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Regulation of Prephenate Dehydratase in Brevibacterium Flavum Downloaded from by Guest on 29 September 2021 /. Biochem., 76, 1103-1111 (1974) Regulation of Prephenate Dehydratase in Brevibacterium flavum Downloaded from https://academic.oup.com/jb/article/76/5/1103/2185327 by guest on 29 September 2021 Shinichi SUGIMOTO and Isamu SHIIO Central Research Laboratories of Ajinomoto Co., Inc., Kawasaki, Kanagawa 244 Received for publication, March 28, 1974 The regulation and some other properties of prephenate dehydratase [prephenate hydro-lyase (decarboxylating), EC 4.2.1.51] of Brevibacterium flavum were studied. Prephenate dehydratase was purified about 38-fold from crude extracts of a tyrosine auxotroph, BX-1. The optimum pH of the reaction was 6.5 in potassium phosphate • buffer. The relationship between the enzyme concentration and activity was linear in the presence of tyrosine but not in its absence. Km for prephenate was 54 pM.. The enzyme was completely inhibited by phenylalanine or by tryptophan, while tyrosine not only reversed the inhibition competitively but also stimulated the enzyme activity about 10-fold. The concentration of tyrosine giving half-maximum activation was 1.6 ^M. Tyrosine affected both the Km value and the maximum reaction velocity. Similarly, phenylalanine and tryptophan were mixed-type inhibitors. The concentration of tryptophan giving 50% inhibition was 25 //M, 10 times that of phenylalanine, 2.5 fiM. None of the tyrosine and phenylalanine analogues tested strongly activated the enzyme, but several analogues inhibited it, as did phenylalanine. The molecular weight which was estimated to be 2.2 xlO5 by gel filtration experi- ments ; this value was not affected by tyrosine or by phenylalanine. On the basis of these results, the regulatory mechanism of aromatic amino acid biosynthesis in B. flavum was discussed. Since some mutants of Brevibacterium flavum by tryptophan, while 3-deoxy-D-arafo>z0-hep- accumulate large amounts of tryptophan (/), tulosonate 7-phosphate synthetase (DAHP syn- phenylalanine, and tyrosine (2), it is of in- thetase*), the first enzyme in the common terest to study the regulatory mechanism of pathway, is synergistically inhibited by phenyl- aromatic amino acid biosynthesis in these or- alanine plus tyrosine and is repressed by tyro- ganisms. It has previously been reported that sine in B. flavum {3, 4). anthranilate synthetase [EC 4.1. 3.27], the first Prephenate dehydratase [prephenate hydro- enzyme in the tryptophan-specific biosynthetic lyase (decarboxylating), EC 4.2.1.51], the first pathway, is strongly inhibited and repressed enzyme in the phenylalanine-specific pathway, has been reported to be regulated by strong * The following abbreviation is used: 3-deoxy-D- end-product (phenylalanine) inhibition in vari- araWwo-heptulosonate 7-phosphate: DAHP. ous bacteria (5—7). However, mutants derived Vol. 76, No. 5, 1974 1103 1104 S. SUGIMOTO and I. SHIIO from B. flavum No. 2247, in which DAHP also contained 500 mg/liter tyrosine for the synthetase had been genetically desensitized to tyrosine auxotroph, BX-1. feedback inhibition by phenylalanine plus tyro- Chemicals—Barium prephenate was pre- sine, over-produced large amounts of phenyl- pared from chorismic acid, as described pre- alanine and tyrosine (2). This result cannot viously (2). Amino acids used were all In- be explained simply by the feedback inhibition form unless otherwise cited and were purchased of prephenate dehydratase by phenylalanine. from Sigma, Calbiochem or Nutritional Bio- A metabolic interlock on prephenate de- chemicals Corp. D-Phenylalanine and D-tyro- Downloaded from https://academic.oup.com/jb/article/76/5/1103/2185327 by guest on 29 September 2021 hydratase has been observed in some bacteria. sine were the products of Nutritional Biochem- For example, prephenate dehydratase of Bacil- icals Corp. and o-hydroxy-DL-phenylalanine, lus subtilis is activated by leucine and methio- m - hydroxy - DL - phenylalanine, p - amino - DL - nine, but inhibited by tryptophan. Further- phenylalanine, w-fluoro-DL-phenylalanine, p- more tyrosine reverses the inhibition by trypto- fluoro - DL - phenylalanine, p - hydroxyphenyl - phan but not that by phenylalanine (8, 9). pyruvic acid, />-aminobenzoic acid, and p- On the other hand, prephenate dehydratase of hydroxybenzoic acid were from Sigma. Mark- Pseudomonas sp. is activated by tyrosine (7). ers used in the gel filtration experiments were These findings also fail to explain the accumu- glutamate dehydrogenase [EC 1.4.1.3] from lation of both tyrosine and phenylalanine as bovine liver, molecular weight 300,000 (18) described above, even if such interactions exist (Nutritional Biochemicals Corp.), catalase [EC in B. flavum. 