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Folate synthesis in plants: purification, kinetic properties, and inhibition of aminodeoxychorismate synthase

Authors :
Andrew D. Hanson
Stéphane Ravanel
Gilles J. Basset
Brian P. Nichols
Fabrice Rébeillé
Tobias Sahr
Laboratoire de physiologie cellulaire végétale (LPCV)
Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)
Direction de Recherche Fondamentale (CEA) (DRF (CEA))
Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA))
Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Horticultural Sciences Department
University of Florida [Gainesville] (UF)
Laboratoire de Physiologie et Biotechnologie Végétale
Institut National de la Recherche Agronomique (INRA)
Department of Biological Sciences [Chicago]
University of Illinois [Chicago] (UIC)
University of Illinois System-University of Illinois System
Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)
University of Florida [Gainesville]
Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)
Source :
Biochemical Journal, Biochemical Journal, 2006, 396, pp.157-162. ⟨10.1042/BJ20051851⟩, Biochemical Journal, Portland Press, 2006, 396, pp.157-162. ⟨10.1042/BJ20051851⟩
Publication Year :
2006
Publisher :
HAL CCSD, 2006.

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 Km values for glutamine and chorismate of 600 and 1.5 μM 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 (Ki values of 10 and 1 μM for dihydrofolate and methotrexate respectively). These findings support the view that the monomeric ADC synthase is a potential target for antifolate drugs.

Details

Language :
English
ISSN :
02646021 and 14708728
Database :
OpenAIRE
Journal :
Biochemical Journal, Biochemical Journal, 2006, 396, pp.157-162. ⟨10.1042/BJ20051851⟩, Biochemical Journal, Portland Press, 2006, 396, pp.157-162. ⟨10.1042/BJ20051851⟩
Accession number :
edsair.doi.dedup.....b69dcf4a0c7d50d39144831507740c0b