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.     

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.     

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 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.     

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.     

大肠杆菌苯丙氨酸生物合成关键酶的串联表达

ａｒｏＧ基因编码的 ３－脱氧－２－阿拉伯庚酮糖－７－磷酸合成酶（ＤＡＨＰ　Ｓｙｎｔｈｅｔａｓｅ　ＤＳ）和 ｐｈｅＡ基因编码的分支酸变位酶／预苯酸脱水酶（Ｃｈｏｒｉｍａｔｅ ｍｕｔａｓｅ／ Ｐｒｅｐｈｅｎａｔｅ　ｄｅｈｙｄｒａｔａｓｅ,ＣＷ／ＰＤ）都是本丙氨酸合成途径中的关键酶,为了通过基因工程手段来增加本丙氨酸生物的产量,在利用高效的原核表达载体ｐＢＶ２２ ０对ｐｈｅＡ基因编码的ＣＭ／ ＰＤ 酶进行了表达的基础上,采用ＰＣＲ方法扩增了抗反馈抑制的ａｒｃＧ基因,进行克隆表达,并与ｐｈｅＡ基因串联,以ＰＲＰＬ－ａｒｏＧ－ＰＬ－ｐｈｅＡ的形式,实现了２种酶基因在大肠杆菌中的表达, ＳＤＳＰＡＧＥ 图谱显示了新增的４３ｋｕ及３５ｋｕ蛋白带,经酶活性测定ＤＳ、ＣＭ／ＰＤ酶的比活分别提高了 ４．６７倍、８０５／１０．７１倍。     

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.     

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.     

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.     

Enzyme Alterations in Tyrosine and Phenylalanine Auxotrophs of Salmonella typhimurium

The enzyme activities specified by the tyrA and pheA genes were studied in wildtype strain Salmonella typhimurium and in phenylalanine and tyrosine auxotrophs. As in Aerobacter aerogenes and Escherichia coli, the wild-type enzymes of Salmonella catalyze two consecutive reactions: chorismate --> prephenate --> 4-hydroxy-phenylpyruvate (tyrA), and chorismate --> prephenate --> phenylpyruvate (pheA). A group of tyrA mutants capable of interallelic complementation had altered enzymes which retained chorismate mutase T activity but lacked prephenate dehydrogenase. Similarly, pheA mutants (in which interallelic complementation does not occur) had one group with altered enzymes which retained chorismate mutase P but lacked prephenate dehydratase. Tyrosine and phenylalanine auxotrophs outside of these categories showed loss of both activities of their respective bifunctional enzyme. TyrA mutants which had mutase T were considerably derepressed in this activity by tyrosine starvation and consequently excreted prephenate. A new and specific procedure was developed for assaying prephenate dehydrogenase activity.     

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.     

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.     

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.     

Structure and function of a complex between chorismate mutase and DAHP synthase: efficiency boost for the junior partner

Chorismate mutase catalyzes a key step in the shikimate biosynthetic pathway towards phenylalanine and tyrosine. Curiously, the intracellular chorismate mutase of Mycobacterium tuberculosis (MtCM; Rv0948c) has poor activity and lacks prominent active‐site residues. However, its catalytic efficiency increases >100‐fold on addition of DAHP synthase (MtDS; Rv2178c), another shikimate‐pathway enzyme. The 2.35 Å crystal structure of the MtCM–MtDS complex bound to a transition‐state analogue shows a central core formed by four MtDS subunits sandwiched between two MtCM dimers. Structural comparisons imply catalytic activation to be a consequence of the repositioning of MtCM active‐site residues on binding to MtDS. The mutagenesis of the C‐terminal extrusion of MtCM establishes conserved residues as part of the activation machinery. The chorismate‐mutase activity of the complex, but not of MtCM alone, is inhibited synergistically by phenylalanine and tyrosine. The complex formation thus endows the shikimate pathway of M. tuberculosis with an important regulatory feature. Experimental evidence suggests that such non‐covalent enzyme complexes comprising an AroQ_δ subclass chorismate mutase like MtCM are abundant in the bacterial order Actinomycetales.     

