Enzymatic Basis for Overproduction of Tryptophan and Its Metabolites in Hansenula polymorpha Mutants

3-Deoxy-d-arabinoheptulosonate 7-phosphate (DAHP) synthetase and anthranilate synthetase are key regulatory enzymes in the aromatic amino acid biosynthetic pathway. The DAHP synthetase activity of Hansenula polymorpha was subject to additive feedback inhibition by phenylalanine and tyrosine but not by tryptophan. The synthesis of DAHP synthetase in this yeast was not repressed by exogenous aromatic amino acids, singly or in combinations. The activity of anthranilate synthetase was sensitive to feedback inhibition by tryptophan, but exogenous tryptophan did not repress the synthesis of this enzyme. Nevertheless, internal repression of anthranilate synthetase probably exists, since the content of this enzyme in H. polymorpha strain 3-136 was double that in the wild-type and less sensitive 5-fluorotryptophan-resistant strains. The biochemical mechanism for the overproduction of indoles by the 5-fluorotryptophan-resistant mutants was due primarily to a partial desensitization of the anthranilate synthetase of these strains to feedback inhibition by tryptophan. These results support the concept that inhibition of enzyme activities rather than enzyme repression is more important in the regulation of aromatic amino acid biosynthesis in H. polymorpha.     

Regulation of a Ligand-Mediated Association-Dissociation System of Anthranilate Synthesis in Clostridium butyricum

The anthranilate synthetase of Clostridium butyricum is composed of two nonidentical subunits of unequal size. An enzyme complex consisting of both subunits is required for glutamine utilization in the formation of anthranilic acid. Formation of anthranilate will proceed in the presence of partially pure subunit I provided ammonia is available in place of glutamine. Partially pure subunit II neither catalyzes the formation of anthranilate nor possesses anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase activity. The enzyme complex is stabilized by high subunit concentrations and by the presence of glutamine. High KCl concentrations promote dissociation of the enzyme into its component subunits. The synthesis of subunits I and II is coordinately controlled with the synthesis of the enzymes mediating reactions 4 and 5 of the tryptophan pathway. When using gel filtration procedures, the molecular weights of the large (I) and small (II) subunits were estimated to be 127,000 and 15,000, respectively. Partially pure anthranilate synthetase subunits were obtained from two spontaneous mutants resistant to growth inhibition by 5-methyltryptophan. One mutant, strain mtr-8, possessed an anthranilate synthetase that was resistant to feedback inhibition by tryptophan and by three tryptophan analogues: 5-methyl-tryptophan, 4- and 5-fluorotryptophan. Reconstruction experiments carried out by using partially purified enzyme subunits obtained from wild-type, mutant mtr-8 and mutant mtr-4 cells indicate that resistance of the enzyme from mutant mtr-8 to feedback inhibition by tryptophan or its analogues was the result of an alteration in the large (I) subunit. Mutant mtr-8 incorporates [(14)C]tryptophan into cell protein at a rate comparable with wild-type cells. Mutant mtr-4 failed to incorporate significant amounts of [(14)C]tryptophan into cell protein. We conclude that strain mtr-4 is resistant to growth inhibition by 5-methyltryptophan because it fails to transport the analogue into the cell. Although mutant mtr-8 was isolated as a spontaneous mutant having two different properties (altered regulatory properties and an anthranilate synthetase with altered sensitivity to feedback inhibition), we have no direct evidence that this was the result of a single mutational event.     

The Arabidopsis thaliana trp5 mutant has a feedback-resistant anthranilate synthase and elevated soluble tryptophan.