1.11.1.6] from bovine liver, molecular weight Preliminary experiments with crude ex- 244,000 (79) (Sigma), alcohol dehydrogenase tract of B. flavum showed that prephenate de- [EC 1.1.1.1] from yeast, molecular weight hydrogenase [EC 1.3.1.12], the first enzyme 150,000 (20) (Boehringer), hexokinase [EC in the tyrosine-specific pathway, is not affected 2.7.1.1] from yeast, molecular weight 96,600 by the end-product, tyrosine, while prephenate (21) (Boehringer), and cytochrome cfrom horse dehydratase is not only inhibited by phenyl- heart, molecular weight 12,400 (22) (Tokyo alanine, but also activated by tyrosine (2). Kasei). Furthermore, the phenylalanine inhibition is Enzyme Assay — Prephenate dehydratase reversed by tyrosine. Since these effects of activity was measured by the spectrophoto- tyrosine on prephenate dehydratase could well metric determination of phenylpyruvate formed explain the phenomenon of tyrosine and phenyl- in the reaction according to Cotton and Gibson alanine accumulation by mutants, prephenate (5). The standard assay system contained 20 dehydratase was further purified from B. flavum //moles of Tris-HCl buffer, pH 7.5, 0.2 //mole and its regulatory and some other properties of barium prephenate, 0.2 //mole of EDTA and were studied, as reported in this paper. enzyme preparation in a total volume of 0.4 ml. The reaction was carried out at 30° for MATERIALS AND METHODS 20 min and stopped by the addition of 0.8 ml of 1N NaOH. The absorbance of phenyl- Organisms and Cultivation—Brevibacterium pyruvate formed in the reaction was measured flavum No. 2247 (ATCC 14067) and its tyrosine at 320 nm. One unit of the enzyme activity auxotroph, BX-1 (1) were aerobically cultured was defined as the amount catalyzing the for- for 24 hr at 30° in a glucose medium (36 g of mation of 1 nmole of phenylpyruvate per min, glucose, 10 g of urea, 1 g of KH2PO4, 0.4 g of taking the molar extinction coefficient as 17,500 MgSO«-7H2O, 10 mg of FeSO4-7H2O, 2 ppm at 320 nm. 2+ Mn , 100 /*g of thiamine-HCl, 1 ml of Mieki,* Protein was determined by the method of 30 fig of d-biotin, 7 ml of 6 N HC1, and dis- Lowry et al. (10). tilled water to a total volume of 1 liter) which Preparation of the Enzyme—After cultiva- tion of the tyrosine auxotroph, BX-1 in the * Mieki is a hydrolysate of soybean meal, a product glucose medium, the cells were harvested, of Ajinomoto Co., Inc. washed twice with 0.2% KC1, suspended in /. Biochem. REGULATION OF PREPHENATE DEHYDRATASE 1105 0.1 M Tris-HCl buffer, pH 7.5 and sonically sulfate. The precipitate was dissolved in a disrupted at 10 kc for 20 min. Crude extract small volume of the same buffer (Fraction V) (Fraction I) obtained by centrifugation of the and used throughout this study unless other- sonicate at 32,000 xg for 30 min at 0° was wise stated. treated with solid ammonium sulfate. The precipitate between 0.25 and 0.60 saturation of RESULTS ammonium sulfate was collected by centrifuga- tion and was dissolved in 0.1 M Tris-HCl buffer, Partial Purification and General Properties pH 7.5 (Fraction II). Fraction II was then of Prephenate Dehydratase from the Tyrosine Downloaded from https://academic.oup.com/jb/article/76/5/1103/2185327 by guest on 29 September 2021 treated with saturated ammonium sulfate so- Auxotroph, BX-1—Since, the substrate of pre- lution and the precipitate between 0.38 and phenate dehydratase, i.e., prephenate, is com- 0.50 saturation of ammonium sulfate was dis- mon to the prephenate dehydrogenase step in solved in a small volume of 0.05 M Tris-HCl the tyrosine-specific pathway, the prephenate buffer, pH 7.5 (Fraction III). Fraction III was dehydratase reaction would be affected by pre- dialyzed against about 200 volumes of the same phenate dehydrogenase, if it was present in buffer at 5° for 16 hr and was then applied to the enzyme preparation. Therefore, prephenate a DEAE-cellulose column (2.5x23 cm) equili- dehydratase was purified from the crude ex- brated with the same buffer. The column tract of the tyrosine auxotroph, BX-1, which was washed with one volume of the same genetically lacked prephenate dehydrogenase. buffer and then linear gradient elution was The results on the partial purification are shown started with the same buffer containing 0.5 M in Table I. Prephenate dehydratase was puri- KC1 at a flow rate of 25 ml/hr. Eight-ml frac- fied about 38-fold from crude extract as de- tions were collected. The fractions containing scribed in "METHODS." The total activity of prephenate dehydratase were pooled and treated Fraction II was higher than that of Fraction with ammonium sulfate to give 0.70 saturation. I, probably due to removal of the inhibitor, The precipitate was dissolved in a small vol- phenylalanine, as mentioned later. Activities ume of the same buffer (Fraction IV). Frac- of DAHP synthetase, chorismate mutase [EC tion IV was passed through a Sephadex G-200 5.4. 99.5], and prephenate dehydrogenase were column (2.5x45 cm) equilibrated with the same not observed at all in the Fraction V prepa- buffer at a flow rate of 10 ml/hr. Two-ml ration, which was used as the enzyme prepa- fractions were collected.
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