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.     

Comparative allostery of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthetase as a molecular basis for classification

The allosteric pattern of control for 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthetase has previously been shown to be strongly conserved among the member species of a given genus in bacteria. The implications of this finding as a procedural tool of bacterial phylogeny were pursued by a study of two organisms, Sporosarcina ureae and Aeromonas formicans, the taxonomic positions of which have been historically controversial. S. ureae has characteristics of both Bacillaceae and Micrococcaceae, and A. formicans has characteristics of both Enterobacteriaceae and pseudomonads. Since the patterns of control for DAHP synthetase in all four of these microbial groups are different from one another but internally homogeneous within each group, the results obtained from the two test organisms were unambiguous. It was concluded that S. ureae is properly classified within Bacillaceae, probably deserving generic rank, and that A. formicans belongs with the family Enterobacteriaceae.     

Comparative allostery of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase as an indicator of taxonomic relatedness in pseudomonad genera.

Recently, an analysis of the enzymological patterning of L-tyrosine biosynthesis was shown to distinguish five taxonomic groupings among species currently named Pseudomonas, Xanthomonas, or Alcaligenes (Byng et al., J. Bacteriol. 144:247--257, 1980). These groupings paralleled with striking consistency those previously defined by ribosomal ribonucleic acid-deoxyribonucleic acid homology relationships. The comparative allostery of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthetase has previously been shown to be a useful indicator of taxonomic relationship at about the level of genus. The comparative allostery of DAHP synthetase was evaluated in relationship to data available from the same pseudomonad species previously studied. Species of Xanthomonas and some named species of Pseudomonas, e.g., P. maltophilia, were unmistakably recognized as belonging to group V, having a DAHP synthetase sensitive to sequential feedback inhibition by chorismate. This control pattern is thus far unique to group V pseudomonads among microorganisms. Group V organisms were also unique in their possession of DAHP synthetase enzymes that were unstimulated by divalent cations. Group IV pseudomonads (P. diminuta) were readily distinguished by the retro-tryptophan pattern of control for DAHP synthetase. Activity for DAHP synthetase was not always recovered in group IV species, e.g., P. vesicularis. The remaining three groups exhibited overlapping patterns of DAHP synthetase sensitivity to both L-phenylalanine and L-tyrosine. Individual species cannot be reliably keyed to group I. II, or III without other data. However, each group overall exhibited a different trend of relative sensitivity to L-tyrosine and L-phenylalanine. Thus, although enzymological patterning of L-tyrosine biosynthesis alone can be used to separate the five pseudomonad groups, the independent assay of DAHP synthetase control pattern can be used to confirm assignments. The latter approach is, in fact, the easiest and most definitive method for recognition of group V (and often of group IV) species.     

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.     

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.     

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.     

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.     

Microbial origin of plant-type 2-keto-3-deoxy-D-arabino-heptulosonate 7-phosphate synthases, exemplified by the chorismate- and tryptophan-regulated enzyme from Xanthomonas campestris

Enzymes performing the initial reaction of aromatic amino acid biosynthesis, 2-keto-3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthases, exist as two distinct homology classes. The three classic Escherichia coli paralogs are AroA(I) proteins, but many members of the Bacteria possess the AroA(II) class of enzyme, sometimes in combination with AroA(I) proteins. AroA(II) DAHP synthases until now have been shown to be specifically dedicated to secondary metabolism (e.g., formation of ansamycin antibiotics or phenazine pigment). In contrast, here we show that the Xanthomonas campestris AroA(II) protein functions as the sole DAHP synthase supporting aromatic amino acid biosynthesis. X. campestris AroA(II) was cloned in E. coli by functional complementation, and genes corresponding to two possible translation starts were expressed. We developed a 1-day partial purification method (>99%) for the unstable protein. The recombinant AroA(II) protein was found to be subject to an allosteric pattern of sequential feedback inhibition in which chorismate is the prime allosteric effector. L-Tryptophan was found to be a minor feedback inhibitor. An N-terminal region of 111 amino acids may be located in the periplasm since a probable inner membrane-spanning region is predicted. Unlike chloroplast-localized AroA(II) of higher plants, X. campestris AroA(II) was not hysteretically activated by dithiols. Compared to plant AroA(II) proteins, differences in divalent metal activation were also observed. Phylogenetic tree analysis shows that AroA(II) originated within the Bacteria domain, and it seems probable that higher-plant plastids acquired AroA(II) from a gram-negative bacterium via endosymbiosis. The X. campestris AroA(II) protein is suggested to exemplify a case of analog displacement whereby an ancestral aroA(I) species was discarded, with the aroA(II) replacement providing an alternative pattern of allosteric control. Three subgroups of AroA(II) proteins can be recognized: a large, central group containing the plant enzymes and that from X. campestris, one defined by a three-residue deletion near the conserved KPRS motif, and one possessing a larger deletion further downstream.     

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 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.     

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.     

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.     

