ki = 5.5mM).