The Two-Isozyme System of 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase in Nicotiana silvestris and Other Higher Plants

Two isozymes of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase, denoted DS-Mn and DS-Co, were identified following DEAE-cellulose chromatography of crude extracts prepared from suspension-cultured cells of Nicotiana silvestris. The strikingly different properties of the isozymes allowed the development of assays for the selective detection of either isozyme in samples containing a mixture of the two. The DS-Mn isozyme required the sulfhydryl reductant, dithiothreitol, for activity and was stimulated by manganese. Activation by dithiothreitol was slow relative to catalysis, accounting for a hysteretic progress curve that was observed when reactions were started with inactive enzyme. The DS-Co isozyme was inhibited by dithiothreitol and required a divalent cation for activity. At optimal cation concentrations of 10 millimolar (magnesium), 0.5 millimolar (cobalt), and 0.5 millimolar (manganese), relative activities obtained were 100, 85, and 20, respectively. The substrate saturation curves with respect to erythrose 4-phosphate differed markedly when the two isozymes were compared. As little as 0.5 millimolar erythrose 4-phosphate saturated DS-Mn, whereas a 10-fold higher concentration was needed for saturation of DS-Co. The pH optimum of DS-Mn was 8.0, while that of the DS-Co isozyme was 8.6. Leaves of both N. silvestris and spinach also exhibited the DS-Mn/DS-Co isozyme arrangement, and the subcellular location of DS-Mn was shown to be the chloroplast compartment. By application of the differential assays for DAHP synthase isozymes, various monocotyledonous and dicotyledonous plants yielded data indicating the general presence of the DS-Mn/DS-Co isozyme pair in higher plants.     

Differentially Regulated Isozymes of 3-Deoxy-d-arabino-Heptulosonate-7-Phosphate Synthase from Seedlings of Vigna radiata [L.] Wilczek

Two isozymes of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) designated DS-Mn and DS-Co were separated from seedlings of Vigna radiata [L.] Wilczek by DEAE-cellulose column chromatography. DS-Mn was activated 2.6-fold by 0.4 millimolar manganese, had an activity optimum of 7.0, and was substrate inhibited by erythrose-4-phosphate (E4P) concentrations in excess of 0.5 millimolar. In contrast, DS-Co had an activity optimum at pH 8.8, required E4P concentrations of at least 4 millimolar to approach saturation, and exhibited an absolute requirement for divalent cation (cobalt being the best). Regulatory properties of the two isozymes differed dramatically. The activity of DS-Mn was activated by chorismate (noncompetitively against E4P and competitively against phosphoenolpyruvate), and was inhibited by prephenate and l-arogenate (competitively against E4P and noncompetitively against phosphoenolpyruvate in both cases). Under standard assay conditions, l-arogenate inhibited the activity of DS-Mn 50% at a concentration of 155 micromolar and was at least 3 times more potent than prephenate on a molar basis. Weak inhibition of DS-Mn by l-tryptophan was also observed, the magnitude of inhibition increasing with decreasing pH. The pattern of allosteric control found for DS-Mn is consistent with the operation of a control mechanism of sequential feedback inhibition governing overall pathway flux. DS-Co was not subject to allosteric control by any of the molecules affecting DS-Mn. However, DS-Co was inhibited by caffeate (3,4-dihydroxycinnamate), noncompetitively with respect to either substrate. The striking parallels between the isozyme pairs of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase and chorismate mutase are noted—one isozyme in each case being tightly regulated, the other being essentially unregulated.     

Evolution of the regulatory isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase present in the Escherichia coli genealogy.