Feedback-Insensitive Mutants of the Gene for the Tyrosine-Inhibited Dahp Synthetase in Yeast

A yeast strain lacking the phenylalanine-sensitive DAHP synthetase exhibits a tyrosine-inhibited phenotype. Reversions from this phenotype were selected. They were the result of mutations affecting the tyrosine-sensitive DAHP synthetase which became insensitive to feedback inhibition, as shown by genetic and enzymatic studies. The feedback-insensitive mutations were located at five different sites scattered on the map of the aro4 gene.     

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.     

Serine 187 is a crucial residue for allosteric regulation of Corynebacterium glutamicum 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase

Corynebacterium glutamicum 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase is sensitive to feedback inhibition by tyrosine. One feedback-insensitive mutant was obtained by in vitro chemical mutagenesis and the mutation was identified as a C-->G mutation at nucleotide 560 causing a Ser(187) to Cys(187) substitution. Replacing Ser(187) with cysteine, tyrosine or phenylalanine by site-directed mutagenesis not only reduced the enzymatic activity but also relieved its feedback inhibition by tyrosine, while Ser(187)Ala exhibited a comparable activity to that of wild-type enzyme and sensitized to allosteric regulation. The His(6)-tagged enzymes were expressed in Escherichia coli and purified to homogeneity by immobilized nickel-ion affinity chromatography. Kinetic analysis showed that tyrosine is a competitive inhibitor of phosphoenol pyruvate, one of the precursors for DAHP biosynthesis.     

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.     

Two components of chorismate mutase in Brevibacterium flavum.

Chorismate mutase of Brevibacterium flavum, a common enzyme in phenylalanine and tyrosine biosynthesis, was separted into two different component, A and B, with molecular weights of 250,000 and 25,000, respectively, by ammonium sulfate fractionation or gel-filtration. Both components were essential for the enzymatic activity. In the presence of the reaction substrate, chorismate, the two components associated reversibly to give an active enzyme complex with a molecular weight of 320,000. Binding sites of the feedback inhibitors, phenylalanine and tyrosine, on the enzyme were localized on component A as determined by hybridization experiments with the wild-type and mutant components. Tyrosine repressed the synthesis of component B much more strongly than that of component A, while phenylalanine did not show any significant repressive effect on either component. The wild-type strain No. 2247 had four times more component A than component B. Elution patterns in gel, DEAE-cellulose or hydroxyapatite column chromatography as well as the disc-gel electrophoretic pattern of chorismate mutase component A and 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthetase activities completely overlapped, suggesting the presence of a bifunctional protein having the two activities. In accord with this suggestion, chorismate mutase as well as DAHP synthetase was insensitive to feedback inhibition by phenylalanine and tyrosine in all the 3-fluorophenylalanine-resistant mutants tested that excreted both phenylalanine and tyrosine. All the phenylalanine and tyrosine double auxotrophs defective in chorismate mutase lacked component B but not A.     

Tyrosine and phenylalanine biosynthesis in Escherichia coli K-12: complementation between different tyrR alleles

Dominance tests in diploids have confirmed that the product of the tyrR gene is involved in a negative control system affecting the synthesis of both 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthetase (tyr) and DAHP synthetase (phe). Some tyrR mutants are derepressed for the synthesis of both DAHP synthetase (tyr) and (phe), whereas others are derepressed for the synthesis of DAHP synthetase (tyr) but overrepressed for the synthesis of DAHP synthetase (phe). Complementation tests between these alleles confirm that they are in the same cistron. The allele causing overrepression of enzyme synthesis is dominant over both the wild type and the derepressing allele in diploids.     

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.     

Repression of 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetase (trp) and enzymes of the tryptophan pathway in Escherichia coli K-12

Mutant strains of Escherichia coli K-12 have been isolated in which the synthesis of 3-deoxy-d-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase (trp) is partially constitutive. The mutation causing derepression is closely linked to aroH [the structural gene for DAHP synthetase (trp)] and occurs in a locus designated aroJ. The aroJ mutation is not recessive in an aroJ(+)/aroJ(-) diploid strain, as the synthesis of DAHP synthetase (trp) is still derepressed in this strain. On the basis of its close linkage to aroH and its continued expression in an aroJ(+)/aroJ(-) diploid, it is postulated that aroJ is an operator locus controlling the expression of the structural gene aroH. In support of this conclusion, the synthesis of anthranilate synthetase is still normally repressible in aroJ(-) strains, whereas, in trpR(-) strains, both DAHP synthetase (trp) and anthranilate synthetase are synthesized constitutively. The synthesis of DAHP synthetase (trp) remains repressible in an operator-constitutive mutant of the tryptophan operon. In two trpS mutants which possess defective tryptophanyl transfer ribonucleic acid synthetase enzymes, neither DAHP synthetase (trp) nor anthranilate synthetase derepress under conditions in which the defective synthetase causes a decrease in growth rate. On the other hand, an effect of the trpS mutant alleles on the level of anthranilate synthetase has been observed in strains which are derepressed for the synthesis of this enzyme, because of a mutation in the gene trpR. Possible explanations for this effect are presented.