[46] Chorismate to tryptophan (Escherichia coli)—anthranilate synthetase, PR transferase, PRA isomerase, InGP synthetase, tryptophan synthetase

Creighton, Thomas E.; Yanofsky, Charles

doi:10.1016/0076-6879(71)17215-1

Cited by 115 publications

(56 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These were then subcultured into the same medium in the presence or absence of 50 g of L-tryptophan per ml and grown to the mid-exponential phase at various temperatures. Anthranilate synthase activity was assayed as described previously (8), and ␤-galactosidase activity was assayed in permeabilized cells as described by Miller (20). One unit of anthranilate synthase specific activity represents the enzyme activity required to synthesize 0.01 nmol of anthranilate in 20 min at 37ЊC per 0.1 ml of a washed, resuspended cell suspension (OD 600 ϭ 0.5).…”

Section: Methodsmentioning

confidence: 99%

A temperature-sensitive trpS mutation interferes with trp RNA-binding attenuation protein (TRAP) regulation of trp gene expression in Bacillus subtilis

1996

View full text Add to dashboard Cite

In Bacillus subtilis, the tryptophan-activated trp RNA-binding attenuation protein (TRAP) regulates expression of the seven tryptophan biosynthetic genes by binding to specific repeat sequences in the transcripts of the trp operon and of the folate operon, the operon containing trpG. Steinberg observed that strains containing a temperature-sensitive mutant form of tryptophanyl-tRNA synthetase, encoded by the trpS1 allele, produced elevated levels of the tryptophan pathway enzymes, when grown at high temperatures in the presence of excess L-tryptophan (W. Steinberg, J. Bacteriol. 117:1023-1034, 1974). We have confirmed this observation and have shown that expression of two reporter gene fusions, trpE-lacZ and trpG-lacZ, is also increased under these conditions. Deletion of the terminator or antiterminator RNA secondary structure involved in TRAP regulation of trp operon expression eliminated the trpS1 effect, suggesting that temperature-sensitive expression was mediated by the TRAP protein. Analysis of expression of mtrB, the gene encoding the TRAP subunit, both by examination of a lacZ translational fusion and by measuring the intracellular levels of TRAP by immunoblotting, indicated that the trpS1-induced increase in trp gene expression was not due to inhibition of mtrB expression or to alteration of the amount of TRAP present per cell. Increasing the cellular level of TRAP by overexpressing mtrB partially counteracted the trpS1 effect, demonstrating that active TRAP was limiting in the trpS1 mutant. We also showed that elevated trp operon expression was not due to increased transcription initiation at the upstream aroF promoter, a promoter that also contributes to trp operon expression. We postulate that the increase in trp gene expression observed in the trpS1 mutant is due to the reduced availability of functional TRAP. This could result from inhibition of TRAP function by uncharged tRNA Trp molecules or by increased synthesis of some other transcript capable of binding and sequestering the TRAP regulatory protein.Expression of seven genes is required for biosynthesis of the amino acid L-tryptophan in Bacillus subtilis (13). Six of these genes are arranged in a cotranscribed gene cluster designated the trpEDCFBA operon, which is located in the aromatic supraoperon. The seventh trp gene, trpG, is within the unlinked folate operon. trpG encodes an amphibolic enzyme required for both p-aminobenzoate and o-aminobenzoate (anthranilate) synthesis (27). Expression of all seven tryptophan biosynthetic genes is regulated in response to the intracellular level of L-tryptophan by the product of the mtrB gene, trp RNA-binding attenuation protein (TRAP) (4,10,21). TRAP regulates transcription of the trp operon by a novel form of transcription attenuation; it regulates TrpG polypeptide synthesis translationally (16,21,33).The leader segment of the trp operon transcript is capable of forming alternative secondary structures (26). One of these structures is a rho-independent transcription terminator which, when formed, dire...

show abstract

Section: Methodsmentioning

confidence: 99%

A temperature-sensitive trpS mutation interferes with trp RNA-binding attenuation protein (TRAP) regulation of trp gene expression in Bacillus subtilis

1996

View full text Add to dashboard Cite

show abstract

“…The ␣ activity of tryptophan synthase was measured using an NADH-coupled assay system (11), and the formation of L-tryptophan * This work was supported by Evangelisches Studienwerk Haus Villigst, Germany. The costs of publication of this article were defrayed in part by the payment of page charges.…”

Section: Methodsmentioning

confidence: 99%

“…E. coli strains W3110 trp C 9830 (F Ϫ ) and W3110 trp D 9923 (D Ϫ ) were a generous gift from Dr. Charles Yanofsky. Enzyme Measurements-PRT activity at substrate concentrations below 75 M was determined fluorimetrically (12), whereas at higher substrate concentrations, a spectrophotometric assay at 278 nm was performed (11). Assays were performed both in the presence and in the absence of 0.1 mg/ml E. coli crude cell extract complementing consecutive "helper" activities but lacking PRT (W3110 trp D 9923 (D Ϫ )).…”

