A single point mutation results in a constitutively activated and feedback-resistant chorismate mutase of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae ARO7 gene product chorismate mutase, a single-branch-point enzyme in the aromatic amino acid biosynthetic pathway, is activated by tryptophan and subject to feedback inhibition by tyrosine. The ARO7 gene was cloned on a 2.05-kilobase EcoRI fragment. Northern (RNA) analysis revealed a 0.95-kilobase poly(A)+ RNA, and DNA sequencing determined a 771-base-pair open reading frame capable of encoding a protein 256 amino acids. In addition, three mutant alleles of ARO7 were cloned and sequenced. These encoded chorismate mutases which were unresponsive to tyrosine and tryptophan and were locked in the on state, exhibiting a 10-fold-increased basal enzyme activity. A single base pair exchange resulting in a threonine-to-isoleucine amino acid substitution in the C-terminal part of the chorismate mutase was found in all mutant strains. In contrast to other enzymes in this pathway, no significant homology between the monofunctional yeast chorismate mutase and the corresponding domains of the two bifunctional Escherichia coli enzymes was found.     

Production of l-Phenylalanine from Starch by Analog-Resistant Mutants of Bacillus polymyxa

p-Fluorophenylalanine-resistant mutants of starch-degrading Bacillus polymyxa ATCC 842, generated by ethyl methanesulfonate mutagenesis followed by incubation with caffeine, overproduced small amounts of l-phenylalanine (l-phe) from starch. A beta-2-thienylalanine-resistant mutant (BT-7) derived from p-fluorophenylalanine mutant (C-4000 FP-4) and resistant to both p-fluorophenylalanine and beta-2-thienylalanine produced 0.5 g of l-phe and 0.15 g of l-tyrosine per liter from 10 g of starch per liter when growing in a minimal medium. trans-Cinnamic acid (CA) was also excreted by both mutants, indicating the possibility of l-phenylalanine ammonia-lyase-induced deamination of l-phe to CA. The amount of l-phe-derived CA detected in BT-7 was less compared with mutant C-4000 FP-4. CA production was induced in the parent only when l-phe was used as a sole nitrogen source. Time of CA production in the two mutants could be delayed by addition of other nitrogen sources, an indication of possible l-phenylalanine ammonia-lyase inhibition or repression. The presence of l-phenylalanine ammonia-lyase in B. polymyxa mutant C-4000 FP-4 was confirmed by assays of cell-free extracts from cells grown in starch minimal medium containing l-phe as the sole nitrogen source. Preliminary studies of the regulation of deoxy-d-arabino-heptulosonate-7-phosphate synthase and prephenate dehydratase in the wild-type strain showed that deoxy-d-arabino-heptulosonate-7-phosphate synthase was subject to feedback inhibition by l-phe, l-tyrosine, and l-tryptophan. Inhibition by each amino acid was to a similar extent singly or in combination at a 0.5 mM level of each amino acid. Prephenate dehydratase was feedback inhibited by l-phe, but not by l-tyrosine or l-tryptophan or both. In the double analog-resistant mutant BT-7, deoxy-d-arabino-heptulosonate-7-phosphate synthase had specific activity similar to that in the wild type, and the enzyme was still subject to feedback inhibition. However, prephenate dehydratase had increased specific activity and it was also insensitive to feedback inhibition by l-phe. The overproduction of aromatic amino acids by BT-7 was thought to be due, at least in part, to deregulation of feedback inhibition of prephenate dehydratase. Chorismate mutase was not subject to feedback inhibition in the wild type and was unaffected in the mutant.     

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.     

