Properties of Glutamate Dehydrogenase and Its Involvement in Alanine Production in a Hyperthermophilic Archaeon, Thermococcus profundus1

Abstract

Thermococcus profundus, a hyperthermophilic archaeon, did not exhibit detectable glutamine synthetase activity, although the organism possessed an extraordinarily high level of glutamate dehydrogenase (GDH), the content of which reached over 10% of total soluble proteins. This GDH was purified to homogeneity. The enzyme had a molecular weight of 263,000 and was composed of six homogeneous subunits of molecular weight 43,000. The enzyme was extremely thermostable with a half life of 1 h at 90 degrees C. Circular dichroism (CD) spectra of the enzyme revealed gradual unfolding of alpha-helices upon exposure to increasing temperature. The enzyme reaction was strongly biased toward glutamate formation. T. profundus excreted L-alanine into the medium, and the concentration reached mM. High activity of alanine aminotransferase (AAT) was present in the cells, while no alanine dehydrogenase activity was detected. The alanine formation may be initiated by ammonia uptake by GDH followed by aminotransfer from glutamate to pyruvate by AAT.

Purification and characterization of a thermostable glutamate dehydrogenase from a thermophilic bacterium isolated from a sterilization drying oven

“…As other GDHs reported to date (4), this enzyme belongs to the Hexameric GDHs group. The apparent molecular mass of the native enzyme and its subunits is only slightly smaller than that found in P. furiosus (10,15,11), P. woesei (2), T. litoralis (7) and T. profundus (8), suggesting that the enzyme is highly conserved in terms of molecular structure among thermophiles and hyperthermophiles. GWE1 GDH is thermoactive, a property shared with enzymes from hyperthermopiles (16), showing an optimal activity at 70 o C which correspond to the temperature of growth of the microorganism.…”

“…In general, GDH enzymes from vertebrates are able to use both coenzymes, whereas the enzymes from bacteria are specific for either NAD + or NADP + (6). To date, several thermostable GDHs have been isolated from different hyperthermophilic microorganims (2,(7)(8)(9); being the most thermostable GDH reported to date the one purified from Pyrococcus furiosus (10,11). So far, microbial GDHs have been purified from many microorganisms, but all of them from natural origin environment.…”

Glutamate Dehydrogenases: Enzymology, Physiological Role and Biotechnological Relevance

“…In this organism, GDH is the first enzyme of the glutamate fermentation via the hydroxyglutarate pathway, and can represent as much as 10% of total protein when grown on glutamate. Very high levels of GDH production has also been reported in some hyperthermophilic archaea like Pyrococcus furiosus or some Thermococcus strains [5,6,71]. These preferentially biosynthetic enzymes represent an exceptionally high percentage of total soluble protein of the cell, in some cases up to 20%, which suggests an important role of these enzymes in these organisms at an extremely high temperature for life.…”

“…The actual physiological reaction of each GDH depends on several factors, as the kinetic constants of the enzyme for its different substrates or the environment where the cell is developed may widely vary. In general, NADP + -GDHs usually operate in the biosynthetic direction, that is, synthesizing glutamate by the assimilation of ammonia into 2-OG [6,31,39,40,45,67,68], whereas NAD + -GDHs have primarily a catabolic function, yielding ammonia and 2-oxoglutarate from the oxidative catabolism of glutamate [23,24,39,68] (Table 1). Sometimes, both enzymes are present in the same organism, and play a different physiological role due to their different kinetic properties or their different time or place of expression [27,51,69,70].…”

“…In plants and microorganisms, the physiological roles of GDH include nitrogen assimilation [2], glutamate catabolism [1,3], but also osmotic balance [4] and tolerance to high temperatures [5,6]. In vertebrates, multiple biochemical pathways involve glutamate, which is also used as a neurotransmitter.…”