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.     

Regulation of aromatic amino acid biosynthesis in phenazine-producing strains of Pseudomonas

Regulation of 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthetase was studied in eight strains of Pseudomonas which synthesize phenazine compounds. Repression studies with individual aromatic amino acids led to the finding that enzyme synthesis was repressed in only one strain, P. aureofaciens B1543p, and by only one amino acid, l-tyrosine. Feedback inhibition by the aromatic amino acids varied from strain to strain in terms of the type of inhibitory control, and the particular acid or acids which inhibited. Prephenate and chorismate, as well as a number of naturally occurring phenazine compounds, inhibited the DAHP synthetase activity to varying degrees.     

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.     

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.     

DAHP Synthetase and Its Control in Corynebacterium glutamicum

Properties of 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthetase from Corynebacterium glutamicum were examined using the cell free extract. The optimum pH for the reaction was broad ranging from 5.5 to 7.0 and the optimum temperature was 37°C. Co²⁺ inhibited the enzyme activity at 20°C, whereas Co²⁺ apparently stimulated the enzyme activity at 37°C because the ion protected the enzyme from inactivation at 37°C. Co²⁺ reversed the inhibition of the enzyme activity by EDTA. The activity of DAHP synthetase was feedback inhibited only weakly by l-phenylalanine, l-tyrosine or l-tryptophan alone, but was strongly inhibited synergistically by l-phenylalanine and l-tyrosine. l-Tryptophan enhanced the inhibition by the pair of l-tyrosine and l-phenylalanine. Maximal inhibition was near 90 % in the simultaneous presence of the three amino acids. Sensitivity of the enzyme to the inhibitors was lost during the purification process of the enzyme or during the reaction at 37°C. Especially sensitivity to l-tryptophan was easily lost. Co²⁺ protected the enzyme from the desensitization. Mutants resistant to p-fluorophenylalanine plus l-tyrosine (or 3-aminotyrosine) had DAHP synthetase which was released from the feedback inhibition by the three amino acids. The formation of the enzyme was not affected by aromatic amino acids.     

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.     

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.     

Biochemistry and genetics of chloramphenicol production

In Streptomyces venezuelae, chloramphenicol is derived by an unusual diversion of chorismate, the branchpoint intermediate of the pathway involved in the biosynthesis of aromatic amino acids. In the chloramphenicol-producing organism, the DAHP synthetase was neither feedback inhibited nor repressed. Chorismate mutase was not repressed or inhibited by the intermediates or end-products of the shikimate-chorismate pathway. However, anthranilate synthetase and prephenate dehydratase are feedback inhibited by tryptophan and phenylalanine, respectively. During growth, when primary metabolism is not perfectly coordinated, decreasing demand for aromatic amino acids results in shunting of chorismate towards chloramphenicol biosynthesis.The endogenous synthesis of chloramphenicol produced by Streptomyces venezuelae is inhibited by the increasing concentration of chloramphenicol in the medium. Arylamine synthetase, the first enzyme involved in chloramphenicol biosynthesis, is repressed by the secreted chloramphenicol, by dl-p-aminophenylalanine and l-threo-p-aminophenylserinol. The excess intracellular chorismate pool is diverted to other aromatic shunt metabolites if biosynthesis of chloramphenicol is inhibited. There appears to be a glutamine binding protein subunit which is shared by several enzymes involved in amination of the aromatic ring of chorismate.Chloramphenicol producing organism also inactivated intracellular chloramphenicol. However, the resistance of the streptomycetes is due to inducible impermeability of the organism to chloramphenicol during antibiotic production. Streptomyces venezuelae is sensitive to chloramphenicol when it is not engaged in antibiotic production. The resistance to and production of chloramphenicol are induced simultaneously.A linkage map for 17 marker loci using Streptomyces venezuelae has been constructed. Restriction enzyme map of a plasmid from the chloramphenicol-producing streptomycetes has also been developed. The role of the plasmid in chloramphenicol biosynthesis and the life-cycle of the Streptomyces venezuelae is not yet understood.     

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.     

Regulated enzymes of aromatic amino acid synthesis: control, isozymic nature, and aggregation in Bacillus subtilis and Bacillus licheniformis

Several regulated enzymes involved in aromatic amino acid synthesis were studied in Bacillus subtilis and B. licheniformis with reference to organization and control mechanisms. B. subtilis has been previously shown (23) to have a single 3-deoxy-d-arabinoheptulosonate 7-phosphate (DAHP) synthetase but to have two isozymic forms of both chorismate mutase and shikimate kinase. Extracts of B. licheniformis chromatographed on diethylaminoethyl (DEAE) cellulose indicated a single DAHP synthetase and two isozymic forms of chorismate mutase, but only a single shikimate kinase activity. The evidence for isozymes has been supported by the inability to find strains mutant in these activities, although strains mutant for the other activities were readily obtained. DAHP synthetase, one of the isozymes of chorismate mutase, and one of the isozymes of shikimate kinase were found in a single complex in B. subtilis. No such complex could be detected in B. licheniformis. DAHP synthetase and shikimate kinase from B. subtilis were feedback-inhibited by chorismate and prephenate. DAHP synthetase from B. licheniformis was also feedback-inhibited by these two intermediates, but shikimate kinase was inhibited only by chorismate. When the cells were grown in limiting tyrosine, the DAHP synthetase, chorismate mutase, and shikimate kinase activities of B. subtilis were derepressed in parallel, but only DAHP synthetase and chorismate mutase were derepressible in B. licheniformis. Implications of the differences as well as the similarities between the control and the pattern of enzyme aggregation in the two related species of bacilli were discussed.     