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

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

Alleviation of feedback inhibition in Saccharomyces cerevisiae aromatic amino acid biosynthesis: quantification of metabolic impact

A quantitative analysis of the impact of feedback inhibition on aromatic amino acid biosynthesis was performed in chemostat cultures of Saccharomyces cerevisiae. Introduction of a tyrosine-insensitive allele of ARO4 (encoding 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase) caused a three-fold increase of intracellular phenylalanine and tyrosine concentrations. These amino acids were not detected extracellularly. However, an over 100-fold increase of the extracellular levels of phenylacetate, phenylethanol and their para-hydroxyl analogues was observed. The total increase of the flux through the aromatic pathway was estimated to be over four-fold. Individual overexpression of either the wild-type or feedback insensitive allele of ARO7 (encoding chorismate mutase had no significant impact. However when they were combined with the Tyr-insensitive ARO4 allele in combination with the Tyr-insensitive ARO4 allele, extracellular concentrations of aromatic compounds were increased by over 200-fold relative to the reference strain, corresponding to a 4.5-fold increase of the flux through the aromatic amino acid biosynthesis pathway. Elimination of allosteric control on these two key reactions in aromatic amino acid metabolism significantly affected intracellular concentrations of several non-aromatic amino acids. This broader impact of amino acid biosynthesis presents a challenge in rational optimization of the production of specific amino acids and derived flavour compounds.     

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.     

HARO7 encodes chorismate mutase of the methylotrophic yeast Hansenula polymorpha and is derepressed upon methanol utilization

The HARO7 gene of the methylotrophic, thermotolerant yeast Hansenula polymorpha was cloned by functional complementation. HARO7 encodes a monofunctional 280-amino-acid protein with chorismate mutase (EC 5.4. 99.5) activity that catalyzes the conversion of chorismate to prephenate, a key step in the biosynthesis of aromatic amino acids. The HARO7 gene product shows strong similarities to primary sequences of known eukaryotic chorismate mutase enzymes. After homologous overexpression and purification of the 32-kDa protein, its kinetic parameters (k(cat) = 319.1 s(-1), n(H) = 1.56, [S](0.5) = 16.7 mM) as well as its allosteric regulatory properties were determined. Tryptophan acts as heterotropic positive effector; tyrosine is a negative-acting, heterotropic feedback inhibitor of enzyme activity. The influence of temperature on catalytic turnover and the thermal stability of the enzyme were determined and compared to features of the chorismate mutase enzyme of Saccharomyces cerevisiae. Using the Cre-loxP recombination system, we constructed mutant strains carrying a disrupted HARO7 gene that showed tyrosine auxotrophy and severe growth defects. The amount of the 0.9-kb HARO7 mRNA is independent of amino acid starvation conditions but increases twofold in the presence of methanol as the sole carbon source, implying a catabolite repression system acting on HARO7 expression.     

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.     

Feedback inhibition of chorismate mutase/prephenate dehydrogenase (TyrA) of Escherichia coli: generation and characterization of tyrosine-insensitive mutants

In order to get insights into the feedback regulation by tyrosine of the Escherichia coli chorismate mutase/prephenate dehydrogenase (CM/PDH), which is encoded by the tyrA gene, feedback-inhibition-resistant (fbr) mutants were generated by error-prone PCR. The tyrA(fbr) mutants were selected by virtue of their resistance toward m-fluoro-D,L-tyrosine, and seven representatives were characterized on the biochemical as well as on the molecular level. The PDH activities of the purified His6-tagged TyrA proteins exhibited up to 35% of the enzyme activity of TyrA(WT), but tyrosine did not inhibit the mutant PDH activities. On the other hand, CM activities of the TyrA(fbr) mutants were similar to those of the TyrA(WT) protein. Analyses of the DNA sequences of the tyrA genes revealed that tyrA(fbr) contained amino acid substitutions either at Tyr263 or at residues 354 to 357, indicating that these two sites are involved in the feedback inhibition by tyrosine.     

