Enzymatic Basis for Overproduction of Tryptophan and Its Metabolites in Hansenula polymorpha Mutants

3-Deoxy-d-arabinoheptulosonate 7-phosphate (DAHP) synthetase and anthranilate synthetase are key regulatory enzymes in the aromatic amino acid biosynthetic pathway. The DAHP synthetase activity of Hansenula polymorpha was subject to additive feedback inhibition by phenylalanine and tyrosine but not by tryptophan. The synthesis of DAHP synthetase in this yeast was not repressed by exogenous aromatic amino acids, singly or in combinations. The activity of anthranilate synthetase was sensitive to feedback inhibition by tryptophan, but exogenous tryptophan did not repress the synthesis of this enzyme. Nevertheless, internal repression of anthranilate synthetase probably exists, since the content of this enzyme in H. polymorpha strain 3-136 was double that in the wild-type and less sensitive 5-fluorotryptophan-resistant strains. The biochemical mechanism for the overproduction of indoles by the 5-fluorotryptophan-resistant mutants was due primarily to a partial desensitization of the anthranilate synthetase of these strains to feedback inhibition by tryptophan. These results support the concept that inhibition of enzyme activities rather than enzyme repression is more important in the regulation of aromatic amino acid biosynthesis in H. polymorpha.     

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.     

Hidden overflow pathway to L-phenylalanine in Pseudomonas aeruginosa

Pseudomonas aeruginosa is representative of a large group of pseudomonad bacteria that possess coexisting alternative pathways to L-phenylalanine (as well as to L-tyrosine). These multiple flow routes to aromatic end products apparently account for the inordinate resistance of P. aeruginosa to end product analogs. Manipulation of carbon source nutrition produced a physiological state of sensitivity to p-fluorophenylalanine and m-fluorophenylalanine, each a specific antimetabolite of L-phenylalanine. Analog-resistant mutants obtained fell into two classes. One type lacked feedback sensitivity of prephenate dehydratase and was the most dramatic excretor of L-phenylalanine. The presence of L-tyrosine curbed phenylalanine excretion to one-third, a finding explained by potent early-pathway regulation of 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase-Tyr (a DAHP synthase subject to allosteric inhibition by L-tyrosine). The second class of regulatory mutants possessed a completely feedback-resistant DAHP synthase-Tyr, the major species (greater than 90%) of two isozymes. Deregulation of DAHP synthase-Tyr resulted in the escape of most chorismate molecules produced into an unregulated overflow route consisting of chorismate mutase (monofunctional), prephenate aminotransferase, and arogenate dehydratase. In the wild type the operation of the overflow pathway is restrained by factors that restrict early-pathway flux. These factors include the highly potent feedback control of DAHP synthase isozymes by end products as well as the strikingly variable abilities of different carbon source nutrients to supply the aromatic pathway with beginning substrates. Even in the wild type, where all allosteric regulation in intact, some phenylalanine overflow was found on glucose-based medium, but not on fructose-based medium. This carbon source-dependent difference was much more exaggerated in each class of regulatory mutants.     

Repression of aromatic amino acid biosynthesis in Escherichia coli K-12

Mutants of Escherichia coli K-12 were isolated in which the synthesis of the following, normally repressible enzymes of aromatic biosynthesis was constitutive: 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthetases (phe and tyr), chorismate mutase T-prephenate dehydrogenase, and transaminase A. In the wild type, DAHP synthetase (phe) was multivalently repressed by phenylalanine plus tryptophan, whereas DAHP synthetase (tyr), chorismate mutase T-prephenate dehydrogenase, and transaminase A were repressed by tyrosine. DAHP synthetase (tyr) and chorismate mutase T-prephenate dehydrogenase were also repressed by phenylalanine in high concentration (10(-3)m). Besides the constitutive synthesis of DAHP synthetase (phe), the mutants had the same phenotype as strains mutated in the tyrosine regulatory gene tyrR. The mutations causing this phenotype were cotransducible with trpA, trpE, cysB, and pyrF and mapped in the same region as tyrR at approximately 26 min on the chromosome. It is concluded that these mutations may be alleles of the tyrR gene and that synthesis of the enzymes listed above is controlled by this gene. Chorismate mutase P and prephenate dehydratase activities which are carried on a single protein were repressed by phenylalanine alone and were not controlled by tyrR. Formation of this protein is presumed to be controlled by a separate, unknown regulator gene. The heat-stable phenylalanine transaminase and two enzymes of the common aromatic pathway, 5-dehydroquinate synthetase and 5-dehydroquinase, were not repressible under the conditions studied and were not affected by tyrR. DAHP synthetase (trp) and tryptophan synthetase were repressed by tryptophan and have previously been shown to be under the control of the trpR regulatory gene. These enzymes also were unaffected by tyrR.     

