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 (Km 34.7 mM) and ammonium (Km 1.05 mM; Ki 0.4 mM) were consistent with an anabolic role. Isophthalate, 2-methyleneglutarate and 2,4-pyridinedicarboxylate were significant inhibitors, with respective Ki values of 6.9, 9.2 and 202.0 microM. The A. niger enzyme 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 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.
Noor
Shahid
S
Biotechnology Group, School of Bioscience and Bioengineering, Indian Institute of Technology Bombay, Mumbai - 400076, India.
Punekar
Narayan S
NS
Biotechnology Group, School of Bioscience and Bioengineering, Indian Institute of Technology Bombay, Mumbai - 400076, India.
D000494:Allosteric Regulation; D001233:Aspergillus nidulans / Q000201:enzymology* / Q000235:genetics; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D002244:Carbon / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D018086:Glutamate Dehydrogenase (NADP+) / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D009584:Nitrogen / Q000378:metabolism
D005971:Glutamates; D007656:Ketoglutaric Acids; D002244:Carbon; D018086:Glutamate Dehydrogenase (NADP+); D009584:Nitrogen