The synthesis of pABA: Coupling between the glutamine amidotransferase and aminodeoxychorismate synthase domains of the bifunctional aminodeoxychorismate synthase from Arabidopsis thaliana.
Aminodeoxychorismate (ADC) synthase in plants is a bifunctional enzyme containing glutamine amidotransferase (GAT) and ADC synthase (ADCS) domains. The GAT domain releases NH(3) from glutamine and the ADCS domain uses NH(3) to aminate chorismate. This enzyme is involved in folate (vitamin B9) biosynthesis. We produced a stable recombinant GAT-ADCS from Arabidopsis. Its kinetic properties were characterized, and activities and coupling of the two domains assessed. Both domains could operate independently, but not at their optimal capacities. When coupled, the activity of one domain modified the catalytic properties of the other. The GAT activity increased in the presence of chorismate, an activation process that probably involved conformational changes. The ADCS catalytic efficiency was 10(4) fold higher with glutamine than with NH(4)Cl, indicating that NH(3) released from glutamine and used for ADC synthesis did not equilibrate with the external medium. We observed that the GAT activity was always higher than that of ADCS, the excess of NH(3) being released in the external medium. In addition, we observed that ADC accumulation retro-inhibited ADCS activity. Altogether, these results indicate that channeling of NH(3) between the two domains and/or amination of chorismate are the limiting step of the whole process, and that ADC cannot accumulate.
Camara
Djeneb
D
Laboratoire de Physiologie Cellulaire Végétale, UMR5168 CNRS-CEA-INRA-Université Joseph Fourier Grenoble I, Institut de Recherches en Technologies et Sciences pour le Vivant, CEA-Grenoble, 17 rue des Martyrs, F-38054 Grenoble Cedex 9, France.
Richefeu-Contesto
Céline
C
Gambonnet
Bernadette
B
Dumas
Renaud
R
Rébeillé
Fabrice
F
D010129:4-Aminobenzoic Acid / Q000378:metabolism*; D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000378:metabolism*; D005492:Folic Acid / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017421:Sequence Analysis; D000637:Transaminases
D011994:Recombinant Proteins; D005492:Folic Acid; D000637:Transaminases; C066599:aminodeoxychorismate synthase; D019731:Carbon-Nitrogen Ligases; D010129:4-Aminobenzoic Acid