Section: Methodsmentioning

confidence: 99%

“…Assays were performed both in the presence and in the absence of 0.1 mg/ml E. coli crude cell extract complementing consecutive "helper" activities but lacking PRT (W3110 trp D 9923 (D Ϫ )). PRAI activity was determined spectrophotometrically at 278 nm using a 0.1 mg/ml concentration of a PRAI-deficient E. coli helper cell extract (W3110 trp C 9830 (F Ϫ )) (11). To determine IGPS activity both a spectroscopic (13) and a fluorimetric assay system (14) were used.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Multifunctional Tryptophan-synthesizing Enzyme

Schwarz

Uthoff

Meyer

et al. 1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

After developing a suitable procedure to produce large amounts of Euglena gracilis as well as a reliable protocol to purify the multifunctional tryptophan-synthesizing enzyme derived from it (Schwarz, T., Bartholmes, P., and Kaufmann, M. (1995) Biotechnol. Appl. Biochem. 22, 179 -190), we here describe structural and catalytic properties of the multifunctional tryptophansynthesizing enzyme. The kinetic parameters k cat of all five activities and K m for the main substrates were determined. The relative molecular weight under denaturing conditions as judged by SDS-polyacrylamide gel electrophoresis is 136,000. Cross-linking as well as gel filtration experiments revealed that the enzyme exists as a homodimer. Neither intersubunit disulfide linkages nor glycosylations were detected. On the other hand, the polypeptide chains are blocked N-terminally. Complete tryptic digestion of the protomer, high pressure liquid chromatography separation of the resulting peptides, and N-terminal sequence analysis of homogenous peaks as judged by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry was performed. Depending on the sequenced peptides, alignments to all entries of the SwissProt data base resulted in both strong sequence homologies to known Trp sequences and no similarities at all. Proteolytic digestion under native conditions using endoproteinase Glu-C uncovered one major cleavage site yielding a semistable, Nterminally blocked fragment with a molecular weight of 119,000. In addition, an increase in ␤-elimination accompanied by a decrease in ␤-replacement activity of the ␤-reaction during proteolysis was observed. In most procaryotes, tryptophan synthase is organized as a tetrameric bienzyme complex consisting of a central ␤ 2 -dimer (TRPS␤) combined with two peripheral ␣-subunits (TRPS␣). The Escherichia coli and Salmonella typhimurium enzymes in particular have been intensively investigated in the past (1, 2). In fungi such as Saccharomyces cerevisiae and Neurospora crassa, tryptophan synthase is a dimer of identical bifunctional polypeptides (3,4,5). In contrast, the enzyme isolated from Euglena gracilis, here designated as multifunctional tryptophan-synthesizing enzyme (MTSE), is capable of catalyzing all reactions shown in Scheme I. Although this discovery was made 2 decades ago (6, 7), no thorough further characterization of this multifunctional enzyme has been published so far, probably due to the excessive expenditure to cultivate sufficient amounts of E. gracilis and the difficulties of purifying MTSE with outdated technology. Using state of the art equipment and materials, we established a reliable and simple procedure to obtain MTSE from E. gracilis (8). Here we present both a reinvestigation of earlier initial findings and new data with respect to structural and functional properties of the enzyme. EXPERIMENTAL PROCEDURESChemicals, Enzymes, and E. coli Strains-If not otherwise stated, all chemicals (reagent or ultrapure grade) were obtained from Sigma (Deisenhofen, Germany) or Merck...

show abstract

“…It is widely applied as a starting material for synthesis of biologically active compounds and medicaments, for production of synthetic dyes and for metal ion determination [1][2][3][4][5][6]. It is the precursor of L-tryptophan in plants, yeast and bacteria [7][8][9] and is also involved in the biosynthesis of phytohormones (auxins) and their precursors [10,11]. Moreover anthranilic acid stimulates milk secretion in female mammals (it is called vitamin L1).…”

Section: Introductionmentioning

confidence: 99%

Studies on equilibrium of anthranilic acid in aqueous solutions and in two-phase systems: Aromatic solvent–water

Zapała

Kalembkiewicz

Sitarz-Palczak

2009

Biophysical Chemistry

View full text Add to dashboard Cite

[46] Chorismate to tryptophan (Escherichia coli)—anthranilate synthetase, PR transferase, PRA isomerase, InGP synthetase, tryptophan synthetase

Cited by 115 publications

References 23 publications

A temperature-sensitive trpS mutation interferes with trp RNA-binding attenuation protein (TRAP) regulation of trp gene expression in Bacillus subtilis

A temperature-sensitive trpS mutation interferes with trp RNA-binding attenuation protein (TRAP) regulation of trp gene expression in Bacillus subtilis

Multifunctional Tryptophan-synthesizing Enzyme

Studies on equilibrium of anthranilic acid in aqueous solutions and in two-phase systems: Aromatic solvent–water

Contact Info

Product

Resources

About