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

• Published in: Microbiology (Society for General Microbiology) 2005-05, Vol.151 (5), p.1409-1419
• Main Authors: Noor, Shahid, Punekar, Narayan S
• Format: Article
• Language: English
• Subjects: Allosteric Regulation; Aspergillus awamori; Aspergillus nidulans; Aspergillus nidulans - enzymology; Aspergillus nidulans - genetics; Aspergillus niger; Aspergillus niger - enzymology; Aspergillus niger - genetics; Aspergillus oryzae; Biological and medical sciences; Carbon - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Fungal; Glutamate Dehydrogenase (NADP+) - chemistry; Glutamate Dehydrogenase (NADP+) - isolation & purification; Glutamate Dehydrogenase (NADP+) - metabolism; Glutamates - metabolism; Growth, nutrition, metabolism, transports, enzymes. Molecular biology; Ketoglutaric Acids - metabolism; Kinetics; Microbiology; Mycology; Nitrogen - metabolism
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.27751-0

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. Abbreviations: GDH, glutamate dehydrogenase; TCA, tricarboxylic acid Details of the gel-filtration results and inhibitor studies are available as supplementary material with the online version of this paper.