Physiological studies on ergot: further studies on the induction of alkaloid synthesis by tryptophan and its inhibition by phosphate

The failure of l-leucine to stimulate ergot alkaloid production in a synthetic medium indicates that the previously observed stimulation by tryptophan and tryptophan analogues does not merely represent a nutritional effect. Tryptophan, but not mevalonate or 5-methyltryptophan, is able to overcome the inhibition of alkaloid synthesis by high levels of inorganic phosphate. Therefore, high phosphate levels seem to limit the synthesis of tryptophan; they may, in addition, prevent induction of alkaloid synthesis by preventing accumulation of tryptophan. Experiments which indicate a 2- to 3-fold temporary increase of intracellular free tryptophan and a 20- to 25-fold increase of tryptophan synthetase activity during the transition period between growth and alkaloid production phase are in agreement with the previously postulated induction of alkaloid synthesis by tryptophan. The latter experiments also indicate 4- to 6-fold repression of this enzyme by tryptophan.     

Purification and properties of dimethylallylpyrophosphate: Tryptophan dimethylallyl transferase,the first enzyme of ergot alkaloid biosynthesis in Claviceps. sp. SD 58

Dimethylallylpyrophosphate:l-tryptophan dimethylallyltransferase (DMAT synthetase), the first pathway-specific enzyme of ergot alkaloid biosynthesis, has been isolated from mycelia of Claviceps sp., strain SD 58, and purified to apparent homogeneity. The enzyme reaction products were identified as l-4-(γ,γ-dimethylallyl)tryptophan and inorganic pyrophosphate. DMAT synthetase is a single subunit protein of molecular weight 70,000–73,000 and has an isoelectric point at pH 5.8. The enzyme is activated by Fe²⁺, Mg²⁺, and particularly Ca²⁺; K_m values for l-tryptophan and dimethylallylpyrophosphate were determined to be 0.067 and 0.2 mm, respectively. Kinetic analysis indicated that the DMAT synthetase reaction proceeds by a sequential rather than a ping-pong mechanism.     

Effect of phosphate on ergot alkaloid synthesis inAspergillus fumigatus

High concentration of inorganic phosphate in the culture medium ofAspergillus fumigatus inhibited ergot alkaloid synthesis. Addition ofl-tryptophan but not mevalonate or 5-methyltryptophan to the above culture restored the alkaloid synthesis to the level found in normal cultures. The decrease in alkaloid synthesis in the fungus accompanies an increase in cell mass, cellular protein and sterol content. Aspartate aminotransferase and alanine aminotransferase activities were significantly increased in the high-phosphate culture. Part of the work was presented at the seminar on “Enzymatic Methods in Mycology” organised by the Czechoslovak Microbiological Society in Brno, Czechoslovakia, in June 1975.     

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.     

Role of endogenous tryptophan during submerged fermentation of ergot alkaloids

Shake flask cultures ofClaviceps paspali (Stev. et Hall) andClaviceps purpurea (Fr.) Tul. on simple synthetic medium have been studied. Both strains grown in the absence of added tryptophan accumulate extra endogenous tryptophan. A certain concentration of cell-pool tryptophan is needed to promote alkaloid synthesis. Alkaloid production commences while tryptophan synthetase activity is increasing. In the alkloid-producing phase cell-pool tryptophan shows a single minimum while the change in level of cell-protein tryptophan is negligible. Alkaloid formation is suggested to reflect a regulatory device to keep endogenous tryptophan balanced. By adding amitrole the alkaloid spectrum is changed. The tryptophan-histidine cross-pathway probably serves a useful function inthe biosynthesis of ergot alkaloids.     

Biosynthesis of ergot alkaloids. Some new results on an old problem (Review)]

The biosynthetic pathway leading from L-tryptophan, mevalonic acid and methionine to the tetracyclic ergoline ring system of the ergot alkaloids in Claviceps species is reviewed. This pathway entails many mechanistically intriguing features. Recent studies are also discussed which reveal the stereochemical course of the isoprenylation of tryptophan and of the N-methylation of dimethylallyltryptophan (DMAT) and which shed some light on the likely steps leading from the open-chain precursors, N-methyl-DMAT to the tricyclic intermediate, chanoclavine-1. Finally, some plans are outlined to probe the evolutionary relationship of ergot alkaloid biosynthesis in fungi to that in higher plants of the family Convolvulaceae.     

