Aromatic Amino Acid Pathway Research Articles

Regulation at metabolic branch points of aromatic amino acid biosynthesis in Brevibacterium flavum.

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 1 5-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.

Production of an aromatic amino acid auxotroph of a trimethoprim-resistant Escherichia coli and deuteration of the aromatic amino acids in its dihydrofolate

Interpretation of the 1H-NMR spectra of Escherichia coli dihydrofolate reductase is complicated by the large number of overlapping resonances due to protonated aromatic amino acids. Deuteration of the aromatic protons of aromatic amino acid residues is one technique useful for simplifying the 1H-NMR spectra. Previous attempts to label the dihydrofolate reductase from over-producing strains of Escherichia coli were not completely successful. This labeling problem was solved by transducing via P1 phage a genetic block into the de novo biosynthetic pathway of aromatic amino acids in a trimethoprim resistant strain of E. coli, MB 3746. A new strain, MB 4065, is a very high level producer of dihydrofolate reductase and requires exogenous aromatic amino acids for growth, therefore allowing efficient labeling of its dihydrofolate reductase with exogenous deuterated aromatic amino acid.

Effect of mutation in the aromatic amino acid pathway on sporulation of Saccharomyces cerevisiae

Mutations in ARO1 and ARO2 genes coding for enzymes involved in the common part of the aromatic amino acid pathway completely block the sporulation of Saccharomyces cerevisiae when in a homozygous state, whereas mutations in all the other genes of the same pathway do not. This effect is not due to the lack of any intermediate metabolite but rather to the accumulation of a metabolite preceding chorismic acid. Shikimic acid or one of its precursors was identified as the possible inhibitor. The presence of the three aromatic amino acids in the sporulation medium restores the ability to undergo meiosis. This seems not to be due to a feedback inhibition of the first enzymes of the pathway but rather to a competition between aromatic amino acids and the inhibitor on a site specific for the meiotic process. The inhibition of sporulation seems to occur at a very early step in meiosis, as indicated by the lack of premeiotic DNA synthesis in aro1 and aro2 mutants.

Purification and properties of chorismate synthase from Bacillus subtilis.

Chorismatic synthase was purified to apparent homogeneity from Bacillus subtilis. The enzyme required NADPH-dependent flavin reductase, Mg2+, NADPH, and flavin (FMN or FAD) for activity. The molecular weight of chorismate synthase was 24,000 as determined by sodium dedecyl sulfate (SDS)-gel electrophoresis. The enzyme was also isolated in a complex form associated with NADPH-dependent flavin reductase and another enzyme of the aromatic amino acid pathway, dehydroquinate synthase. On SDS-gel electrophoresis, this form was resolved into three bands with molecular weights of 13,000, 17,000, and 24,000. The enzyme complex was easily dissociated and the dissociation resulted in a change in the chromatographic properties of NADPH-dependent flavin reductase which was no longer retained on phosphocellulose whereas chorismate synthase was still adsorbed. Chorismate synthase activity was linear with time and protein concentration, whereas partially purified preparations showed a significant lag period before the reaction took place. Moreover, crude or partially purified enzyme preparations were completely inactivated by dilution and the activity could be recovered by addition of flavin reductase. A possible role of NADPH-dependent flavin reductase in the activation and regulation of chorismate synthase activity is discussed.

Mis-regulation of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase does not account for growth inhibition by phenylalanine in Agmenellum quadruplicatum.