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.     

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.     

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.     

Pseudomonas aeruginosa possesses two novel regulatory isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase

In Pseudomonas aeruginosa the initial enzyme of aromatic amino acid biosynthesis, 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase, has been known to be subject to feedback inhibition by a metabolite in each of the three major pathway branchlets. Thus, an apparent balanced multieffector control is mediated by L-tyrosine, by L-tryptophan, and phenylpyruvate. We have now resolved DAHP synthase into two distinctive regulatory isozymes, herein denoted DAHP synthase-tyr (Mr = 137,000) and DAHP synthase-trp (Mr = 175,000). DAHP synthase-tyr comprises greater than 90% of the total activity. L-Tyrosine was found to be a potent effector, inhibiting competitively with respect to both phosphoenolpyruvate (Ki = 23 microM) and erythrose 4-phosphate (Ki = 23 microM). Phenylpyruvate was a less effective competitive inhibitor: phosphoenolpyruvate (Ki = 2.55 mM) and erythrose 4-phosphate (Ki = 1.35 mM). DAHP synthase-trp was found to be inhibited noncompetitively by L-tryptophan with respect to phosphoenolpyruvate (Ki = 40 microM) and competitively with respect to erythrose 4-phosphate (Ki = 5 microM). Chorismate was a relatively weak competitive inhibitor: phosphoenolpyruvate (Ki = 1.35 mM) and erythrose 4-phosphate (Ki = 2.25 mM). Thus, each isozyme is strongly inhibited by an amino acid end product and weakly inhibited by an intermediary metabolite.     

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.     

Control of aromatic acid biosynthesis in Bacillus subtilis: sequenial feedback inhibition

Nester, E. W. (University of Washington, Seattle), and R. A. Jensen. Control of aromatic acid biosynthesis in Bacillus subtilis: sequential feedback inhibition. J. Bacteriol. 91:1594-1598. 1966.-The three major end products of aromatic acid synthesis, tyrosine, phenylalanine, and tryptophan, were tested for their ability to inhibit the first enzymes of the three terminal branches of the pathway as well as the enzyme common to both tyrosine and phenylalanine synthesis. Tyrosine inhibits the activity of prephenate dehydrogenase and also prephenate dehydratase to a limited extent. Phenylalanine inhibits the activity of prephenate dehydratase and, at much higher concentrations, prephenate dehydrogenase. Tryptophan inhibits the activity of anthranilate synthetase and, to some extent, prephenate dehydrogenase and prephenate dehydratase. Chorismate mutase is not inhibited by either 1 mm tyrosine or 1 mm phenylalanine when these are present singly or together in the reaction mixture. The significance of the feedback control of the terminal branches to the feedback control of that part of the pathway common to the synthesis of all three amino acids is discussed.     

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.     

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.     

Aromatic amino acid biosynthesis in Alcaligenes eutrophus H16. I. Properties and regulation of 3-deoxy-d-arabino heptulosonate 7-phosphate synthase.

Properties and regulation of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHP-synthase), EC4.1.2.15, from Alcaligenes eutrophus H16 were investigated. DAHP synthase was unstable during manipulations such as dialysis, dilution, ammonium sulfate fractionation, chromatography on DEAE-cellulose or Sephadex G-200. For kinetic measurements Sephadex G-25 treated crude extracts were used. The enzyme was not affected by thiol reagents, EDTA or divalent metal ions. The activation energy, deltaH, amounted to 16100 cal/mole. Between pH 7.2 and pH 8.2 there was little change of enzyme activity. The Km-values for the two substrates were found to be 0.043 mM phosphoenolpyruvate and 0.055 mM erythrose-4-phosphate. DAHP-synthase was inhibited by 0.5 mM phenylalanine for 60% and by 0.5 mM tyrosine for 20%. In the presence of both amino acids cumulative inhibition occurred amounting to about 70%. No other amino acid exerted inhibitory effects. A repression of DAHP-synthase by the aromatic amino acids was not observed. Some other strains of hydrogen bacteria were included in this study. The DAHP synthase from strain 12/60/X and Corynebacterium autotrophicum 7C was unregulated. The enzyme from strain 33/X was subject to retro-tyrosine inhibition and from strain 3/2, H1 and H20 were subject to cumulative inhibition.