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

Abstract

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 nigerandAspergillus nidulans. TheA. nigerNADP-GDH was characterized at length and its kinetic interaction constants with glutamate (Km34·7 mM) and ammonium (Km1·05 mM;Ki0·4 mM) were consistent with an anabolic role. Isophthalate, 2-methyleneglutarate and 2,4-pyridinedicarboxylate were significant inhibitors, with respectiveKivalues of 6·9, 9·2 and 202·0 μM. TheA. nigerenzyme showed allosteric properties and a sigmoid response (nH=2·5) towards 2-oxoglutarate saturation. The co-operative behaviour was a feature common to NADP-GDH fromAspergillus awamori,A. nidulansandAspergillus oryzae. NADP-GDH may therefore be a crucial determinant in adjusting 2-oxoglutarate flux between the tricarboxylic acid cycle and glutamate biosynthesis in aspergilli.