Physiological status of submergedClaviceps during enzymic assembly of ergot alkaloids

Ergot alkaloids are formed only for arelatively brief time during the lifespan of the culture and under conditions of reduced proliferation. They cannot be taken for waste products of general metabolism. Ergot strains are capable of carrying out all the simple steps of the anthranilic acid—tryptophan cycles. Alkaloids influence activities of certain enzymes of primary metabolism in the ergot mycelium,e.g. tryptophan synthetase, acetyl-CoA carboxylase, citrate synthase, isocitrate lyase, and malate synthase. Ergot alkaloids do not belong to a group of physiologically inert secondary metabolites. A tentative scheme of the enzymic assembly of the ergoline nucleus is presented. The increased yield of ergoline alkaloids may be attributed to the following points: (1) Unbalnced growth of the culture. (2) Support of competition of fatty acids and alkaloid biosynthesis for acetyl-CoA. (3) Decreased activities of tricarboxylic acid and glyoxylate cycles. (4) Positive association between the rate of protein turnover and alkaloid formation. (5) Stimulation of both tryptophan synthesis and degradation via kynurenine—anthranilate. (6) Regulation of tryptophan-histidine cross-pathway. (7) Continuous control of the alkaloid level during fermentation.     

Effect of Tween 80 on alkaloid-producing cultures ofClaviceps paspali

Addition of Tween 80 to submerged cultures ofClaviceps paspali (Stevens and Hall) growing in a simple defined medium increased biomass formation and caused a temporary change in alkaloid synthesis intensity. The Tween-supplemented culture reached maximal alkaloid yields four days earlier than the control. The shift of alkaloid production was associated with a shift of organic acids and amino acids in the cell-pool. Thus the maximal formation of alkaloids was characterized by a decrease in the amount of citric acid, by the disappearance of succinic, fumaric and oxaloacetic acids and by increased accumulation of methionine, cysteine, alanine and histidine. The slow alkaloid synthesis was accompanied by a relatively high content of citric and succinic acids in the cell-pool and by the absence of methionine. The data are consistent with the hypothesis that ergot alkaloids participate in the regulation of the metabolism of cultures.     

Separation of Two Forms of Anthranilate Synthetase from 5-Methyltryptophar Susceptible and -Resistant Cultured Solarium tuberosumCells

Potato cell suspension cultures (Solanum tuberosumL. cv. Merrimack) have been selected which are resistant to growth inhibition by D,L-5-methyltryptophan. Anthranilate synthetase activity in crude extracts from resistant cells was less sensitive to feedback inhibition by L-tryptophan and D,L-5-methyltryptophan than the activity from the sensitive line. This altered feedback control apparently accounts for the cell's resistance to growth inhibition since there is a 48-fold increase in free tryptophan in one of the resistant cell lines. Preparative polyacrylamide gel electro-phoresis separated feedback-sensitive and -resistant forms of anthranilate synthetase in extracts from both 5-methyltryptophan-susceptible and -resistant cells, with a predominance of the corresponding form in the respective cell type. The anthranilate synthetase activity from the 5-methyltryptophan-resistant line was inactivated more slowly by incubation of crude extracts at 50°C than the activity from the sensitive line. These results suggest the presence of two isoenzymes of anthranilate synthetase in cultured potato cells.     

Antimetabolite Action of 5-Methyltryptophan in Bacillus subtilis

The roles of anthranilate and histidine in the regulation of tryptophan synthesis were implicated by observations of antagonistic relationships with 5-methyltryptophan in Bacillus subtilis.     

Some characteristics of tryptophan uptake in Claviceps species

Tryptophan serves as a precursor for the biosynthesis of alkaloids in the ergot fungus, Claviceps purpurea (Fries) Tulasne, and also is believed to act as an inducer of the enzymes necessary for alkaloid production. The characteristics of the transport system responsible for the accumulation of tryptophan in ergot mycelium were investigated, with the goal of clarifying the complex relationships among tryptophan uptake, size of the free intracellular pool of tryptophan, and alkaloid production. The characteristics of tryptophan uptake were studied by pulse feeding radioactively labeled tryptophan to cultures of Claviceps species, strain SD-58, which represented a variety of ages and nutritional states. Tryptophan accumulation in strain SD-58 is mediated by an energy-requiring system which exhibits specificity for neutral aromatic and aliphatic l-amino acids, is pH and temperature dependent, and shows saturation at high substrate concentrations. Tryptophan transport is a function of the intracellular concentration of free tryptophan, the nitrogen deficiency of the mycelium, the rate of growth, and alkaloid production, which were measured in Claviceps strain SD-58 growth in several culture media, some of which promoted alkaloid production and some of which did not. The results indicate that the initial velocity of tryptophan transport is not directly related to alkaloid production.     