Significance of Altered Carbon Flow in Aromatic Amino Acid Synthesis: an Approach to the Isolation of Regulatory Mutants in Pseudomonas aeruginosa

Pseudomonas aeruginosa displays a native resistance to a variety of inhibitory compounds, including many analogues of amino acids, purines, and pyrimidines. Therefore, it has been difficult to isolate analogue-resistant regulatory mutants which have been so valuable in other microbial species for the study of enzyme control mechanisms and for the study of amino acid transport and its regulation. However, we have found that increased sensitivity to growth inhibition by analogues can be demonstrated by manipulation of the nutritional environment. When P. aeruginosa is grown with fructose as the nutritional source of carbon and energy, the cells become sensitive to growth inhibition by beta-2-thienylalanine and p-amino-phenylalanine, analogues of phenylalanine and tyrosine, respectively. Thus, mutants were isolated which are resistant to growth inhibition by beta-2-thienylalanine and p-amino-phenylalanine when fructose is the carbon source, and many of the beta-2-thienylalanine-resistant mutants overproduce phenylalanine. Several lines of evidence suggest that the increased sensitivity to growth inhibition by analogues of phenylalanine and tyrosine reflects a decreased rate of synthesis of aromatic amino acids or their precursors when fructose is the carbon source. This general approach promises to be valuable in the study of regulatory phenomena in microorganisms which, like P. aeruginosa, are naturally resistant to many metabolite analogues.     

Novel mutations in the pheA gene of Escherichia coli K-12 which result in highly feedback inhibition-resistant variants of chorismate mutase/prephenate dehydratase.

The bifunctional enzyme chorismate mutase/prephenate dehydratase (EC 5.4.99.5/4.2.1.51), which is encoded by the pheA gene of Escherichia coli K-12, is subject to strong feedback inhibition by L-phenylalanine. Inhibition of the prephenate dehydratase activity is almost complete at concentrations of L-phenylalanine greater than 1 mM. The pheA gene was cloned, and the promoter region was modified to enable constitutive expression of the gene on plasmid pJN302. As a preliminary to sequence analysis, a small DNA insertion at codon 338 of the pheA gene unexpectedly resulted in a partial loss of prephenate dehydratase feedback inhibition. Four other mutations in the pheA gene were identified following nitrous acid treatment of pJN302 and selection of E. coli transformants that were resistant to the toxic phenylalanine analog beta-2-thienylalanine. Each of the four mutations was located within codons 304 to 310 of the pheA gene and generated either a substitution or an in-frame deletion. The mutations led to activation of both enzymatic activities at low phenylalanine concentrations, and three of the resulting enzyme variants displayed almost complete resistance to feedback inhibition of prephenate dehydratase by phenylalanine concentrations up to 200 mM. In all four cases the mutations mapped in a region of the enzyme that has not been implicated previously in feedback inhibition sensitivity of the enzyme.     

Metabolic Influences on Tyrosine Excretion in Bacillus subtilis

The biosynthetic pathway for tyrosine synthesis is regulated both by repression of enzyme synthesis and by feedback inhibition of enzyme activity in Bacillus subtilis. Nevertheless, wild-type cells produce significantly more tyrosine than is required for protein synthesis, and part of this is excreted into the medium. Alteration of nutritional and other environmental conditions of cultivation strongly influenced the amount of tyrosine excretion. The excretion of tyrosine by wild-type cells was compared with that of a regulatory mutant having a feedback-insensitive prephenate dehydrogenase. Tyrosine excretion varied directly with the in vitro activity of prephenate dehydrogenase and inversely with temperature in the two strains. The regulatory mutant excreted five times as much tyrosine as the wild type at all growth temperatures examined. The carbon source used for growth significantly influenced the level of tyrosine excretion. The specific activity of prephenate dehydrogenase was also affected by the carbon source. Incorporation studies with isotopic tyrosine and fluorometric determinations of tyrosine concentrations extractable in hot water were used to measure operationally the tyrosine pools in the mutant and wild-type strains. The effects of various environmental conditions on the synthesis and excretion of tyrosine led to the conclusion that metabolic controls governing end product contrations exist which are completely independent of regulation by feedback inhibition and repression.     

Novel mutations in the pheA gene of Escherichia coli K-12 which result in highly feedback inhibition-resistant variants of chorismate mutase/prephenate dehydratase.