The evolutionary history of isozymes for 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase has been constructed in a phylogenetic cluster of procaryotes (superfamily B) that includes Escherichia coli. Members of superfamily B that have been positioned on a phylogenetic tree by oligonucleotide cataloging possess one or more of four distinct isozymes of DAHP synthase. DAHP synthase-0 is insensitive to feedback inhibition, while DAHP synthase-Tyr, DAHP synthase-Trp, and DAHP synthase-Phe are sensitive to feedback inhibition by L-tyrosine, L-tryptophan, and L-phenylalanine, respectively. The evolutionary history of this isozyme family can be deduced within superfamily B by using a cladistic methodology of maximum parsimony (R. A. Jensen, Mol. Biol. Evol. 2:92-108, 1985). DAHP synthase-0 was found in Acinetobacter species and in Oceanospirillum minutulum, organisms that also possess DAHP synthase-Tyr. These two isozymes were apparently present in a common ancestor that predated the evolutionary divergence of contemporary superfamily B sublineages. DAHP synthase-0 is postulated to have been the evolutionary forerunner of DAHP synthase-Trp. The newly evolved DAHP synthase-Trp is postulated to have possessed sensitivity to feedback inhibition by chorismate as well as by L-tryptophan, chorismate sensitivity having been retained in rRNA group I pseudomonads (minor sensitivity), group V pseudomonads (very sensitive), and Lysobacter enzymogenes (ultrasensitive). Organisms constituting the enteric lineage of the phylogenetic tree (including a cluster of four Oceanospirillum species) have all lost the chorismate sensitivity of DAHP synthase-Trp. The absence of DAHP synthase-Phe in the Oceanospirillum cluster of organisms supports the previous conclusion that DAHP synthase-Phe evolved recently within superfamily B, being present only Escherichia coli and its close relatives.     

Evidence from Solanum tuberosum in Support of the Dual-Pathway Hypothesis of Aromatic Biosynthesis

Key branchpoint enzymes of aromatic amino acid biosynthesis, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DS) and chorismate mutase (CM), have previously been shown to exist as separate compartmentalized isozymes in the chloroplasts and cytosol of tobacco, sorghum and spinach. Although additional examples of plants containing these isozyme pairs are accumulating, some studies in the literature report the presence of only the single plastidic DS or CM enzyme. Such apparent exceptions contradict the universality of pathway organization existing in higher plants that is implied by the dual-pathway hypothesis of aromatic biosynthesis. Since potato (Solanum tuberosum) exemplifies a case where only a single species of both DS and CM have been reported, we selected this system for further analysis. The DS-Mn and DS-Co isozyme pair, exhibiting all of the differential properties described in Nicotiana silvestris, have now been identified in S. tuberosum. Likewise, partial purification via DEAE-cellulose chromatography revealed two isozymes of CM in disks excised from tubers of S. tuberosum. The differential regulatory properties of these isozymes were comparable to the CM-1 and CM-2 isozymes of N. silvestris.     

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.     

Enzymological basis for herbicidal action of glyphosate

The effects of 1 millimolar glyphosate (N-[phosphonomethyl]glycine) upon the activities of enzymes of aromatic amino acid biosynthesis, partially purified by ion-exchange chromatography from mung bean seedings (Vigna radiata [L.] Wilczek), were examined. Multiple isozyme species of shikimate dehydrogenase, chorismate mutase, and aromatic aminotransferase were separated, and these were all insensitive to inhibition by glyphosate. The activities of prephenate dehydrogenase and arogenate dehydrogenase were also not sensitive to inhibition. Two molecular species of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase were resolved, one stimulated several-fold by Mn²⁺ (DAHP synthase-Mn), and the other absolutely dependent upon the presence of Co²⁺ for activity (DAHP synthase-Co). Whereas DAHP synthase-Mn was invulnerable to glyphosate, greater than 95% inhibition of DAHP synthase-Co was found in the presence of glyphosate. Since Co²⁺ is a V_max activator with respect to both substrates, glyphosate cannot act simply by Co²⁺ chelation because inhibition is competitive with respect to erythrose-4-phosphate. The accumulation of shikimate found in glyphosate-treated seedlings is consistent with in vivo inhibition of both 5-enolpyruvylshikimic acid 3-phosphate synthase and one of the two DAHP synthase isozymes. Aromatic amino acids, singly or in combination, only showed a trend towards reversal of growth inhibition in 7-day seedlings of mung bean. The possibilities are raised that glyphosate may act at multiple enzyme targets in a given organism or that different plants may vary in the identity of the prime enzyme target.     