The determinant step in ergot alkaloid biosynthesis by an endophyte of perennial ryegrass

Many cool-season grasses harbor fungal endophytes in the genus Neotyphodium, which enhance host fitness, but some also produce metabolites--such as ergovaline--believed to cause livestock toxicoses. In Claviceps species the first step in ergot alkaloid biosynthesis is thought to be dimethylallyltryptophan (DMAT) synthase, encoded by dmaW, previously cloned from Claviceps fusiformis. Here we report the cloning and characterization of dmaW from Neotyphodium sp. isolate Lp1, an endophyte of perennial ryegrass (Lolium perenne). The gene was then disrupted, and the mutant failed to produce any detectable ergovaline or simpler ergot and clavine alkaloids. The disruption was complemented with the C. fusiformis gene, which restored ergovaline production. Thus, the biosynthetic role of DMAT synthase was confirmed, and a mutant was generated for future studies of the ecological and agricultural importance of ergot alkaloids in endophytes of grasses.     

Cell-pool tryptophan phases in ergot alkaloid fermentation

Three cell-pool tryptophan phases are recorded as characteristics of the alkaloid fermentation byClaviceps paspali grown on a simple defined medium without tryptophan. Within the early phase designated “tryptophan down” the alkaloid-biosynthetic activity of the mycelium attains the maximum, protein synthesis is reduced and extracellular proteases are formed. Cell-pool tryptophan level (b) drops, tryptophan synthetase activity (c) intensifies and sums of logb+logc after different time intervals remain constant. In the subsequent “tryptophan up” phase tryptophan level (b) increases, alkaloid yield (a) becomes a function of time and reaches the top level still tolerable by tryptophan synthetase. The difference of the logb—logc is constant. The tryptophan synthetase diminishes its activity simultaneously with the alkaloid-biosynthetic activity of the mycelium. The district between the “tryptophan down” and “tryptophan up” phase is an especially promising target for the investigation of the regulation of alkaloid formation and continuous fermentation of these compounds. During the third, i.e. “tryptophan over” phase, cell-pool tryptophan accumulates and attains a concentration exerting a negative effect on the alkaloid biosynthesis.     

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.     

Effect of the exogenous tryptophan on biosynthesis of ergot alkaloids in Penicillium sizovae

Various concentrations of exogenous L and D-tryptophan as well as of their analogue D,L-6-methyltryptophan were added to the growth medium of Penicillium sizovae during its inoculation and after the active growth of the fungus was over. The authors studied the effect of these compounds on the accumulation of exocellular alkaloids and biomass as well as on the synthesis of proteins, the content of free tryptophan in the cells, and the activity of tryptophan synthetase. As was shown in experiments using labeled tryptophan, this amino acid is a direct precursor of alkaloids in the culture.     

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.     

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.     

Expression of 5-Methyltryptophan Resistance in Plants Regenerated from Resistant Cell Lines of Datura innoxia

Seventy-nine 5-methyltryptophan-resistant cell lines have been selected from haploid Datura innoxia Mill. cell cultures by plating suspensions in agar medium containing a growth inhibitory concentration of 5-methyltryptophan. Mutagen treatment increased the frequency of resistance. The eleven variants tested posses an altered anthranilate synthase less sensitive to feedback inhibition by tryptophan. All five of the variants which were analyzed for free amino acids contained elevated levels of free tryptophan (8 to 30 times the wild type level). None of the selected cell lines were auxin-autotrophic. Resistance to 5-methyltryptophan, altered anthranilate synthase, and high free tryptophan (4 to 44 times) were also expressed in leaves of plants regenerated from the variant lines and in cultures reinitiated from the resistant plants. These results show that the amino acid overproduction phenotype can be selected at the cellular level of organization and be expressed identically in whole plants regenerated from the selected cells.     

Relationship of indole-3-acetic acid and tryptophan concentrations in normal and 5-methyltryptophan-resistant cell lines of wild carrots

A 5-methyltryptophan(5-MT)-resistant cell line of wild carrot (Daucus carota L.), W001, that exhibited auxin-independent callus growth, was found to accumulate indole-3-acetic acid (IAA) and tryptophan (trp). Anthranilate-synthetase activity in W001 cell extract was less sensitive to feedback inhibition by trp than in the original 5-MT-sensitive cell lines. It is hypothesized that the resistant enzyme allowed more trp synthesis and accumulation which, in turn, affected the IAA concentration in the cell. Since carrot cultures cannot regenerate in the presence of exogenous auxin, the elevated IAA concentration in W001 may be responsible for its drastically reduced capacity to regenerate. The relationship between trp and IAA levels was further investigated by examining the effect of 2,4-dichlorophenoxy acetic acid (2,4-D) on the endogenous concentration of trp and IAA. In general, the IAA level was reduced but the trp concentration was elevated when 2,4-D was present in the culture medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 5-MT 5-methyltryptophan - 5-MT^r 5-MT-resistant - 5-MT^s 5-MT-sensitive - trp tryptophan