The growth of the blue-green bacterium, Agmenellum quadruplicatum, is inhibited in the presence of l-phenylalanine. This species has a single, constitutively synthesized 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthetase. l-Phenylalanine inhibits DAHP synthetase non-competitively with respect to both substrate reactants. Other aromatic amino acids do not inhibit the activity of DAHP synthetase. A common expectation for branch-point enzymes such as DAHP synthetase is a balanced pattern of feedback control by all of the ultimate end products. It seemed likely that growth inhibition might equate with defective regulation within the branched aromatic pathway. Accordingly, the possibility was examined that mis-regulation of DAHP synthetase by l-phenylalanine in wild-type cells causes starvation for precursors of the other aromatic end products. However, the molecular basis for growth inhibition cannot be attributed to l-phenylalanine inhibition of DAHP synthetase for the following reasons: (i) DAHP synthetase enzymes from l-phenylalanine-resistant mutants are more, rather than less, sensitive to feedback inhibition by l-phenylalanine. (ii) Shikimate not only fails to antagonize inhibition, but is itself inhibitory. (iii) Neither the sensitivity nor the completeness of l-phenylalanine inhibition of the wild-type enzyme in vitro appears sufficient to account for the potent inhibition of growth in vivo by l-phenylalanine. The dominating effect of l-phenylalanine in the control of DAHP synthetase appears to reflect a mechanism that prevents rather than causes growth inhibition by l-phenylalanine. The alteration of the control of DAHP synthetase in mutants selected for resistance to growth inhibition by l-phenylalanine did indicate that the cause for this metabolite vulnerability can be localized within the aromatic amino acid pathway. Apparently, an aromatic intermediate (between shikimate and the end products) accumulates in the presence of l-phenylalanine, causing toxicity by some unknown mechanism. It is concluded that phenylpyruvate, potentially formed by transamination of l-phenylalanine, is an unlikely cause of growth inhibition. Although several significant questions remain unanswered, our results suggest that single-effector control of DAHP synthetase, the first regulatory enzyme activity of a branched pathway, may be more appropriate than it would seem a priori.

Genes and isoenzymes controlling the first step in the aromatic amino acid biosynthesis in Schizosaccharomyces pombe

1. 1. Crude extracts of Schizosaccharomyces pombe wild-type were assayed in order to characterize the first enzyme of the aromatic amino acid pathway, 3-deoxy- d- arabino-heptulosonate-7-phosphate synthase (7-phospho-2-keto-3-deoxy- d- arabino-heptonate- d-erythrose-4-phosphate lyase (pyruvate-phosphorylating), EC 4.1.2.15) (DAHP synthase). Optimal conditions for activity have been found with a 0.1 M phosphate buffer (pH 6.5). Co 2+ and Mn 2+ stimulate activity whereas EDTA inhibits strongly. Inhibition by EDTA is reversed by Co 2+. The enzyme activity is feedback inhibited by tyrosine and phenylalanine but not by tryptophan. 2. 2. On analyzing the inhibition pattern in the activity peak obtained by Sephadex G-100 filtration, two overlapping regions with different degrees of inhibition by phenylalanine and tyrosine were found. 3. 3. DAHP synthase mutants were obtained and characterized. Aro2-C mutants show a reduced growth on minimal medium containing phenylalanine (1 mM) and aro1-C mutants grow poorly on minimal medium supplemented with tyrosine. In vitro the DAHP synthase activity of aro2-C mutants is inhibited to a greater extent by phenylalanine than in the wild-type, and aro-C mutants are more inhibited by tyrosine compared to the wild-type. 4. 4. Linkage relationships assign all aro1-C mutants to one locus, and all aro2-C mutants to another locus. The two genes are unlinked. 5. 5. Double mutants obtained from aro1-C × aro2-C crosses require tyrosine, phenylalanine, tryptophan and 4-aminobenzoate for growth. They do not grow on minimal medium or minimal medium supplemented with only one or with any combination of two aromatic amino acids plus 4-aminobenzoate. In vitro they show no DAHP synthase activity. 6. 6. These results show that in S. pombe the first step in the biosynthesis of aromatic amino acids is catalyzed by two isoenzymes.

Allosteric inhibition of [formula omitted] 7-phosphate synthetase by tyrosine, tryptophan and phenylpyruvate in Pseudomonas aeruginosa

1. 1. In Pseudomonas aerginosa the allosteric control of 3-deoxy- d -arabino- heptulosonate 7-phosphate (DAHP) synthetase, EC 4.1.2.15, by tyrosine has been known for some time. However, a more detailed analysis of the enzyme in crude extracts revealed sensitivity to inhibition by tryptophan and phenylpyruvate in addition to tyrosine. It is concluded that a single enzyme exists bearing three inhibitor specificities. Testing of various inhibitor combinations revealed no synergistic or antagonistic effects of aromatic metabolites. Combinations of the three inhibitors produced cummulative or less-than-cumulative effects. 2. 2. Tyrosine and phenylpyruvate were both capable of producing more than 90% inhibition at saturating concentrations of substrates, but tyrosine was about 18 times more potent than phenylpyruvate on a molar basis. although inhibition by tryptophan was limited to a maximum of 25–30%, relatively low concentrations were effective. It is concluded that inhibition by either tyrosine or tryptophan is competitive with respect to phosphoenolpyruvate and noncompetitive with respect to erythrose-4- P. On the other hand, phenylpyruvate inhibition appears to be competitive with respect to erythrose-4- P and noncompetitive with respect to phosphoenolpyruvate. 3. 3. Hence, one metabolite of each terminal branch of the aromatic amino acid pathway exhibits allosteric influence upon the activity of DAHP synthetase, an observation in accord with expectations for balanced participation of multiple end products in the regulation of a branched pathway. The quantitative effects actually realized in vivo would depend in complex fashion upon the intracellular concentrations of tyrosine, tryptophan and phenylpyruvate and upon the intracellular concentrations of erythrose-4- P and phosphoenolpyruvate. 4. 4. A variety of physiological experiments with wild type and mutant cells led to the conclusion that the synthesis of DAPH synthetase is not controlled by repression in P. aeruginosa.