Regulation of Aromatic Amino Acid Biosynthesis and Production of Tyrosine and Phenylalanine m Brevibacterium flavum

In Brevibacterium flavum, prephenate dehydratase in the phenylalanine specific biosynthetic pathway was strongly inhibited by phenylalanine and activated by tyrosine. Furthermore. the inhibition by phenylalanine was completely reversed by tyrosine. Inhibition by tyrosine of prephenate dehydrogenase in the tyrosine specific pathway was very weak. Overall regulation mechanism of the aromatic amino acid biosynthesis in B. flavum was proposed on the bases of these results and the previous findings on 3-deoxy-D-arabino-heptulosonate-7- phosphate synthetase(DAHP synthetase*) of the common pathway and on anthranilate synthetase of the tryptophan specific pathway. Two types of m-fluorophenylalanine(mFP) resistant mutants which accumulated phenylalanine alone or both phenylalanine and tyrosine, respectively, were derived. The accumulation in the former mutants was inhibited by tyrosine, but that in the latter was affected neither by tyrosine nor by phenylalanine. DAHP synthetase of the latter mutants had been desensitized from the synergistic feedback inhibition by tyrosine and phenylalanine, while prephenate dehydratase of the former mutants had been desensitized in the feedback inhibition by phenylalanine. Tyrosine auxotroph accumulated phenylalanine under tyrosine limitation and its accumulation was inhibited by the excessive addition of tyrosine. Phenylalanine auxotroph accumulated tyrosine under phenylalanine limitation and its accumulation was inhibited by the excessive addition of phenylalanine. These results in vivo strongly supported the proposed regulation mechanism in which synthesis of phenylalanine in preference to tyrosine was assumed.     

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.     

Enzymological basis for growth inhibition by L-phenylalanine in the cyanobacterium Synechocystis sp. 29108

The pattern of allosteric control in the biosynthetic pathway for aromatic amino acids provides a basis to explain vulnerability to growth inhibition by l-phenylalanine (0.2 mM or greater) in the unicellular cyanobacterium Synechocystis sp. 29108. We attribute growth inhibition to the hypersensitivity of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase to feedback inhibition by l-phenylalanine. Hyperregulation of this initial enzyme of aromatic biosynthesis depletes the supply of precursors needed for biosynthesis of l-tyrosine and l-tryptophan. Consistent with this mechanism is the total reversal of phenylalanine inhibition by a combination of tyrosine and tryptophan. Inhibited cultures also contained decreased levels of phycocyanin pigments, a characteristic previously correlated with amino acid starvation in cyanobacteria. l-Phenylalanine is a potent noncompetitive inhibitor (with both substrates) of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase, whereas l-tyrosine is a very weak inhibitor. Prephenate dehydratase also displays allosteric sensitivity to phenylalanine (inhibition) and to tyrosine (activation). Both 2-fluoro and 4-fluoro derivatives of phenylalanine were potent analog antimetabolites, and these were used in addition to l-phenylalanine as selective agents for resistant mutants. Mutants were isolated which excreted both phenylalanine and tyrosine, the consequence of an altered 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase no longer sensitive to feedback inhibition. Simultaneous insensitivity to l-tyrosine suggests that l-tyrosine acts as a weak analog mimic of l-phenylalanine at a common binding site. Prephenate dehydratase in the regulatory mutants was unaltered. Surprisingly, in view of the lack of regulation in the tyrosine branchlet of the pathway, such mutants excrete more phenylalanine than tyrosine, indicating that l-tyrosine activation dominates l-phenylalanine inhibition of prephenate dehydratase in vivo. In mutant Phe r19 the loss in allosteric sensitivity of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase was accompanied by a threefold increase in specific activity. This could suggest that existence of a modest degree of repression control (autogenous) over 3-deoxy-d-arabinoheptulosonate synthase, although other explanations are possible. Specific activities of chorismate mutase, prephenate dehydratase, shikimate/nicotinamide adenine dinucleotide phosphate dehydrogenase, and arogenate/nicotinamide adenine dinucleotide phosphate dehydrogenase in mutant Phe r19 were identical with those of the wild type.     