Dehydroquinate synthase in Bacillus subtilis. An enzyme associated with chorismate synthase and flavin reductase.

Dehydroquinate synthase, the enzyme which catalyzes the conversion of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) to 5-dehydroquinate, has been purified from Bacillus subtilis in association with chorismate synthase and NADPH-dependent flavin reductase. The enzyme was only active when associated with chorismate synthase, whereas the flavin reductase could be separated from the complex with retention of dehydroquinate synthase activity. The enzyme requires NAD and either Co2+ or Mn2+ for maximal activity. The activity was completely inhibited by EDTA. The Km of the enzyme for DAHP, NAD, and Co2+ were estimated to be 1.3 X 10(-4), 5.5 X 10(-5), and 5.5 X 10(-5) M, respectively. Enzyme activity was completely inhibited by NADH and the inhibition was not reversed by the addition of NAD, NADPH and NADP were not inhibitory. The enzyme was unstable to heat and lost all activity at 55 degrees C. A protein fraction which did not adsorb to phosphocellulose was found to inhibit the enzyme.     

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.     

Chorismate Mutase Isoenzymes from Selected Plants and Their Immunological Comparison with the Isoenzymes from Sorghum bicolor

The isoenzyme pattern of chorismate mutase (EC 5.4.99.5) was examined by diethylaminoethyl-cellulose chromatography in a wide variety of plants. All plants contained a regulated form of chorismate mutase (CM-1), and most contained an additional, unregulated form (CM-2). The regulatory properties of CM-1 differed significantly between plants. Antisera prepared against CM-1 and CM-2 from Sorghum bicolor were used to test immunological cross reaction of chorismate mutases from other plants. There was a high degree of similarity between chorismate mutase isoenzymes from Sorghum bicolor and Zea mays and some with Hordeum vulgare, but all other species studied were antigenically distinct from sorghum. No homology between the structure of CM-1 and CM-2 was detected within any species.     

Changes in the activity of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase in suspension-cultured cells of Vitis

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15) is the first enzyme in the shikimate pathway, which leads to the biosynthesis of the aromatic amino acids. These amino acids are utilized as precursors for the synthesis of some secondary metabolites. The relationship between the accumulation of anthocyanin and the activity of DAHP synthase in suspension cultures of Vitis hybrid (Bailey Alicante A) was investigated. The activity of the plastidic isozyme, designated DS-Mn, was very low throughout the culture of cells. However, the activity of the cytosolic isozyme, designated DS-Co, increased transiently and then decreased after transfer of cells to fresh medium, reaching minimum levels during the logarithmic phase. Thereafter, the activity of DS-Co increased rapidly prior to the accumulation of anthocyanin. When phosphate was removed from the culture medium, growth of cells was limited and rapid accumulation of anthocyanin occurred, coincident with the termination of cell division. The activity of phenylalanine ammonia-lyase continued to increase from day 1 and the activity of DS-Co in phosphate-free culture also was 1.6-fold greater than that in the control culture on day 1, while the activity of DS-Mn was unaffected by this treatment. These results suggest a close correlation between the activity of DS-Co and the biosynthesis of anthocyanin.     

Purification and characterization of chorismate mutase isoenzymes from ruta graveolens l.