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 tyrosine and phenylalanine biosynthesis in Salmonella.

Several types of 4-fluorophenylalanine resistant mutants were isolated. In one type of mutant DAHP synthetase (tyr) and prephenate dehydrogenase were coordinately derepressed. The mutation was linked to aroF and tyrA and was cis- dominant by merodiploid analysis, thus confirming that it is an operator constitutive mutation (tyrOc). A second type of mutation showed highly elevated levels of tyrosine pathway enzymes which were not repressed by L-tyrosine. It was unlinked to tyrA and aroF, and was trans-recessive in merodiploids. These properties were attributed to a mutation in a regulator gene, tyrR (linked to pyr F), that resulted in altered or non-functional aporepressor. Hence tyrO, tyrA, and aroF constitute an operon regulated by tyrR. In a third type of mutation chorismate mutase P-prephenate dehydratase was highly elevated. It was not linked to pheA, was located in the 95--100 min region of the Salmonella chromosome, and was recessive to the wild type gene in merodiploids. A mutation was, therefore, indicated in a regulatory gene, pheR, which specified an aporepressor for regulating pheA. DAHP synthetase (phe), specified by aroG, was not regulated by pheR, but was derepressed in one of the tyrR mutants, suggesting that as in Escherichia coli tyrR may regulate DAHP synthetase(phe) and DAHP synthetase (tyr) with the same aporepressor. A novel mutation in chorismate mutase is described.     

A Bifunctional Enzyme in Pseudomonas aeruginosa: a New Pattern in the Organization of Enzymes Concerned with Phenylalanine and Tyrosine Biosynthesis

Two isozymes of chorismate mutase (CA mutase(1) and CA mutase(2)) and two isozymes of prephenate dehydratase (PPA dehydratase(1) and PPA dehydratase(2)) have been found in Pseudomonas aeruginosa. The activities CA mutase(2)-PPA dehydratase(2) catalyzing phenylalanine biosynthesis have been purified almost 40-fold and were found to be associated as a bifunctional enzyme or an enzyme complex. The enzymes specific for tyrosine biosynthesis did not appear to manifest such physical association. Thus, the organization of enzymes concerned with phenylalanine and tyrosine biosynthesis in P. aeruginosa is unique and is unlike most other organisms. Single site mutants have been isolated which have lost both CA mutase(2)-PPA dehydratase(2) activities resulting in a requirement for phenylalanine for growth. Single site revertants of these mutants regained both these activities simultaneously and were able to grow on minimal medium. A mutant, r(6), was also isolated which had normal CA mutase(2) but lacked PPA dehydratase(2) activity.     

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.     

Yeast allosteric chorismate mutase is locked in the activated state by a single amino acid substitution

Chorismate mutase, a branch-point enzyme in the aromatic amino acid pathway of Saccharomyces cerevisiae, and also a mutant chorismate mutase with a single amino acid substitution in the C-terminal part of the protein have been purified approximately 20-fold and 64-fold from overproducing strains, respectively. The wild-type enzyme is activated by tryptophan and subject to feedback inhibition by tyrosine, whereas the mutant enzyme does not respond to activation by tryptophan nor inhibition by tyrosine. Both enzymes are dimers consisting of two identical subunits of Mr 30,000, each one capable of binding one substrate and one activator molecule. Each subunit of the wild-type enzyme also binds one inhibitor molecule, whereas the mutant enzyme lost this ability. The enzyme reaction was observed by 1H NMR and shows a direct and irreversible conversion of chorismate to prephenate without the accumulation of any enzyme-free intermediates. The kinetic data of the wild-type chorismate mutase show positive cooperativity toward the substrate with a Hill coefficient of 1.71 and a [S]0.5 value of 4.0 mM. In the presence of the activator tryptophan, the cooperativity is lost. The enzyme has an [S]0.5 value of 1.2 mM in the presence of 10 microM tryptophan and an increased [S]0.5 value of 8.6 mM in the presence of 300 microM tyrosine. In the mutant enzyme, a loss of cooperativity was observed, and [S]0.5 was reduced to 1.0 mM. This enzyme is therefore locked in the activated state by a single amino acid substitution.