Cross pathway regulation: effect of histidine on the synthesis and activity of enzymes of aromatic acid biosynthesis in Bacillus subtilis

l-Histidine and, to a lesser degree, l-phenylalanine at concentrations of 10(-4)m inhibit the growth of leaky mutants (bradytrophs) of Bacillus subtilis that are deficient in the synthesis of p-hydroxyphenylpyruvate, the first intermediate specific to tyrosine synthesis. The inhibition can be overcome by growth factor amounts of l-tyrosine and p-hydroxyphenylpyruvate. Histidine and phenylalanine are capable of inhibiting the synthesis of tyrosine in several ways, and the major physiological effect which results in growth inhibition has not been established. Both l-histidine and l-phenylalanine inhibit the activity of prephenate dehydrogenase at concentrations about 100-fold higher than the inhibitory concentration of l-tyrosine. Histidine also appears to repress the synthesis of prephenate dehydrogenase because a histidine bradytroph growing in histidine-supplemented medium has a twofold lower level of this enzyme than the same cells growing in unsupplemented medium. These same two amino acids also inhibit the growth of a bradytroph deficient in dehydroquinate synthetase, an early enzyme in the pathway of tyrosine, phenylalanine, and tryptophan synthesis. The inhibition is overcome by a combination of tyrosine and phenylalanine. Histidine-resistant derivatives of both the prephenate dehydrogenase and dehydroquinate synthetase-deficient strains, which simultaneously have gained resistance to phenylalanine, have been isolated. Most of these resistant mutants synthesize additional tyrosine compared with the parent strain. One class of resistant mutants excretes tyrosine and has a number of enzymes of aromatic acid synthesis which are no longer repressible by any combination of the aromatic amino acids. Tyrosine inhibits the growth of histidine bradytrophs. Histidine, at growth factor levels, overcomes the inhibition.     

Phenylalanine and tyrosine biosynthesis in Escherichia coli K-12: mutants derepressed for 3-deoxy-D-arabinoheptulosonic acid 7-phosphate synthetase (phe), 3-deoxy-D-arabinoheptulosonic acid 7-phosphate synthetase (tyr), chorismate mutase T-prephenate dehydrogenase, and transaminase A

Mutant strains of Escherichia coli have been isolated in which the synthesis of 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthetase (phe) is derepressed, in addition to those enzymes of tyrosine biosynthesis previously shown to be controlled by the gene tyrR. The major enzyme of the terminal pathway of phenylalanine biosynthesis chorismate mutase-prephenate dehydratase is not derepressed in these strains. Genetic analysis of the mutants shows that the mutation or mutations causing derepression map close to previously reported tyrR mutations. A study of one of the mutations has shown it to be recessive to the wild-type allele in a diploid strain. It is proposed that the tyrR gene product is involved in the regulation of the synthesis of DAHP synthetase (phe) as well as the synthesis of DAHP synthetase (tyr), chorismate mutase-prephenate dehydrogenase, and transaminase A.     

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.     

Regulatory control of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthetase in Streptomyces antibioticus

Streptomyces antibioticus possesses a tryptophan-inhibitable 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthetase whose synthesis is also repressed by L-tryptophan. Studies of the DAHP synthetase obtained by ammonium sulfate fractionation of a crude extract derived from S. Antibioticus revealed that the enzymic activity was only partially inhibited by tryptophan. Inhibition of the DAHP synthetase activity was strongly pH dependent at values below 7.0. A number of tryptophan analogues was noted to inhibit the enzyme; by contrast, other aromatic amino acid end products failed to affect DAHP synthetase activity. Chorismic acid, a key intermediate in aromatic amino acid biosynthesis, was ineffective as an inhibitor when used alone; however, if supplied with L-tryptophan, a further reduction of DAHP synthetase activity (15--25%) was routinely observed.     

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.     