The first step of tryptophan biosynthesis is catalyzed by anthranilate synthase (AS), which is normally subject to feedback inhibition by tryptophan. Three independent trp5 mutants defective in the Arabidopsis thaliana AS alpha subunit structural gene ASA1 were identified by selection for resistance to the herbicidal compound 6-methylanthranilate. In all three mutants these biochemical changes are caused by a single amino acid substitution from aspartate to asparagine at residue position 341. Compared with the enzyme from wild-type plants, the tryptophan concentration causing 50% inhibition of AS activity in the trp5 mutant increased nearly 3-fold, the apparent Km for chorismate decreased by approximately 50%, and the apparent Vmax increased 60%. As a consequence of altered AS kinetic properties, the trp5 mutants accumulated 3-fold higher soluble tryptophan than wild-type plants. However, even though the soluble tryptophan levels were increased in trp5 plants, the concentrations of five tryptophan biosynthetic proteins remained unchanged. These data are consistent with the hypothesis that the reaction catalyzed by A. thaliana AS is rate limiting for the tryptophan pathway and that accumulation of tryptophan biosynthetic enzymes is not repressed by a 3-fold excess of end product.     

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.     

Mutant Strains of Escherichia coli K-12 Exhibiting Enhanced Sensitivity to 5-Methyltryptophan

Eighteen mutants (designated MT(s)), isolated in Escherichia coli K-12, showed increased sensitivity to inhibition of growth by 5-methyltryptophan. All mutants were also much more sensitive to 4-methyltryptophan and 7-azatryptophan but exhibited near normal sensitivity to 5-fluorotryptophan and 6-fluorotryptophan. All of the mutations were linked to the trp operon. Their locations within the trp operon were established by deletion mapping. There was good agreement between the map position of an MT(s) mutation and a lowered activity of one of the tryptophan pathway enzymes. Three mutants, one of which contained a mutation that mapped within the trpE gene, were deficient in their ability to use glutamine as an amino donor in the formation of anthranilic acid. Another trpE mutation led to the production of an anthranilate synthetase with an increased sensitivity to feedback inhibition by tryptophan.     

Production of l-Tryptophan by Azaserine-resistant Mutants of Brevibacterium flavum

Azaserine-resistant mutants derived from a 5-fluorotryptophan-resistant, l-tryptophan-producing mutant of Brevibacterium flavum, accumulated 10.3 g/liter of l-tryptophan at maximum. The production increased to 11.4 g/liter when l-serine was added. In the mutant, only anthranilate synthase among enzymes of the tryptophan-specific bio synthetic pathway increased in activity to a 2-fold higher level than that in the parent strain, No. 187. Sensitivity of anthranilate synthase to the feedback inhibition was not altered by the mutation. Activity of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, the first common enzyme for aromatic amino acid biosynthesis, also increased 2.7-fold and was less sensitive to the feedback inhibition by phenylalanine and tyrosine. Tryptophan transport activity in strain A-100 was similar as that in the parent. Azaserine inhibited anthranilate synthase activity by 50% at 0.075 mm. The inhibition was of a mixed type with respect to both the two substrates. Anthranilate synthase of strain A-100 was inhibited in a similar manner to that of the parent.     

Free tryptophan pool and tryptophan biosynthetic enzymes in Saccharomyces cerevisiae

The free tryptophan pool and the levels of two enzymes of tryptophan biosynthesis (anthranilate synthase and indoleglycerolphosphate synthase) have been determined in a wild type strain of Saccharomyces cerevisiae and in mutants with altered regulatory properties.The tryptophan pool of wild type cells growing in minimal medium is 0.07 mole per g dry weight. Addition of anthranilate, indole or tryptophan to the medium produces a fifteen- to forty-fold increase in tryptophan pool, but causes no repression of the biosynthetic enzymes. Inclusion of 5-methyltryptophan in the growth medium causes a reduction in growth rate and a derepression of the biosynthetic enzymes, and this is shown here not to be correlated with a decrease in the free tryptophan pool.Mutants with an altered anthranilate synthase showing decreased sensitivity to inhibition by l-tryptophan or by the analogue dl-5-methyltryptophan have a tryptophan pool far higher than the wild type strain, but no repression of indoleglycerolphosphate synthase was observed. Mutants with an anthranilate synthase more sensitive to tryptophan inhibition show a slightly reduced tryptophan pool, but no derepression of indoleglycerolphosphate synthase was found.A mutant with constitutively derepressed levels of the biosynthetic enzymes shows a considerably increased tryptophan pool. Addition of 5-methyltryptophan to the growth medium of non-derepressible mutants causes a decrease in growth rate accompanied by a decrease in the tryptophan pool.Abbreviations CDRP 1-(o-carboxyphenylamino)-1-deoxyribulosephosphate - paba paraaminobenzoic acid - PRA N-(5-phosphoribosyl)-anthranilate - tRNA transfer ribonucleic acid; trp1 to trp5 refer to the structural genes for corresponding tryptophan biosynthetic enzymes     