Chorismate mutase from Streptomyces aureofaciens: a heat-stable enzyme.

Chorismate mutase from Streptomyces aureofaciens was purified 12-fold. This enzyme preparation did not show any activity when tested for anthranilate synthetase, prephenate dehydrogenase, or prephenate dehydratase. The catalytic activity of chorismate mutase has a broad optimum between pH 7 and 8. The initial velocity data followed regular Michaelis-Menten kinetics with a K(m) of 5.3 x 10(-4) M, and the molecular weight of the enzyme was determined by sucrose gradient centrifugation to be 50,000. Heat inactivation of chorismate mutase, which occurs above temperatures of 60 C, is reversible. The enzyme activity can be restored even when chorismate mutase is treated at the temperature of a boiling-water bath for 15 min. Heat-denatured and renatured enzymes showed the same Michaelis constant and the same molecular weight as the native enzyme. l-Phenylalanine, l-tyrosine, l-tryptophan, and metabolites of the aromatic amino acid pathway were tested as potential modifiers of chorismate mutase activity. The activity of the enzyme was inhibited by none of these substances. Chorismate mutase of S. aureofaciens was not repressed in cells grown in minimal medium supplemented with l-phenylalanine, l-tyrosine, or l-tryptophan.

3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase of Streptomyces aureofaciens Tü 24. I. Partial purification and properties.

Abstract 1. 1. 3-Deoxy- d -arabino- heptulosonate-7-phosphate synthase ( 7-phospho-2-oxo-3-deoxy- d -arabino- heptonate d -erythrose-4-phosphate-lyase (pyruvate phosphorylating), EC 4.1.2.15, DAHP synthase), the first enzyme of the aromatic amino acid pathway, has been purified 170 fold from Streptomyces aureofaciens Tu 24. DAHP synthase is a single enzyme with a molecular weight of 97 000. 2. 2. The enzyme has a broad pH stability optimum between pH 6 and 8. The catalytic activity has a pH optimum of 7. EDTA causes a partial inactivation of the enzyme, which can be reversed by Co 2+ . The activation energy of the enzymatic reaction was estimated to be 16 500 cal/mole. Heat inactivation occurs above 50°. Phosphoenolpyruvate protects the enzyme against heat inactivation, whereas the presence of erythrose 4-phosphate increases the rate of inactivation. 3. 3. The initial velocity data follow regular Michaelis-Menten kinetics without any detectable kinetic evidence for subunit interaction. On the basis of kinetic experiments the mechanism of enzyme action is ping-pong, and the first substrate is phosphoenolpyruvate. The absolute Michaelis constants of the enzyme have been determined to be 3·10 −4 M for phosphoenolpyruvate and 1.6·10 −4 M for erythrose 4-phosphate. 4. 4. Quantitative determination of phosphoenolpyruvate and erythrose 4-phosphate by the use of a DAHP synthase preparation of Streptomyces aureofaciens is described. The method shows a deviation of ±0.7% from the theoretical values.

