Publications

• Chemical genetic identification of glutamine phosphoribosylpyrophosphate amidotransferase as the target for a novel bleaching herbicide in Arabidopsis.

  Walsh T.A., Bauer T., Neal R., Merlo A.O., Schmitzer P.R., Hicks G.R., Honma M., Matsumura W., Wolff K., Davies J.P.

  A novel phenyltriazole acetic acid compound (DAS734) produced bleaching of new growth on a variety of dicotyledonous weeds and was a potent inhibitor of Arabidopsis (Arabidopsis thaliana) seedling growth. The phytotoxic effects of DAS734 on Arabidopsis were completely alleviated by addition of ade ... >> More

  A novel phenyltriazole acetic acid compound (DAS734) produced bleaching of new growth on a variety of dicotyledonous weeds and was a potent inhibitor of Arabidopsis (Arabidopsis thaliana) seedling growth. The phytotoxic effects of DAS734 on Arabidopsis were completely alleviated by addition of adenine to the growth media. A screen of ethylmethanesulfonate-mutagenized Arabidopsis seedlings recovered seven lines with resistance levels to DAS734 ranging from 5-to 125-fold. Genetic tests determined that all the resistance mutations were dominant and allelic. One mutation was mapped to an interval on chromosome 4 containing At4g34740, which encodes an isoform of glutamine phosphoribosylamidotransferase (AtGPRAT2), the first enzyme of the purine biosynthetic pathway. Sequencing of At4g34740 from the resistant lines showed that all seven contained mutations producing changes in the encoded polypeptide sequence. Two lines with the highest level of resistance (125-fold) contained the mutation R264K. The wild-type and mutant AtGPRAT2 enzymes were cloned and functionally overexpressed in Escherichia coli. Assays of the recombinant enzyme showed that DAS734 was a potent, slow-binding inhibitor of the wild-type enzyme (I(50) approximately 0.2 microm), whereas the mutant enzyme R264K was not significantly inhibited by 200 microm DAS734. Another GPRAT isoform in Arabidopsis, AtGPRAT3, was also inhibited by DAS734. This combination of chemical, genetic, and biochemical evidence indicates that the phytotoxicity of DAS734 arises from direct inhibition of GPRAT and establishes its utility as a new and specific chemical genetic probe of plant purine biosynthesis. The effects of this novel GPRAT inhibitor are compared to the phenotypes of known AtGPRAT genetic mutants. << Less

  Plant Physiol. 144:1292-1304(2007) [PubMed] [EuropePMC]

• Purification and properties of glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis.

  Wong J.Y., Bernlohr D.A., Turnbough C.L., Switzer R.L.

  A procedure for the rapid and efficient purification of glutamine phosphoribosylpyrophosphate amidotransferase to better than 98% homogeneity from depressed Bacillus subtilis cells is described. The molecular weight of the subunit was estimated to be about 50 000. The purified enzyme exhibits micr ... >> More

  A procedure for the rapid and efficient purification of glutamine phosphoribosylpyrophosphate amidotransferase to better than 98% homogeneity from depressed Bacillus subtilis cells is described. The molecular weight of the subunit was estimated to be about 50 000. The purified enzyme exhibits microheterogeneity on electrophoresis on highly resolving polyacrylamide gels; it is suggested that this heterogeneity results from limited proteolytic modification of the native subunit. The native enzyme exists in equilibrium among tetrameric, dimeric, and monomeric forms. The influence of enzyme concentration and the presence of substrates and allosteric inhibitors on this equilibrium are described. There is no simple correlation between allosteric inhibition and stabilization of dimeric or tetrameric states. The amino acid composition of the amidotransferase is reported; presence of a 4Fe-4S center in the enzyme was described previously. Preparation of inactive apoprotein by treatment with 1,10-phenanthroline and general characteristics of the apoprotein are presented. << Less

  Biochemistry 20:5669-5674(1981) [PubMed] [EuropePMC]

• Glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli. Purification and properties.

  Messenger L.J., Zalkin H.

  Glutamine 5-phosphoribosylamine:pyrophosphate phosphoribosyltransferase (amidophosphoribosyl-transferase) has been purified to homogeneity from Escherichia coli. The molecular weight of the native enzyme was 194,000 by sedimentation equilibrium centrifugation and 224,000 by gel filtration. A subun ... >> More

  Glutamine 5-phosphoribosylamine:pyrophosphate phosphoribosyltransferase (amidophosphoribosyl-transferase) has been purified to homogeneity from Escherichia coli. The molecular weight of the native enzyme was 194,000 by sedimentation equilibrium centrifugation and 224,000 by gel filtration. A subunit Mr = 57,000 was estimated by gel electrophoresis in sodium dodecyl sulfate. Cross-linking experiments gave species of Mr = 57,000, 117,000, and 177,000. A trimer or tetramer of identical subunits is indicated for the native enzyme. Highly active E. coli amidophosphoribosyl-transferase lacks significant nonheme iron. Enzyme activity was not enhanced by addition of iron salts and sulfide. Amidophosphoribosyltransferase exhibited both NH3- and glutamine-dependent activities. Glutaminase activity was detected in the absence of other substrates. Both glutamine- and NH3-dependent activities were subject to end product inhibition by purine 5'-ribonucleotides. AMP and GMP, in combination, gave synergistic inhibition. AMP and GMP exhibited positive cooperativity. In addition, GMP promoted cooperativity for saturation by 5-phosphoribosyl-1-pyrophosphate. Glutamine utilization was inhibited by NH3, suggesting that the amide of glutamine is transferred to the NH3 site prior to amination of 5-phosphoribosyl-1-pyrophosphate. The glutamine-dependent activity was selectively inactivated by the glutamine analogs L-2-amino-4-oxo-5-chloropentanoic acid and 6-diazo-5-oxo L-norleucine (DON) and by iodoacetamide. Incorporation of 1 eq of DON/subunit (Mr = 57,000) caused complete inactivation of the glutamine-dependent activity, thus providing evidence for one glutamine site per monomer and for the functional identity of the subunits. Following alkylation with iodoacetamide, carboxymethylcysteine was the only modified amino acid isolated from an acid hydrolysate. The glutamine-dependent activity was sensitive to oxidation. Inactivation by exposure to air was reversed by incubation with high concentrations of dithiothreitol. << Less

  J. Biol. Chem. 254:3382-3392(1979) [PubMed] [EuropePMC]