Regulation of tryptophan metabolism in Coprinus cinereus: Isolation and characterisation of mutants resistant to 5-fluoroindole

171 mutations conferring resistance to the indole analogue 5-fluoroindole (5 FI) were isolated in the filamentous basidiomycete fungus Coprinus cinereus. 5 FI is thought to be toxic because it is converted intracellularly to 5-fluorotryptophan (5 FT) which feedback inhibits the first enzyme of the tryptophan biosynthetic pathway, anthranilate synthase. Mutations were assigned to five loci, iar-1-iar-5 on the basis of functional analyses and mapping experiments. iar-5 mutations mapped in the anthranilate synthase structural gene and gave rise to an enzyme feedback resistant to tryptophan and its analogue. Mutants at other loci had regulatory changes. iar-1 and iar-3 mutants had elevated levels of two pathway enzymes measured (anthranilate synthase and tryptophan synthase) and were cross resistant to analogues of other aromatic amino acids suggesting that the entire aromatic pathway was derepressed. iar-3 mutants were unable to degrade metabolically derived typtophan to anthranilic acid unlike iar-1 mutants which excreted high levels of anthranilic acid. iar-2 mutants appeared to have a constitutive degradative pathway. iar-4 mutants had a blocked degradative pathway and unusual levels of tryptophan pathway enzymes.Abbreviations 5 FI 5-fluoroindole - 5 FT 5-fluorotryptophan - pFP para-fluorophenylalanine - mFT meta-fluoro-tyrosine     

Anthranilate synthase/anthranilate 5-phosphoribosyl 1-pyrophosphate phosphoribosyltransferase from Aerobacter aerogenes

1. Anthranilate synthase and phosphoribosyltransferase from Aerobacter aerogenes purify simultaneously and sediment together on sucrose gradients, showing that they occur as an enzyme aggregate. Both activities of the intact aggregate are subject to inhibition by tryptophan. 2. By using appropriate auxotrophic mutants it was shown that an intact active enzyme aggregate is formed when the components come from separate mutant strains. An intact active aggregate can also be formed when one component is from Escherichia coli and the other from A. aerogenes. 3. Phosphoribosyltransferase of A. aerogenes is active when not in an aggregate with anthranilate synthase, but is not subject to tryptophan inhibition, indicating that the inhibitor site is on the anthranilate synthase component. 4. Anthranilate synthase can be active and sensitive to tryptophan inhibition when complexed with an inactive phosphoribosyltransferase. 5. Kinetic studies on the anthranilate synthase activity show that tryptophan is a competitive inhibitor with respect to chorismate and a non-competitive inhibitor with respect to either glutamine or NH(4) (+) ions. This is consistent with a sequential mechanism of the ordered type in which chorismate is the first reactant.     