The bifunctional enzyme chorismate mutase/prephenate dehydratase (EC 5.4.99.5/4.2.1.51), which is encoded by the pheA gene of Escherichia coli K-12, is subject to strong feedback inhibition by L-phenylalanine. Inhibition of the prephenate dehydratase activity is almost complete at concentrations of L-phenylalanine greater than 1 mM. The pheA gene was cloned, and the promoter region was modified to enable constitutive expression of the gene on plasmid pJN302. As a preliminary to sequence analysis, a small DNA insertion at codon 338 of the pheA gene unexpectedly resulted in a partial loss of prephenate dehydratase feedback inhibition. Four other mutations in the pheA gene were identified following nitrous acid treatment of pJN302 and selection of E. coli transformants that were resistant to the toxic phenylalanine analog beta-2-thienylalanine. Each of the four mutations was located within codons 304 to 310 of the pheA gene and generated either a substitution or an in-frame deletion. The mutations led to activation of both enzymatic activities at low phenylalanine concentrations, and three of the resulting enzyme variants displayed almost complete resistance to feedback inhibition of prephenate dehydratase by phenylalanine concentrations up to 200 mM. In all four cases the mutations mapped in a region of the enzyme that has not been implicated previously in feedback inhibition sensitivity of the enzyme.     

Molecular cloning and characterization of ARO7-OSM2, a single yeast gene necessary for chorismate mutase activity and growth in hypertonic medium

Summary The chorismate mutase structural gene, ARO7, which is necessary for both phenylalanine and tyrosine biosynthesis was cloned by complementation in yeast. Genetic analysis showed that ARO7 was identical to a gene necessary for growth in hypertonic medium, OSM2, which mapped nearby. After restriction mapping and subcloning of the plasmid, the cloned gene was used to detect mRNA levels in several growth conditions. Enzyme activities were measured in various genotypes. At our level of detection ARO7-OSM2 is a low level constitutively expressed gene.     

A cis/trans Test of the Effect of the First Enzyme for Histidine Biosynthesis on Regulation of the Histidine Operon

Previous studies showed that when triazolalanine was added to a derepressed culture of a histidine auxotroph, repression of the histidine operon occurred as though histidine had been added (6). However, when triazolalanine was added to a derepressed culture of a strain with a mutation in the first gene of the histidine operon which rendered the first enzyme for histidine biosynthesis resistant to inhibition by histidine, repression did not occur. The studies reported here represent a cis/trans test of this effect of mutations to feedback resistance. Using specially constructed merodiploid strains, we were able to show that the wild-type allele is dominant to the mutant (feedback resistant) allele and that the effect operates in trans. We conclude that the enzyme encoded by the first gene of the histidine operon exerts its regulatory effect on the operon not by acting locally at its site of synthesis, but by acting as a freely diffusible protein.     

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.     

Mutation analysis of the feedback inhibition site of aspartokinase III of Escherichia coli K-12 and its use in L-threonine production.

Aspartokinase III (AKIII), one of three isozymes of Escherichia coli K-12, is inhibited allosterically by L-lysine. This enzyme is encoded by the lysC gene and has 449 amino acid residues. We analyzed the feedback inhibition site of AKIII by generating various lysC mutants in a plasmid vector. These mutants conferred resistance to L-lysine and/or an L-lysine analogue on their host. The inhibitory effects of L-lysine on and heat tolerance of 14 mutant enzymes were examined and DNA sequencing showed that the types of mutants were 12. Two hot spots, amino acid residue positions 318-325 and 345-352, were detected in the C-terminal region of AKIII and these enzyme regions may be important in L-lysine-mediated feedback inhibition of AKIII. Feedback resistant lysC relieved on L-threonine hyper-producing strain, B-3996, from reduced L-threonine productivity by addition of L-lysine, and furthermore increased L-threonine productivity even when no addition of L-lysine. It suggested that the bottleneck of L-threonine production of B-3996 was AK and feedback resistant lysC was effective because of the strict inhibition by cytoplasmic L-lysine.     

Regulation of the tryptophan synthetic enzymes in Clostridium butyricum

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