Regulation of the Escherichia coli tryptophan operon by early reactions in the aromatic pathway

7-Methyltryptophan (7MT) or compounds which can be metabolized to 7MT, 3-methylanthranilic acid (3MA) and 7-methylindole, cause derepression of the trp operon through feedback inhibition of anthranilate synthetase. Tyrosine reverses 3MA or 7-methylindole derepression, apparently by increasing the amount of chorismic acid available to the tryptophan pathway. A mutant isolated on the basis of 3MA resistance (MAR 13) was found to excrete small amounts of chorismic acid and to have a feedback-resistant phenylalanine 3-deoxy-d-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase. Genetic evidence indicates that the mutation conferring 3MA resistance and feedback resistance is very closely linked to aroG, the structural gene for the DAHP synthetase (phe). Since feedback inhibition of anthranilate synthetase by l-tryptophan (or 7MT) is competitive with chorismic acid, alterations in growth conditions (added tyrosine) or in a mutant (MAR 13) which increase the amount of chorismic acid available to the tryptophan pathway result in resistance to 7MT derepression. Owing to this competitive nature of tryptophan feedback inhibition of anthranilate synthetase by chorismic acid, the early pathway apparently serves to exert a regulatory influence on tryptophan biosynthesis.     

Mutants of Escherichia coli with an altered tyrosyl-transfer ribonucleic acid synthetase

We have isolated several mutants defective in the gene for tyrosyl-transfer ribonucleic acid (tRNA) synthetase (tyrS). One of these mutants is described in detail. It was isolated as a tyrosine auxotroph with defects both in the tyrosyl-tRNA synthetase and in the tyrosine biosynthetic enzyme, prephenate dehydrogenase. It also had derepressed levels of the tyrosine-specific 3-deoxy-d-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase. The latter finding suggested that a wild-type tyrS gene was required for repression of the tyrosine biosynthetic enzymes. The following results demonstrated that this hypothesis was not correct. (i) When the defective tyrS gene was transferred to another strain, the tyrosine-specific DAHP synthetase in that strain was not derepressed, and (ii) two other mutants with defective tyrosyl-tRNA synthetases had repressed levels of the tyrosine biosynthetic enzymes. The tyrS gene was located near minute 32 on the Escherichia coli chromosome by interrupted mating experiments.     

Regulation of the tryptophan synthetic enzymes in Clostridium butyricum

Experiments concerned with the regulation of the tryptophan synthetic enzymes in anaerobes were carried out with a strain of Clostridium butyricum. Enzyme activities for four of the five synthetic reactions were readily detected in wild-type cells grown in minimal medium. The enzymes mediating reactions 3, 4, and 5 were derepressed 4- to 20-fold, and the data suggest that these enzymes are coordinately controlled in this anaerobe. The first enzyme of the pathway, anthranilate synthetase, could be derepressed approximately 90-fold under these conditions, suggesting that this enzyme is semicoordinately controlled. Mutants resistant to 5-methyl tryptophan were isolated, and two of these were selected for further analysis. Both mutants retained high constitutive levels of the tryptophan synthetic enzymes even in the presence of repressing concentrations of tryptophan. The anthranilate synthetase from one mutant was more sensitive to feedback inhibition by tryptophan than the enzyme from wild-type cells. The enzyme from the second mutant was comparatively resistant to feedback inhibition by tryptophan. Neither strain excreted tryptophan into the culture fluid. Tryptophan inhibits anthranilate synthetase from wild-type cells noncompetitively with respect to chorismate and uncompetitively with respect to glutamine. The Michaelis constants calculated for chorismate and glutamine are 7.6 x 10(-5)m and 6.7 x 10(-5)m, respectively. The molecular weights of the enzymes estimated by zonal centrifugation in sucrose and by gel filtration ranged from 24,000 to 89,000. With the possible exception of a tryptophan synthetase complex, there was no evidence for the existence of other enzyme aggregates. The data indicate that tryptophan synthesis is regulated by repression control of the relevant enzymes and by feedback inhibition of anthranilate synthetase. That this enzyme system more closely resembles that found in Bacillus than that found in enteric bacteria is discussed.     

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.     

Allostery of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase in Clostridium: another conserved generic characteristic

Enzymological studies were done to characterize the allosteric control of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthetase in three species of Clostridium. Allosteric control was identified as feedback inhibition by phenylalanine and was qualitatively similar for the DAHP synthetases of C. butyricum, C. acetobutylicum, and C. tetanomorphum. Quantitative differences in the enzymology and kinetics of allosteric control distinguished C. tetanomorphum from C. butyricum and C. acetobutylicum. Crude extracts contained apparent proteolytic activity which could be fractionated from DAHP synthetase. The proteolytic activity was more labile than DAHP synthetase in extracts and was progressively inactivated by serial freeze-thaw treatments. Protease activity was at least partially inhibited by phenylmethylsulfonyl-fluoride. The method of comparative allostery of DAHP synthetase distinguishes the genera Bacillus and Clostridium, each having a strongly conserved pattern of regulation for DAHP synthetase. The data reinforce previous conclusions that allosteric control patterns governing the activity of DAHP synthetase are stable, reliable generic characteristics.     