Tryptophan Analog Resistance Mutations in Chlamydomonas Reinhardtii

Forty single gene mutations in Chlamydomonas reinhardtii were isolated based on resistance to the compound 5'-methyl anthranilic acid (5-MAA). In other organisms, 5-MAA is converted to 5'-methyltryptophan (5-MT) and 5-MT is a potent inhibitor of anthranilate synthase, which catalyzes the first committed step in tryptophan biosynthesis. The mutant strains fall into two phenotypic classes based on the rate of cell division in the absence of 5-MAA. Strains with class I mutations divide more slowly than wild-type cells. These 17 mutations map to seven loci, which are designated MAA1 to MAA7. Strains with class II mutations have generation times indistinguishable from wild-type cells, and 7 of these 23 mutations map to loci defined by class I mutations. The remainder of the class II mutations map to 9 other loci, which are designated MAA8-MAA16. The maa5-1 mutant strain excretes high levels of anthranilate and phenylalanine into the medium. In this strain, four enzymatic activities in the tryptophan biosynthetic pathway are increased at least twofold. These include the combined activities of anthranilate phosphoribosyl transferase, phosphoribosyl anthranilate isomerase, indoleglycerol phosphate synthetase and anthranilate synthase. The slow growth phenotypes of strains with class I mutations are not rescued by the addition of tryptophan, but the slow growth phenotype of the maa6-1 mutant strain is partially rescued by the addition of indole. The maa6-1 mutant strain excretes a fluorescent compound into the medium, and cell extracts have no combined anthranilate phosphoribosyl transferase, phosphoribosyl anthranilate isomerase and indoleglycerol phosphate synthetase activity. The MAA6 locus is likely to encode a tryptophan biosynthetic enzyme. None of the other class I mutations affected these enzyme activities. Based on the phenotypes of double mutant strains, epistatic relationships among the class I mutations have been determined.     

Two single-base-pair substitutions causing desensitization to tryptophan feedback inhibition of anthranilate synthase and enhanced expression of tryptophan genes of Brevibacterium lactofermentum.

A 5-fluorotryptophan-resistant mutant, termed 1041, was isolated from Brevibacterium lactofermentum AJ12036. The anthranilate synthase of 1041 was insensitive to feedback inhibition by tryptophan, and the specific activities of the anthranilate synthase and anthranilate phosphoribosyltransferase of 1041 were 29- and 23-fold higher than those in parental strain AJ12036, respectively. A single-base change (adenine to cytosine) that resulted in a Ser-to-Arg substitution was found in the trpE structural gene of 1041. This substitution was identified as the cause of the desensitization to feedback inhibition by tryptophan of anthranilate synthase in 1041. Another substitution (guanine to adenine) was found at a position in which a mutation would destabilize the rho-independent terminator structure within the putative attenuator. The enhanced synthesis of tryptophan enzymes in 1041 could be caused by this substitution in the attenuator.     

New approach to tryptophan production by Escherichia coli: genetic manipulation of composite plasmids in vitro.

For the purpose of studying the production of L-tryptophan by Escherichia coli, the deletion mutants of the trp operon (trpAE1) were transformed with mutant plasmids carrying the trp operon whose anthranilate synthase and phosphoribosyl anthranilate transferase (anthranilate aggregate), respectively, had been desensitized to tryptophan inhibition. In addition to release of the anthranilate aggregate from the feedback inhibition required for plasmids such as pSC101 trp.I15, the properties of trp repression (trpR) and tryptophanase deficiency (tnaA) were both indispensable for host strains such as strain Tna (trpAE1 trpR tnaA). The gene dosage effects on tryptophan synthase activities and on production of tryptophan were assessed. A moderate plasmid copy number, approximately five per chromosome, was optimal for tryptophan production. Similarly, an appropriate release of the anthranilate aggregate from feedback inhibition was also a necessary step to ward off the metabolic anomaly. If the mutant plasmid pSC101 trp-I15 was further mutagenized (pSC101 trp.I15.14) and then transferred to Tna cells, an effective enhancement of tryptophan production was achieved. Although further improvement of the host-plasmid system is needed before commercial production of tryptophan can be realized by this means, a promising step toward this goal has been established.     

Differences between the anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferases of Salmonella typhimurium strains LT2 and LT7.

The anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferases (PRT), coded by the second structural gene (trpB) of the tryptophan (trp) operon in strains LT2 and LT7 of Salmonella typhimurium, differ from each other in a number of parameters. These include the apparent Km values for their substrates anthranilic acid and 5-phosphoribosylpyrophosphate, thermostability, sensitivity to substrate inhibition by anthranilic acid, as well as end-product inhibition by tryptophan and specific activity. The PRT of strain LT7 further differs from that of strain LT2 in that its apparent Km for 5-phosphoribosylpyrophosphate is three to seven times higher when associated with anthranilate synthase in the enzyme complex which catalyses the first two steps of tryptophan biosynthesis than in its free uncomplexed form, which the PRT of strain LT2 shows the same apparent Km for this substrate in both its free and complexed forms. These results confirm and extend the finding of Stuttard (1975) that strains LT2 and LT7 differ genetically form each other at a single site within region II of the trpB gene.     

Action of tryptophan analogues in Saccharomyces cerevisiae

In an analysis of the effects of various tryptophan and indole analogues in Saccharomyces cerevisiae we determined the mechanisms by which they cause growth inhibition: 4-Methyltryptophan causes a reduction in protein synthesis and a derepression of the tryptophan enzymes despite of the presence of high internal levels of tryptophan. This inhibition can only be observed in a mutant with increased permeability to the analogue. These results are consistent with but do not prove an interference of this analogue with the charging of tryptophan onto tRNA. 5-Methyltryptophan causes false feedback inhibition of anthranilate synthase, the first enzyme of the tryptophan pathway. This inhibits the further synthesis of tryptophan and results in results in tryptophan limitation, growth inhibition and derepression of the enzymes. Derepression eventually allows wild type cells to partially overcome the inhibitory effect of the analogue. 5-Fluoroindole is converted endogenously to 5-fluorotryptophan by tryptophan synthase. Both endogenous and externally supplied 5-fluorotryptophan are incorporated into protein. This leads to intoxication of the cells due to the accumulation of faulty proteins. 5-Fluorotryptophan also causes feedback inhibition of anthranilate synthase and reduces the synthesis of tryptophan which would otherwise compete with the analogues in the charging reaction. Indole acrylic acid inhibits the conversion of indole to tryptophan by tryptophan synthase. This results in a depletion of the tryptophan pool which, in turn, causes growth inhibition and derepression of the tryptophan enzymes.Abbreviations cpm counts per minute - OD optical density at 546 nm - TCA trichloro acetic acid - tRNA transfer ribonucleic acid; trp1 to trp5 refer to the structural genes for the corresponding tryptophan biosynthetic enzymes - trpl^– res. trp1^± refer to mutant strains synthesizing completely resp. partially defective enzymes     

Dominant expression of an amino-acid uptake deficiency in carrot somatic fusion hybrids

Somatic hybrids were selected previously by their ability to grow in medium containing normally inhibitory levels of the two amino acid analogs aminoethylcysteine (AEC) and 5-methyltryptophan (5MT) following fusion of protoplasts from a cell strain resistant to AEC with protoplasts resistant to 5MT. The hybrid nature of the selected clones was shown by several criteria including the presence of another resistance, azetidine-2-carboxylate (A2C), carried by one of the parental strains which was not selected for in the initial hybrid selection scheme. The characterization presented here shows that the AEC resistance in the parental strain, as well as the two somatic hybrids, was due to decreased AEC uptake. Also the 5MT resistance in the hybrids, as in the parent was caused by a feedback altered form of the tryptophan biosynthetic control enzyme, anthranilate synthase which leads to increases in free tryptophan. The A2C resistance was caused by the accumulation of free proline by a mechanism which has not been studied. These studies confirm that AEC resistance caused by decreased uptake can be expressed dominantly in protoplast fusion hybrids.Abbreviations A2C Azetidine-2-carboxylate - AEC Aminoethylcysteine - 5MT 5-methyltryptophan     

Tryptophan biosynthesis in Saccharomyces cerevisiae: control of the flux through the pathway.

