title: Folate synthesis in plants: purification, kinetic properties, and inhibition of aminodeoxychorismate synthase.
abstract: pABA (p-aminobenzoate) is a precursor of folates and, besides esterification to glucose, has no other known metabolic fate in plants. It is synthesized in two steps from chorismate and glutamine, the first step being their conversion into glutamate and ADC (4-aminodeoxychorismate). In Escherichia coli, two proteins forming a heterodimeric complex are required for this reaction, but, in plants and lower eukaryotes, a single protein is involved. The Arabidopsis enzyme was expressed in E. coli and was purified to homogeneity. The monomeric enzyme (95 kDa) catalyses two reactions: release of NH3 from glutamine (glutaminase activity) and substitution of NH3 for the hydroxy group at position 4 of chorismate (ADC synthase activity). The kinetic parameters of the plant enzyme are broadly similar to those of the bacterial complex, with K(m) values for glutamine and chorismate of 600 and 1.5 microM respectively. As with the bacterial enzyme, externally added NH3 was a very poor substrate for the plant enzyme, suggesting that NH3 released from glutamine is preferentially channelled to chorismate. The glutaminase activity could operate alone, but the presence of chorismate increased the efficiency of the reaction 10-fold, showing the interdependency of the two domains. The plant enzyme was inhibited by dihydrofolate and its analogue methotrexate, a feature never reported for the prokaryotic system. These molecules were inhibitors of the glutaminase reaction, competitive with respect to glutamine (K(i) values of 10 and 1 microM for dihydrofolate and methotrexate respectively). These findings support the view that the monomeric ADC synthase is a potential target for antifolate drugs.
authors.affiliation: Laboratoire de Physiologie Cellulaire Végétale, UMR5168 CNRS-CEA-INRA-Université Joseph Fourier Grenoble I, Département Réponse et Dynamique Cellulaires, CEA-Grenoble, 17 rue des Martyrs, F-38054 Grenoble Cedex 9, France.
authors.forename: Tobias
authors.initials: T
authors.lastname: Sahr
authors.forename: Stéphane
authors.initials: S
authors.lastname: Ravanel
authors.forename: Gilles
authors.initials: G
authors.lastname: Basset
authors.forename: Brian P
authors.initials: BP
authors.lastname: Nichols
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Hanson
authors.forename: Fabrice
authors.initials: F
authors.lastname: Rébeillé
mesh_terms: D010129:4-Aminobenzoic Acid / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002827:Chorismic Acid / Q000378:metabolism; D004926:Escherichia coli; D005492:Folic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005973:Glutamine / Q000378:metabolism; D007700:Kinetics; D008727:Methotrexate / Q000494:pharmacology; D019289:Pyruvic Acid / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013379:Substrate Specificity; D000637:Transaminases
chemical_list: D029681:Arabidopsis Proteins; D011993:Recombinant Fusion Proteins; D005973:Glutamine; C010920:dihydrofolate; D019289:Pyruvic Acid; D005492:Folic Acid; D000637:Transaminases; C066599:aminodeoxychorismate synthase; D019731:Carbon-Nitrogen Ligases; D002827:Chorismic Acid; D010129:4-Aminobenzoic Acid; D008727:Methotrexate