Two isoenzymes of chorismate mutase (EC 5.4.99.5), designated as CM-1 and CM-2, were isolated and partially purified from suspension-cultured cells of Ruta gravelens by DEAE-sephacel chromatography and gel filtration. 60–72% of the total activity measured after DEAE-sephacel chromatography were obtained as CM-1 and 28–40% were CM-2 activity. CM-1 was inhibited by phenylalanine (K₁ = 4 · 10^?6 M) and tyrosine (K₁ = 8. 10^?6M) and activated by tryptophan. In contrast, CM-2 was not influenced by these three amino acids. The molecular weights estimated by gel filtration on SEPHADEX G-150 were 56000 for CM-1 and 45000 for CM-2, respectively. Both isoenzymes were stable at ?20°C, but exhibited different behaviour during thermal inactivation and different optima of reaction temperature. CM-1 catalysed the reaction at a pH optimum of pH 7.8 and CM-2 showed a broad optimum between 6–10. The K_m-values for chorismic acid were determined to be 1.1 mM for CM-1 and 0.5 mM for CM-2. The isoenzymes showed different behaviour to inhibitors of sulfhydryl groups. There were no differences in all parameters of chorismate mutase examined for two various cell lines of Ruta graveolens.     

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.     

Clues from Xanthomonas campestris about the evolution of aromatic biosynthesis and its regulation

The recent placement of major Gram-negative prokaryotes (Superfamily B) on a phylogenetic tree (including, e.g., lineages leading to Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus) has allowed initial insights into the evolution of the biochemical pathway for aromatic amino acid biosynthesis and its regulation to be obtained. Within this prokaryote grouping, Xanthomonas campestris ATCC 12612 (a representative of the Group V pseudomonads) has played a key role in facilitating deductions about the major evolutionary events that shaped the character of aromatic biosynthesis within this grouping. X. campestris is like P. aeruginosa (and unlike E. coli) in its possession of dual flow routes to both L-phenylalanine and L-tyrosine from prephenate. Like all other members of Superfamily B, X. campestris possesses a bifunctional P-protein bearing the activities of both chorismate mutase and prephenate dehydratase. We have found an unregulated arogenate dehydratase similar to that of P. aeruginosa in X. campestris. We separated the two tyrosine-branch dehydrogenase activities (prephenate dehydrogenase and arogenate dehydrogenase); this marks the first time this has been accomplished in an organism in which these two activities coexist. Superfamily B organisms possess 3-deoxy-D-arabino-heptulosonate 7-P (DAHP) synthase as three isozymes (e.g., in E. coli), as two isozymes (e.g., in P. aeruginosa), or as one enzyme (in X. campestris). The two-isozyme system has been deduced to correspond to the ancestral state of Superfamily B. Thus, E. coli has gained an isozyme, whereas X. campestris has lost one. We conclude that the single, chorismate-sensitive DAHP synthase enzyme of X. campestris is evolutionarily related to the tryptophan-sensitive DAHP synthase present throughout the rest of Superfamily B. In X. campestris, arogenate dehydrogenase, prephenate dehydrogenase, the P-protein, chorismate mutase-F, anthranilate synthase, and DAHP synthase are all allosteric proteins; we compared their regulatory properties with those of enzymes of other Superfamily B members with respect to the evolution of regulatory properties. The network of sequentially operating circuits of allosteric control that exists for feedback regulation of overall carbon flow through the aromatic pathway in X. campestris is thus far unique in nature.     

Differential Activities of Chorismate Mutase Isozymes in Tubers and Leaves of Solanum tuberosum L