Metabolic Interlock: THE ROLE OF THE SUBORDINATE TYPE OF ENZYME IN THE REGULATION OF A COMPLEX PATHWAY

Abstract The activity of chorismate mutase plays a strategic role in mediating the flow of chorismate between the synthesis of tyrosine and phenylalanine on the one hand, and of tryptophan and a number of aromatic vitamins on the other hand. A gene specifying chorismate mutase in strain 23 of Bacillus subtilis was introduced into the genetic background of strain 168 by deoxyribonucleate transformation. The newly introduced chorismate mutase has a specific activity which is 10 times greater than that of strain 168. This hybrid strain was used to study the effect of the altered activity of chorismate mutase upon the routing of common pathway precursors into the divergent terminal branches of the aromatic amino acid pathway of biosynthesis. 1. Compared to strain 168, the hybrid strain (carrying chorismate mutase-H) was more resistant to growth inhibition by low concentrations of d-tyrosine and less resistant to growth inhibition by low concentrations of 5-methyltryptophan. The hybrid strain was resistant to a weak inhibition of growth which may occur in derivatives of strain 168 in the presence of the combination of tryptophan and tyrosine. The hybrid strain also possesses an increased capacity for incorporation of exogenous shikimate. 2. Chorismate mutase was product-inhibited by prephenate, a finding which we suggest is physiologically significant—a part of the pattern of sequential feedback inhibition which regulates the aromatic pathway in B. subtilis. 3. The considerable ability of the hybrid strain to adjust to the unbalancing influence of chorismate mutase-H is attributed to the compensatory capabilities of allosteric and repressive controls existing in the pathway. Accordingly, when these regulatory controls in the terminal branches of the pathway were lost by mutation, the full potential of an alteration in level of chorismate mutase to unbalance the flow of common precursors to the terminal branches of the pathway was unmasked. The dependence of the aromatic pathway in B. subtilis upon the regulatory enzymes in its terminal branches for the appropriate function of chorismate mutase leads us to describe this chorismate mutase as an example of a subordinate enzyme. 4. Hybrid strains of B. subtilis are widely distributed, often inadvertently. The properties of hybrid strains explain a number of findings in the literature.

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

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.

Regulation of enzyme synthesis in the aromatic amino acid pathway of Bacillus subtilus.

The control of the synthesis of certain key enzymes of aromatic amino acid biosynthesis was studied. Tyrosine represses the first enzyme of the 3-deoxy-d-arabino heptulosonic acid 7-phosphate pathway, DAHP synthetase, as well as shikimate kinase and chorismate mutase about fivefold in cultures grown under conditions limiting the synthesis of the aromatic amino acids. A mixture of tyrosine and phenylalanine represses twofold further. Tryptophan does not appear to be involved in the control of these enzymes. The specific activity of at least one early enzyme, dehydroquinase, remains essentially constant under a variety of nutritional supplementations. Two enzymes in the terminal branches are repressed by the amino acids they help to synthesize: prephenate dehydrogenase can be repressed fourfold by tyrosine, and anthranilate synthetase can be repressed over 200-fold by tryptophan. There is no evidence that phenylalanine represses prephenate dehydratase. Regulatory mutants have been isolated in which various enzymes of the pathway are no longer repressible. One class is derepressed for several of the prechorismate enzymes, as well as chorismate mutase and prephenate dehydrogenase. In another mutant, several enzymes of tryptophan biosynthesis are no longer repressible. Thus, the rate of synthesis of enzymes at every stage of the pathway is under control of various aromatic amino acids. Tyrosine and phenylalanine control the synthesis of enzymes involved in the synthesis of the three aromatic amino acids. Each terminal branch is under the control of its end product.

Comparative regulation of isoenzymic 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetases in microorganisms.

The independent control of regulatory isoenzymes by different metabolites constitutes one well-known pattern of control in branched metabolic pathways. This pattern was previously found to be widely distributed in the aromatic amino acid pathway of microorganisms in the case of the first enzyme of the sequence, 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthetase. The comparative stability of the isoenzymes as well as the effect of aromatic amino acids in the growth medium upon the levels of the individual isoenzymes were shown for Salmonella typhimurium. Several lines of evidence are discussed to demonstrate the strong reliance of Escherichia coli upon the phenylalanine-sensitive isoenzyme for the ordinary biosynthetic needs of wild-type strains. The frequent occurrence of "dominant" isoenzyme species which resist repressive effects of the inhibitory end products was noted. The lack of an obligatory correlation of the level of an isoenzyme activity and the synthesis of the end product which specifically controls its activity is used to discount the possibility that each isoenzyme might feed a unique and separate metabolic pool of end-product precursor. An isoenzymic DAHP synthetase sensitive to feedback inhibition by low levels of tryptophan was fractionated from tyrosine- and phenylalanine-sensitive isoenzymes in cell-free extracts of Neurospora crassa.