Purification and Properties of 3-Deoxy-
 <scp>d</scp>
 -Arabinoheptulosonic Acid-7-Phosphate Synthetase (phe) from a λ
 aroG
 +
 Transductant of
 Escherichia coli

Simpson, Richard J.; Davidson, Barrie E.; Dopheide, Theo A.A.; Andrews, S.; Pittard, J

doi:10.1128/jb.107.3.798-805.1971

Cited by 18 publications

(7 citation statements)

References 16 publications

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“…The three DAHP synthase isoenzymes were separated on DEAE-cellulose with a phosphate gradient, but it was necessary to stabilize the phenylalanine-inhibitable DAHP synthase with 1 mM enolpyruvate-P in the eluting gradient. Stabilization of DAHP synthase isoenzymes by enolpyruvate-P during chromatography and against heat denaturation has been reported previously (25,27) and is in accord with evidence for an ordered sequential mechanism in DAHP synthase (tyr) with obligatory binding of enolpyruvate-P to the enzyme prior to binding of the second substrate, erythrose-4-P (8).…”

Section: Resultssupporting

confidence: 79%

3-Deoxy-D-arabino-heptulosonic acid 7-phosphate synthase mutants of Salmonella typhimurium

DeLeo¹,

Sprinson²

1975

J Bacteriol

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The first committed step of aromatic amino acid biosynthesis in Salmonella typhimurium was shown to be catalyzed by three isoenzymes of 3-deoxy-Darabino-heptulosonic acid 7-phosphate (DAHP) synthase. Mutations in each of the genes specifying the isoenzymes were isolated and mapped. aroG, the structural gene for the phenylalanine-inhibitable isoenzyme, was linked to gal, and aroH, the structural gene for the tryptophan-inhibitable isoenzyme, was linked to aroE. aroF, the structural gene for the tyrosine-inhibitable isoenzyme, was linked to pheA and tyrA, which specify the phenylalanineand tyrosinespecific branch-point enzymes, respectively. The phenylalanine-inhibitable isoenzyme was the predominant DAHP synthase in wild-type cells, and only the tyrosine-inhibitable isoenzyme was completely repressed, as well as inhibited, by low levels of its allosteric effector. The DAHP synthase isoenzymes were separated by chromatography on diethylaminoethyl-cellulose with a phosphate gradient which contained enolpyruvate phosphate to protect the otherwise unstable phenylalanine-inhibitable isoenzyme. No cross-inhibition of either the tyrosineor phenylalanine-inhibitable isoenzyme was observed at inhibitor concentrations up to 1 mM. The tryptophan-inhibitable isoenzyme was partially purified from extracts of a strain lacking the other two isoenzymes and shown to be inhibited about 30% by 1 mM tryptophan. A preliminary study of interference by tryptophan in the periodate-thiobarbiturate assay for DAHP suggested a combined effect of tryptophan and erythrose 4-phosphate, or an aldehydic compound resulting from degradation of erythrose 4-phosphate by periodate.

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Section: Resultssupporting

confidence: 79%

3-Deoxy-D-arabino-heptulosonic acid 7-phosphate synthase mutants of Salmonella typhimurium

DeLeo¹,

Sprinson²

1975

J Bacteriol

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“…In the operator-constitutive mutant SG12 grown at 250C (13), activity of DAHP synthase is twice as high as found at 37°C (10), and activity of DAHP synthase (tyr) is 85% of the total ( Table 1). The levels obtained were about 35-fold higher than in wild-type cells grown in minimal medium and approximated those of DAHP synthase (phe) found by Simpson et al (23) structural gene for the phenylalanine isoenzyme into the genome of specialized transducing phage lambda. We used the procedure described previously (10) as a starting point for purifying DAHP synthase (tyr) to homogeneity.…”

Section: Discussionsupporting

confidence: 64%

Properties of tyrosine-inhibitable 3-deoxy-d-arabinoheptulosonic acid-7-phosphate synthase from Salmonella

Hu¹,

Sprinson²

1977

J Bacteriol

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Tyrosine-inhibitable 3-deoxy-D-arabinoheptulosonic acid-7-phosphate (DAHP) synthase was purified to homogeneity without significant loss of sensitivity to inhibition by tyrosine from an operator-constitutive strain (tyrOc) of Salmonella. The enzyme had an apparent molecular weight of 76,000 by gel filtration and a subunit molecular weight of 40,000 by sodium dodecyl sulfate-gel electrophoresis and by reaction with dimethyl suberimidate. It had an isoelectric point of 4.68. Inhibition by L-tyrosine showed a Hill coefficient of 1.8 at pH 7.0, suggesting cooperative interaction between tyrosine-binding sites, and was competitive with phosphoenol pyruvate and noncompetitive with erythrose-4phosphate. Tyrosine-inhibitable 3-deoxy-n-arabinoheptulosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15) has been extensively purified from an operator-constitutive (tyrO) strain of Salmonella typhimurium (13). Since gel electrophoresis of this preparation indicated minor impurities (10), we undertook a further purification of the enzyme and developed a procedure for preparing it in homogeneous form. We also studied its allosteric properties and subunit structure. (This report is in partial fulfillment of the requirements for the Ph.D. degree, submitted by C. Y. H., 1974.) MATERIALS AND METHODS Materials. Barium DAHP was prepared according to Sprinson et al. (24) and was converted to the potassium salt. Monocyclohexylammonium phosphoenolpyruvate (PEP) was prepared according to the method of Clark and Kirby (5) and used directly. Monocyclohexylammonium phosphoenol a-ketobutyrate was prepared according to Bondinell and Sprinson (3). Barium chorismate was prepared according to Gibson (12). Erythrose-4-phosphate (E4P) was prepared according to the method of Ballou and MacDonald (2). Hemoglobin was a gift from R. Benesch. Barium prephenate was isolated from accumulation media of strain tyrAl9 according to the method of Dayan and Sprinson (9). Dimethyl suberimidate was prepared according to the method of Davies and Stark (7). The following compounds were gifts: monocyclohexylammonium (Z)-phosphoenol-3-fluoropyruvate and a-(dihydroxyphosphinyl methyl)-acrylic acid from G. L. Kenyon (25); and fosfomycin from D. Hendlin (4, 15). All other materials used were obtained commercially and used without further purification.

