Induction, isolation, and some properties of the NADPH-dependent glutamate dehydrogenase from the nonheterocystous cyanobacterium Phormidium laminosum

Abstract

The level of the NADPH-dependent glutamate dehydrogenase activity (EC 1.4.1.4) from nitrate-grown cells of the thermophilic non-N2-flxing cyanobacterium Phormidium laminosum OH-1-p.Cl1 could be significantly enhanced by the presence of ammonium or nitrite, as well as by L-methionine-DL-sulfoximine and other sources of organic nitrogen (L-Glu, L-Gln, and methylamine). The enzyme was purified more than 4,400-fold by ultracentrifugation, ion-exchange chromatography, and affinity chromatography, and at 30°C it showed a specific activity of 32.9 gimol of NADPH oxidized per min per mg of protein. The purified enzyme showed no aminotransferase activity and catalyzed the amination of 2-oxoglutarate preferentially to the reverse catabolic reaction. The enzyme was very specific for its substrates 2-oxoglutarate (K,m = 1.25 mM) and NADPH (Km = 64 ,uM), for which hyperbolic kinetics were obtained. However, negative cooperativity (Hill coefficient h = 0.89) and [SJ0.5 of 18.2 mM were observed for ammonium. The mechanism of the aminating reaction was of a random type with independent sites. The purified enzyme showed its maximal activity at 60°C (Ea = 5.1 kcal/ mol [21.3 kJ/mol]) and optimal pH values of 8.0 and 7.5 when assayed in Tris hydrochloride and potassium phosphate buffers, respectively. The native molecular mass of the enzyme was about 280 kilodaltons. The possible physiological role of the enzyme in ammonia assimilation is discussed.

Physiological role and regulation of glutamate dehydrogenase inProchlorococcussp. strain MIT9313

“…We obtained K m values of 43.72 mM for ammonium (aminating activity) and 56.45 mM for glutamate (deaminating activity). These results are consistent with previous studies on cyanobacteria (Martínez‐Bilbao et al. , 1988) and if we take into account the reported cyanobacterial pools of ammonium (0.64–2 µM, calculated from Boussiba et al.…”

“…We obtained K m values of 43.72 mM for ammonium (aminating activity) and 56.45 mM for glutamate (deaminating activity). These results are consistent with previous studies on cyanobacteria (Martínez-Bilbao et al, 1988) and if we take into account the reported cyanobacterial pools of ammonium (0.64-2 mM, calculated from Boussiba et al, 1984) and glutamate (24.5-182.3 mM; Zahradnícková et al, 2007), they might suggest a catabolic role for NADPH-GDH in Prochlorococcus. Km have also been determined for GDH in other organisms by other teams, and in particular in the cases of Km for ammonium and L-glutamate, there exists a great diversity.…”

Coral Uptake of Inorganic Phosphorus and Nitrogen Negatively Affected by Simultaneous Changes in Temperature and pH

“…One hypothesis to explain this result is that ammonium-assimilating enzymes (GS, GOGAT, and glutamate dehydrogenase GDH) [25], [80], [82]-[84] in corals are stimulated by seawater acidification, possibly through the impact of acidification on the intracellular pH [85]. For example, the activity of GS, GOGAT, and GDH are influenced by pH in an actinobacteria [86] and a cyanobacteria [87], with pH T optima of 7.0 (GS), 7.2-7.6 (GOGAT), and 7.2-7.5 (GDH). In the coral S. pistillata , GDH aminating activity is also pH-dependent, as it increased when pH T was decreased between 8.1 and 7.3 [25].…”

Ammonium-assimilating enzymes and their regulation in wild and NADP-glutamate dehydrogenase-deficient strains of the ectomycorrhizal fungus Hebeloma cylindrosporum

“…Wild mycelia grown on ammonium containing medium supplemented with MSX were able to grow normally, indicating that NADP-GDH alone is able to assure ammonium assimilation (Fig, IA), Tbe apparent glutamate-inducible nature of the NADP-GDH (Tab. 1) has been reported for the ascomycete Sphaerostilbe repens , the basidiomycete Coprinus cinereus (Moore 1981) or the cyanobacteritim Phormidium laminosum (Martinez-Bilbao et al, 1988). Opposite results were found in Neurospora crassa (Hernandez et al, 1983) and Candida nitratophila (Hipkin et al 1990) since higher activities were found when organisms were grown on ammonium as nitrogen source than when grown on glutamate.…”