Tryptophan Synthetase Activity Research Articles

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.

Control of Tryptophan Biosynthesis in Plant Tissue Cultures: Lack of Repression of Anthranilate and Tryptophan Synthetases by Tryptophan

AbstractTobacco (cv. Xanthi and cv. Wisconsin 38), rice, carrot, tomato, and soybean tissue cultures were grown in liquid media containing L‐tryptophan. The addition of tryptophan increased the cellular tryptophan levels greatly (12–2500 fold), but did not lower appreciably the levels of two tryptophan biosynthetic enzymes, anthranilate synthetase and tryptophan synthetase. However, the addition of 50 μM tryptophan to the crude enzyme extract completely inhibited the anthranilate synthetase activity while 1 mM tryptophan inhibited the tryptophan synthetase activity by only 10–20°/o. This information indicates that tryptophan biosynthesis is controlled by the feedback inhibition of anthranilate synthetase by tryptophan and not by repression of enzyme synthesis.All of the species had significant enzyme levels. Anthranilate synthetase activity could not be detected in extracts from cells grown on tryptophan unless the extracts were first passed through two G‐25 Sephadex columns with a short 30 °C warming step in between, a procedure shown to remove an inhibitor of the enzyme.

Regulation of Tryptophan Biosynthetic Enzymes in Neurospora crassa

The formation of enzymatic activities involved in the biosynthesis of tryptophan in Neurospora crassa was examined under various conditions in several strains. With growth-limiting tryptophan, the formation of four enzymatic activities, anthranilic acid synthetase (AAS), anthranilate-5-phosphoribosylpyrophosphate phosphoribosyl transferase (PRAT), indoleglycerol phosphate synthetase (InGPS), and tryptophan synthetase (TS) did not occur coordinately. AAS and TS activities began to increase immediately, whereas PRAT and InGPS activities began to increase only after 6 to 12 hr of incubation. In the presence of amitrole (3-amino-1,2,4-triazole), the formation of TS activity in a wild-type strain was more greatly enhanced than were AAS and InGPS activities. With a tr-3 mutant, which ordinarily exhibits an elevated TS activity, amitrole did not produce an increase in TS activity greater than that observed on limiting tryptophan. With tr-3 mutants, the increased levels of TS activity could be correlated with the accumulation of indoleglycerol in the medium; prior genetic blocks which prevented or reduced the synthesis of indoleglycerol also reduced the formation of TS activity. The addition of indoleglycerol to cultures of a double mutant (tr-1, tr-3) which could not synthesize indoleglycerol markedly stimulated the production of TS activity but not PRAT activity; the production of TS activity reached the same level with limiting or with excess tryptophan. A model explaining these and other related observations on enzyme formation in N. crassa is proposed.

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.

The B Protein of Escherichia coli Tryptophan Synthetase: I. EFFECTS OF SULFHYDRYL MODIFICATION ON ENZYMATIC ACTIVITIES AND SUBUNIT INTERACTION

Abstract Sulfhydryl modification of the B protein of Escherichia coli tryptophan synthetase has been carried out to investigate the role of sulfhydryl residues in the active center of this protein and in its interaction with the A protein. Both N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) react with two sulfhydryls per monomer of apo-B protein and one sulfhydryl per monomer of the holo-B protein, which contains 1 mole of pyridoxal phosphate per monomer. The serine deaminase activity and the tryptophan synthetase activity are both largely lost after the reaction of one sulfhydryl in the holo-B protein with DTNB. Similar results are obtained after the reaction of one sulfhydryl in the apo-B protein with NEM. In contrast, after the reaction of one sulfhydryl in the holo-B protein with NEM, the serine deaminase activity is somewhat enhanced, whereas, the tryptophan synthetase activity is largely lost. Kinetic comparisons of the NEM-modified holo-B protein with the untreated holo-B protein show that the Km for l-serine is largely unchanged, but that both the Km for indole and the Vmax in the tryptophan synthetase reaction are changed by modification. Since one sulfhydryl in the B protein is fully protected from NEM modification by pyridoxal phosphate and a second sulfhydryl is partially protected from modification in the holo-B protein by the substrate l-serine, both of these sulfhydryl residues may be located in the active center of the protein. The reactive sulfhydryl residue in the holo-B protein is also protected by interaction with the A protein. However, this sulfhydryl is not necessary for the interaction of the A and B proteins since the holo-B protein modified with 1 mole of NEM still associates normally with the A protein and facilitates its hydrolysis of indole-3-glycerol phosphate.