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“…DAHPS was previously shown to follow an ordered sequential kinetic mechanism with respect to PEP and E4P, with PEP binding first. , In those studies, the experimental initial velocities were well fitted by kinetic mechanisms that implicitly assumed that all the active sites were equivalent. However, the Hill coefficients of ∼2 for E4P binding with some metal cofactors and for allosteric inhibition by phenylalanine − suggest that the subunits are nonequivalent under some conditions. Similarly, in the current study, the substrate dependence of activity was well modeled assuming equivalent active sites (Figures and ), though it was necessary to include two kinetically distinct active sites when DAHP oxime was present (Figures –), supported by the observation of two structurally distinct subunits in the crystal structure (Figure ).…”

Section: Resultsmentioning

confidence: 99%

“…Similarly, allosteric inhibition by phenylalanine has a Hill coefficient of ∼2, though phenylalanine does not bind in the active site. 6,[33][34][35]48 Stoichiometry of Inhibitor Binding. It was not possible to definitively establish the stoichiometry of inhibitor binding under all conditions.…”

mentioning

confidence: 99%

Potent Inhibition of 3-Deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) Synthase by DAHP Oxime, a Phosphate Group Mimic

Balachandran¹,

Heimhalt²,

Liuni

et al. 2016

Biochemistry

147

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3-Deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthase catalyzes the first step in the shikimate pathway. It catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to form DAHP. The kinetic mechanism was rapid equilibrium sequential ordered ter ter, with the essential divalent metal ion, Mn, binding first, followed by PEP and E4P. DAHP oxime, in which an oxime group replaces the keto oxygen, was a potent inhibitor, with K = 1.5 ± 0.4 μM, though with residual activity at high inhibitor concentrations. It displayed slow-binding inhibition with a residence time, t, of 83 min. The crystal structure revealed that the oxime functional group, combined with two crystallographic waters, bound at the same location in the catalytic center as the phosphate group of the tetrahedral intermediate. DAHP synthase has a dimer-of-dimers homotetrameric structure, and DAHP oxime bound to only one subunit of each tight dimer. Inhibitor binding was competitive with respect to all three substrates in the subunits to which it bound. DAHP oxime did not overlap with the metal binding site, so the cause of their mutually exclusive binding was not clear. Similarly, there was no obvious structural reason for inhibitor binding in only two subunits; however, changes in global hydrogen/deuterium exchange showed large scale changes in protein dynamics upon inhibitor binding. The k value for the residual activity at high inhibitor concentrations was 3-fold lower, and the apparent K value decreased at least 10-fold. This positive cooperativity of binding between DAHP oxime in subunits B and C, and E4P in subunits A and D appears to be the dominant cause for incomplete inhibition at high inhibitor concentrations. In spite of its lack of obvious structural similarity to phosphate, the oxime and crystallographic waters acted as a small, neutral phosphate mimic.

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Purification and Properties of 3-Deoxy- d -Arabinoheptulosonic Acid-7-Phosphate Synthetase (phe) from a λ aroG ⁺ Transductant of Escherichia coli

Cited by 18 publications

References 16 publications

3-Deoxy-D-arabino-heptulosonic acid 7-phosphate synthase mutants of Salmonella typhimurium

3-Deoxy-D-arabino-heptulosonic acid 7-phosphate synthase mutants of Salmonella typhimurium

Properties of tyrosine-inhibitable 3-deoxy-d-arabinoheptulosonic acid-7-phosphate synthase from Salmonella

Potent Inhibition of 3-Deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) Synthase by DAHP Oxime, a Phosphate Group Mimic

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Purification and Properties of 3-Deoxy- d -Arabinoheptulosonic Acid-7-Phosphate Synthetase (phe) from a λ aroG + Transductant of Escherichia coli

Cited by 18 publications

References 16 publications

3-Deoxy-D-arabino-heptulosonic acid 7-phosphate synthase mutants of Salmonella typhimurium

3-Deoxy-D-arabino-heptulosonic acid 7-phosphate synthase mutants of Salmonella typhimurium

Properties of tyrosine-inhibitable 3-deoxy-d-arabinoheptulosonic acid-7-phosphate synthase from Salmonella

Potent Inhibition of 3-Deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) Synthase by DAHP Oxime, a Phosphate Group Mimic

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Purification and Properties of 3-Deoxy- d -Arabinoheptulosonic Acid-7-Phosphate Synthetase (phe) from a λ aroG ⁺ Transductant of Escherichia coli