Enzyme derepression and feedback inhibition of the first enzyme are the regulatory mechanisms demonstrated for the tryptophan pathway in Saccharomyces cerevisiae. The relative contributions of the two mechanisms to the control of the flux through the pathway in vivo were analyzed by (i) measuring feedback inhibition of anthranilate synthase in vivo, (ii) determining the effect of regulatory mutations on the level of the tryptophan pool and the flux through the pathway, and (iii) varying the gene dose of individual enzymes of the pathway at the tetraploid level. We conclude that the flux through the pathway is adjusted to the rate of protein synthesis by means of feedback inhibition of the first enzyme by the end product, tryptophan. The synthesis of the tryptophan enzymes could not be repressed below a basal level by tryptophan supplementation of the media. The enzymes are present in excess. Increasing or lowering the concentration of individual enzymes had no noticeable influencing on the overall flux to tryptophan. The uninhibited capacity of the pathway could be observed both upon relieving feedback inhibition by tryptophan limitation and in feedback-insensitive mutants. It exceeded the rate of consumption of the amino acid on minimal medium by a factor of three. Tryptophan limitation caused derepression of four of the five tryptophan enzymes and, as a consequence, led to a further increase in the capacity of the pathway. However, because of the large reserve capacity of the "repressed" pathway, tryptophan limitation could not be imposed on wild-type cells without resorting to the use of analogs. Our results, therefore, suggest that derepression does not serve as an instrument for the specific regulation of the flux through the tryptophan pathway.     

Inheritance and Stability of 5-Methyltryptophan Resistance in Datura innoxia Selected In vitro

The resistance to 5-methyltryptophan (5MT) in Datura innoxiaplants regenerated from selected cultures was maintained forover 10 years and was inherited in progeny as if controlledby a single, nuclear, dominant gene. The resistance was causedby a feedback altered anthranilate synthase leading to highfree tryptophan levels. (Received August 18, 1995; Accepted February 5, 1996)     

Mechanism of 3-Methylanthranilic Acid Derepression of the Tryptophan Operon in Escherichia coli

3-Methylanthranilic acid (3MA) inhibits growth and causes derepression of the tryptophan biosynthetic enzymes in wild-type strains of Escherichia coli. Previous reports attributed this effect to an inhibition of the conversion of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate to indole-3-glycerol phosphate and a consequent reduction in the concentration of endogenous tryptophan. Our studies have shown that 3MA-resistant mutants linked to the tryptophan operon have a feedback-resistant anthranilate synthetase; mutants with an altered indole-3-glycerol phosphate synthetase were not found. 3MA or 7-methylindole can be metabolized to 7-methyltryptophan, and 3MA, 7-methylindole, and 7-methyltryptophan lead to derepression of the tryptophan operon. Furthermore, 3MA-resistant mutants are also resistant to 7-methylindole derepression. These results strongly suggest that the primary cause of derepression by 3MA is through its conversion to 7-methyltryptophan, which can inhibit anthranilate synthetase, thereby decreasing the concentration of endogenous tryptophan. Unlike 5- or 6-methyltryptophan, 7-methyltryptophan does not appear to function as an active corepressor.     

Study of anthranilate synthase function by replacement of cysteine 84 using site-directed mutagenesis

Cysteine 84 was replaced by glycine in Serratia marcescens anthranilate synthase Component II using site-directed mutagenesis of cloned trpG. This replacement abolished the glutamine-dependent anthranilate synthase activity but not the NH3-dependent activity of the enzyme. The mutation provides further evidence for the role of active site cysteine 84 in the glutamine amide transfer function of anthranilate synthase Component II. By the criteria of circular dichroism, proteolytic inactivation, and feedback inhibition the mutant and wild type enzymes were structurally similar. The NH3-dependent anthranilate synthase activity of the mutant enzyme supported tryptophan synthesis in media containing a high concentration of ammonium ion.