Chromatography on DEAE cellulose equilibrated with Pipes buffer resolved three forms of chorismate mutase (CM) from tubers and leaves of Solanum tuberosum: CM-1A and CM-1B were activated by tryptophan and inhibited by phenylalanine and tyrosine; CM-2 was unaffected by these aromatic amino acids. When compared to freshly excised discs, 3 day old tuber discs demonstrated a 4.5-fold increase in CM-1 activity following wounding. By contrast, CM-2 activity levels were not affected by this treatment. In aged tuber discs the CM-1:CM-2 activity ratio was 9:1. However, in green leaves the CM-1:CM-2 activity ratio was 1:4 suggesting organ specific regulation for the expression of these isozymes. The CM-1 isozymes isolated from both tubers and leaves shared similar native molecular weight values of 55,000, K_m values of 40 to 56 micromolar, and inhibition by phenylalanine (110-145 micromolar concentrations required for 50% inhibition) and tyrosine (50-70 micromolar concentrations required for 50% inhibition). The resolution of CM-1 into two forms occurred only in the presence of Pipes buffer. When this buffer was replaced with Aces, Bes, imidazole or Tris, only a single peak of CM-1 activity was observed. In these buffers CM-2 eluted as a shoulder on the CM-1 peak. Analytical isoelectric focusing of the CM-1 fraction followed by assay of the gel yielded only one form of CM-1 with an isoelectric point of 5.0. Gel filtration studies with Pipes buffer yielded molecular weights of 60,000 for both CM-1A and CM-1B indicating these forms are not the result of aggregation. The two forms of CM-1 may be artifacts generated by Pipes buffer.     

Chorismate mutase isoenzymes from Sorghum bicolor: purification and properties

Two forms of chorismate mutase (EC 5.4.99.5), designated as CM-1 and CM-2, have been detected in etiolated seedlings of Sorghum bicolor after DEAE-cellulose chromatography. CM-1 and CM-2 contained 44 and 56%, respectively, of the total activity measured after DEAE-cellulose chromatography. CM-1 was activated by tryptophan and inhibited by phenylalanine and tyrosine. In contrast, CM-2 was insensitive to all three aromatic amino acids. CM-1 and CM-2 were purified 1389- and 1018-fold, respectively, by anion exchange, hydrophobic, and dye matrix chromatography. The molecular weights estimated by gel filtration on Sephacryl S-200 were 56,000 for CM-1 and 48,000 for CM-2. Subunit molecular weights of the two forms were estimated by sodium dodecyl sulfate-gel electrophoresis at 36,000 and 51,000 for CM-1 and CM-2, respectively. Tryptophan was required for the stability of CM-1 at all stages of purification. Both isoenzymes were stable at 0 or -20 degrees C and had broad pH optima (6-10 for CM-1 and 7.5-9.5 for CM-2).     

Differential expression of DAHP synthase and chorismate mutase in various organs of Brassica juncea and the effect of external factors on enzyme activity

The multibranched shikimic acid pathway was discovered as the biosynthetic route to the aromatic amino acids phenylalanine, tyrosine and tryptophan and a host of other secondary metabolites. An extensive body of work is available on the characterization of various enzymes of this pathway in order to understand the underlying mechanisms of aromatic amino acid biosynthesis and secondary metabolism in higher plants. In the present investigation, selective assays, based on feedback regulation patterns and divalent cation requirements, were used to monitor the isozyme profiles of two of the key regulatory enzymes of this pathway. 3-Deoxy- d -arabino heptulosonate-7-phosphate synthase (DAHP synthase/DS) (EC 4.1.2.15) and chorismate mutase (CM) (EC 5.4.99.4) have been characterized from different vegetative and reproductive organs of Brassica juncea cv. Pusa Bold. An attempt has also been made to investigate the effect of external factors, such as light and wounding on the regulation of these enzymes. The results reveal differential expression of DAHP synthase and CM in various organs of Brassica and an adaptability of plants to various stresses by up or down regulation of these enzymes.     

Comparison of chorismate mutase isozyme patterns in selected plants

A wide variety of plants have been assayed to determine if they contain three isozymes of chorismate mutase (EC 5.4.99.5) as does alfalfa (Medicago sativa L.) or two isozymes, as does mung bean (Phaseolus aureus). The isozymes were separated by disc electrophoresis. All anthophyta with the exception of some closely related Leguminosae contained three isozymes of chorismate mutase. The one coniferophyta (a pine), and pterophyta (a fern) and one microphyllophyta (a Selaginella) assayed contained two isozymes of chorismate mutase. All plants assayed contained measurable chorismate mutase levels and at least two isozymes of chorismate mutase.     

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.