Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon-nitrogen interface

NADP-dependent glutamate dehydrogenase (NADP-GDH) mediates fungal ammonium assimilation through reductive synthesis of glutamate from 2-oxoglutarate. By virtue of its position at the interface of carbon and nitrogen metabolism, biosynthetic NADP-GDH is a potential candidate for metabolic control. In order to facilitate characterization, a new and effective dye-affinity method was devised to purify NADP-GDH from two aspergilli, Aspergillus niger and Aspergillus nidulans. The A. niger NADP-GDH was characterized at length and its kinetic interaction constants with glutamate (K-m 34(.)7 mM) and ammonium (K-m 1(.)05 mM; K-i 0(.)4 mM) were consistent with an anabolic role. Isophthalate, 2-methyleneglutarate and 2,4-pyridinedicarboxylate were significant inhibitors, with respective K-i values of 6(.)9, 9(.)2 and 202(.)0 mu M. The A. niger enzyme showed allosteric properties and a sigmoid response (n(H) = 2(.)5) towards 2-oxoglutarate saturation. The co-operative behaviour was a feature common to NADP-GDH from Aspergillus awamori, A. nidulans and Aspergillus oryzae. NADP-GDH may therefore be a crucial determinant in adjusting 2-oxoglutarate flux between the tricarboxylic acid cycle and glutamate biosynthesis in aspergilli.