Studies on tryptophan synthetases from various strains of Bacillus subtilis.

1. Tryptophan synthetase B of three strains of Bacillus subtilis was prepared from ;exo-protoplastic' and ;endo-protoplastic' fractions; the enzyme from ;exo-protoplastic' fraction was purified 30- to 120-fold by ammonium sulphate precipitation and DEAE-cellulose column chromatography; the latter step separated this enzyme from tryptophan synthetase A, tryptophanase and proteolytic enzymes, but the purified preparations were not stable. 2. The activity of tryptophan synthetase B did not depend on the presence of tryptophan synthetase A. 3. Tryptophan synthetases B of the strains tested differed in their utilization of 2- and 7-methylindole as compared with indole; this suggests that these tryptophan synthetases B are not identical.

Effect of gene dosage on tryptophan synthetase activity in Saccharomyces cerevisiae.

Effect of gene dosage on tryptophan synthetase activity in Saccharomyces cerevisiae.

Regulation of factors that influence the in vitro stability of tryptophan synthetase from yeast.

The activity of tryptophan synthetase in crude extracts from Saccharomyces cerevisiae is stable if the cells are cultured in a complex medium, but extremely unstable if they are cultured in a minimal medium. The difference is not the result of different inherent properties of the enzyme formed in the two cultures. Rather, there are at least two kinds of macromolecular factors that influence the stability, one inactivating tryptophan synthetase and the other protecting against this inactivation. The abundance of these factors varies with the composition of the culture medium, which accounts for the differences observed.

Tryptophan synthetase in shoot and root tissue of pea seedlings

The occurrence of tryptophan synthetase in excised tomato root cultures (Lycopersicon esculentum Mill. var. Red River) and pea seedlings (Pisum sativum L. var. Alaska) was examined using cell-free extracts. No tryptophan synthetase activity could be detected in the former. Using serine-14C and indole-14C, with extracts from peas, it is shown by chromatographic and radioautographic methods that tryptophan is the product of the reaction, that both the substrates are necessary, and that pyridoxal phosphate is a necessary cofactor. Inhibition by metal ions, lability, thermolability, and various other properties are described. Partial purification was effected by gel filtration. The enzyme activity in pea root extracts, as measured by indole utilization per unit fresh weight, is much lower than in the enzyme preparations from terminal buds. A system causing destruction of indole, other than tryptophan synthetase, is also present in crude root extracts.

Altered Protein Formation as a Result of Suppression in Neurospora Crassa

The mutant td201 of Neurospora crassa is mutated in the trp-3 locus and forms an altered tryptophan synthetase. A suppressor mutation, su2-6, in this mutant, unlinked to the trp-3 locus, results in the production of wild-type tryptophan synthetase activity, which accounts for the alleviation of the tryptophan or indole requirement. This enzyme activity is associated with a protein physically dissimilar to the wild-type enzyme. A second altered protein, a serologically cross-reacting material is also formed in the suppressed mutant, in addition to the altered enzyme normally formed by the td201 mutant. Normal growth, equivalent to that of wild type, is not restored in the suppressed mutant even with tryptophan supplementation. The relationship of the data to possible mechanisms of suppression is discussed.

The Role of Pyridoxal Phosphate in the Aldolytic Activity of Tryptophan Synthetase from Neurospora crassa

The Role of Pyridoxal Phosphate in the Aldolytic Activity of Tryptophan Synthetase from Neurospora crassa

An increase in alkaline phosphatase in an [formula omitted] system derived from [formula omitted

There have been a number of reports that cell-free preparations from micro-organisms are capable of enzyme synthesis. Increases in the activity of tryptophan synthetase of Neurospora crassa ( Wainwright, 1959) and in the β-galactosidase of Escherichia coli ( Nisman and Fukuhara, 1959; Kameyama and Novelli, 1960) were the first to be described; more recently, the formation of tryptophan synthetase of E. coli ( Yura, et al., 1962) and amylase of Bacillus subtilis ( Oishi, et al., 1962) by particulate and soluble fractions have been reported. The present communication describes a similar observation with respect to alkaline phosphatase of B. subtilis and presents evidence concerning requirements and the effects of inhibitors. The requirement for a supernatant fraction from a genetically competent strain is discussed.

