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

Biotechnology Group, School of Bioscience and Bioengineering, Indian Institute of Technology Bombay, Mumbai – 400076, India

Correspondence
Narayan S. Punekar
nsp{at}iitb.ac.in

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 µM. 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.

Details of the gel-filtration results and inhibitor studies are available as supplementary material with the online version of this paper.

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