Corynebacterium glutamicum contains 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases that display novel biochemical features

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 2.5.1.54) catalyzes the first step of the shikimate pathway that finally leads to the biosynthesis of aromatic amino acids phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr). In Corynebacterium glutamicum ATCC 13032, two chromosomal genes, NCgl0950 (aroF) and NCgl2098 (aroG), were located that encode two putative DAHP synthases. The deletion of NCgl2098 resulted in the loss of the ability of C. glutamicum RES167 (a restriction-deficient strain derived from C. glutamicum ATCC 13032) to grow in mineral medium; however, the deletion of NCgl0950 did not result in any observable phenotypic alteration. Analysis of DAHP synthase activities in the wild type and mutants of C. glutamicum RES167 indicated that NCgl2098, rather than NCgl0950, was involved in the biosynthesis of aromatic amino acids. Cloning and expression in Escherichia coli showed that both NCgl0950 and NCgl2098 encoded active DAHP synthases. Both the NCgl0950 and NCgl2098 DAHP synthases were purified from recombinant E. coli cells and characterized. The NCgl0950 DAHP synthase was sensitive to feedback inhibition by Tyr and, to a much lesser extent, by Phe and Trp. The NCgl2098 DAHP synthase was slightly sensitive to feedback inhibition by Trp, but not sensitive to Tyr and Phe, findings that were in contrast to the properties of previously known DAHP synthases from C. glutamicum subsp. flavum. Both Co2+ and Mn2+ significantly stimulated the NCgl0950 DAHP synthase's activity, whereas Mn2+ was much more stimulatory than Co2+ to the NCgl2098 DAHP synthase's activity.     

Feedback regulation of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase from a marine bacterium, Vibrio MB22

A marine bacterium, Vibrio MB22, has been studied to determine the pattern of feedback regulation of the first enzyme unique to the biosynthesis of the aromatic amino acids, 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthetase. The crude extract was used to study response of the enzyme to various salts as well as possible feedback inhibitors. Ethylenediaminetetraacetic acid was found to be inhibitory to enzyme activity, and only CoCl(2), of the salts tested, allowed full recovery as well as apparent stimulation of the DAHP synthetase activity. The DAHP synthetase activity was inhibited solely by the aromatic amino acids, tyrosine, tryptophan, and phenylalanine, of the possible effectors tested. Further work demonstrated the existence of three isozymes of DAHP synthetase, each primarily inhibited by one of the aromatic amino acids.     

Regulation of the aromatic pathway in the cyanobacterium Synechococcus sp. strain Pcc6301 (Anacystis nidulans).

A pattern of allosteric control for aromatic biosynthesis in cyanobacteria relies upon early-pathway regulation as the major control point for the entire branched pathway. In Synechococcus sp. strain PCC6301 (Anacystis nidulans), two enzymes which form precursors for L-phenylalanine biosynthesis are subject to control by feedback inhibition. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (first pathway enzyme) is feedback inhibited by L-tyrosine, whereas prephenate dehydratase (enzyme step 9) is feedback inhibited by L-phenylalanine and allosterically activated by L-tyrosine. Mutants lacking feedback inhibition of prephenate dehydratase excreted relatively modest quantities of L-phenylalanine. In contrast, mutants deregulated in allosteric control of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase excreted large quantities of L-phenylalanine (in addition to even greater quantities of L-tyrosine). Clearly, in the latter mutants, the elevated levels of prephenate must overwhelm the inhibition of prephenate dehydratase by L-phenylalanine, an effect assisted by increased intracellular L-tyrosine, an allosteric activator. The results show that early-pathway flow regulated in vivo by 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase is the dominating influence upon metabolite flow-through to L-phenylalanine. L-Tyrosine biosynthesis exemplifies such early-pathway control even more simply, since 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase is the sole regulatory enzyme subject to end-product control by L-tyrosine.