Pilot plant batch production of Neurospora crassa

AbstractA 50‐gal. growth chamber is described together with its air filter and humidifier. The unit was designed especially for the production of Neurospora crassa, and was found to be effective in producing bulk amounts of mycelia with a high specific activity of tryptophan synthetase.

The Role of Zinc in the Growth of Plant Tissue Cultures

Klein, Richard M., Emerita M. Caputo, and Barbara A. Witterholt. (New York Bot. Gard., N. Y., N. Y.) The role of zinc in the growth of plant tissue cultures. Amer, Jour. Bot. 49(4): 323–327. Illus. 1962.—Zinc-sufficient and zinc-deficient auxin-auxotrophic callus and auxin-prototrophic crown-gall cultures of Parthenocissus tricuspidatus were examined for their growth capacity and ability to synthesize tryptophan. Growth capacity decreased rapidly when zinc ion was withheld and tryptophan synthetase activity in vitro was reduced. Growth of –Zn crown-gall tissues was stimulated by either auxin or tryptophan and growth of –Zn callus cultures was stimulated by tryptophan.

THE ROLE OF ZINC IN THE GROWTH OF PLANT TISSUE CULTURES

Klein, Richard M., Emerita M. Caputo, and Barbara A. Witterholt. (New York Bot. Gard., N. Y., N. Y.) The role of zinc in the growth of plant tissue cultures. Amer, Jour. Bot. 49(4): 323–327. Illus. 1962.—Zinc‐sufficient and zinc‐deficient auxin‐auxotrophic callus and auxin‐prototrophic crown‐gall cultures of Parthenocissus tricuspidatus were examined for their growth capacity and ability to synthesize tryptophan. Growth capacity decreased rapidly when zinc ion was withheld and tryptophan synthetase activity in vitro was reduced. Growth of –Zn crown‐gall tissues was stimulated by either auxin or tryptophan and growth of –Zn callus cultures was stimulated by tryptophan.

Mediation of the Genetic Control of Protein Synthesis in Cell-free Extracts of Neurospora crassa

I HAVE recently demonstrated the development of tryptophan synthetase activity by cell-free extracts from conidia of wild-type Neurospora crassa. The process appeared to be one of synthesis of enzyme protein, and was associated with the net increase in the protein content of the system1. Similar extracts prepared from the td 2 tryptophan-less mutant strain neither contained nor developed any detectable tryptophan synthetase activity, but did catalyse a net synthesis of protein.

ON THE DEVELOPMENT OF INCREASED TRYPTOPHAN SYNTHETASE ENZYME ACTIVITY BY CELL-FREE EXTRACTS OF NEUROSPORA CRASSA

A cell-free extract from conidia of Neurospora crassa developed increased tryptophan synthetase activity when incubated with a mixture of amino acids, a source of energy, and inorganic salts. Fifteen individual acids were required for development of maximal increases of enzymic activity. Either hexose diphosphate or adenosine triphosphate could serve as energy source. Diphosphopyridine nucleotide, polyvinyl acetate, and magnesium and calcium ions were required. The addition of ribonuclease enzyme, ethionine, fluorophenylalanine, thienylalanine, or various antibiotics prevented development of increased tryptophan synthetase activity. The increase of activity was accompanied by a net synthesis of protein.

Tryptophan Synthetase Activity in Pea Seedling Extracts.

Tryptophan Synthetase Activity in Pea Seedling Extracts.

ON THE DEVELOPMENT OF INCREASED TRYPTOPHAN SYNTHETASE ENZYME ACTIVITY BY CELL-FREE EXTRACTS OF NEUROSPORA CRASSA

A cell-free extract from conidia of Neurospora crassa developed increased tryptophan synthetase activity when incubated with a mixture of amino acids, a source of energy, and inorganic salts. Fifteen individual acids were required for development of maximal increases of enzymic activity. Either hexose diphosphate or adenosine triphosphate could serve as energy source. Diphosphopyridine nucleotide, polyvinyl acetate, and magnesium and calcium ions were required. The addition of ribonuclease enzyme, ethionine, fluorophenylalanine, thienylalanine, or various antibiotics prevented development of increased tryptophan synthetase activity. The increase of activity was accompanied by a net synthesis of protein.

Restoration of Tryptophan Synthetase Activity in Escherichia coli by Suppressor Mutations

Restoration of Tryptophan Synthetase Activity in <i>Escherichia coli</i> by Suppressor Mutations