Extremely thermostable glutamate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus furiosus

Abstract

The hyperthermophilic archaebacterium Pyrococcus furiosus contains high levels of NAD(P)-dependent glutamate dehydrogenase activity. The enzyme could be involved in the first step of nitrogen metabolism, catalyzing the conversion of 2-oxoglutarate and ammonia to glutamate. The enzyme, purified to homogeneity, is a hexamer of 290 kDa (subunit mass 48 kDa). Isoelectric-focusing analysis of the purified enzyme showed a pl of 4.5. The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate but utilizes both NADH and NADPH as cofactors. The purified enzyme reveals an outstanding thermal stability (the half-life for thermal inactivation at 100°C was 12 h), totally independent of enzyme concentration.P. furiosus glutamate dehydrogenase represents 20% of the total protein; this elevated concentration raises questions about the roles of this enzyme in the metabolism of P..furiosus.Recently we reported the purification of an NAD(P)-dependent glutamate dehydrogenase from the thermophilic archaebacterium Sulfolobus so@ztaricus [l, 21. The enzyme of this microorganism is probably involved in the first step of ammonia assimilation by the following reaction: glutamate + NAD(P)+ + H 2 0 + 2-oxoglutarate + NH,' + NAD(P)H + H + .The glutamate dehydrogenase from S . solfutaricus presents some interesting properties; it has a double coenzyme specificity, is strictly specific for L-glutamate and 2-oxoglutarate and its thermal stability is a function of the enzyme concentration [2]. This latter property is in good agreement with the observed self-associating behaviour of the purified enzyme.Among archaebacterial glutamate dehydrogenases, the only data available for comparison are restricted to the halophilic phenotype [3]. Therefore, it is difficult to extrapolate from our observations on S. solfituricus glutamate dehydrogenase to obtain general rules for thermal adaptation in this class of enzymes and general knowledge regarding nitrogen metabolism in archaebacteria.To provide more information about the enzymological properties of archaebacterial glutamate dehydrogenases and to extend our investigations towards more thermostable forms of this enzyme, we attampted to purify glutamate dehydrogenase from the so-called 'hyperthermophiles'. These remarkable microorganisms, recently discovered, grow optimally at temperatures near 100 "C [4]. They all are archaebacteria and most of them are strict anaerobes and depend on the reduction of elemental sulfur for growth. So far, hyperthermophiles -___ have been classified into three distinct genera, Pyrodiictium, Pyrobaculurn and Pyrococcus. As a source of hyperthermophilic glutamate dehydrogenase we used Pyrococcus furiosus [5], one of the most interesting members of this last genus because of its relatively high cell yield and rapid growth rate. The microorganism grows optimally at 10O'C by a fermentative-type metabolism and is a strict heterotroph which utilizes both simple and complex carbohydrates and produces only H2 and C 0 2 as detectable products. P. furiosus reduces eleme...

Crystallization of the NAD(P)-dependent glutamate dehydrogenase from the hyperthermophilePyrococcus furiosus

“…The cell dimensions are a = b = 167.2, c = 172.9 A and the cell volume is 4.83 x 106/~3. Gel-filtration studies have shown that this GluDH is hexameric (Consalvi et al, 1991) and since it shows sequence similarity (34% identity over 413 equivalenced positions in the sequence alignment) with that of the hexameric GluDH from C. symbiosum this indicates that the subunits are arranged in 32 symmetry. Given the subunit Mr of 47 000, (Maras et al, 1994) if the asymmetric unit of the cell contains a hexamer the corresponding value for the Vm would be 2.14A 3 Da -1.…”

“…The most well studied of these hyperthermophilic microorganisms is Pyrococcus furiosus, which grows at temperatures up to 376 K (Klump et al, 1992). The GIuDH from this organism is extremely stable with a half-life at 373 K of 12h (Consalvi et al, 1991;.…”

“…Such insights might suggest how proteins might be redesigned for novel commercial applications. In this regard, studies of GIuDH have become a model system for the analysis of thermostability since this enzyme is abundant in these organisms and has been widely studied from a variety of species (Consalvi et al, 1991, Maras et al, 1992, DiRuggiero et al, 1993, Maras et al, 1994.…”

“…The elutant containing GluDH activity was concentrated through a YM-30 Diaflo membrane in an ultrafiltration cell (Amicon) and dialysed against 400 ml of 0.1 M KH2PO4 at pH 6. The purified protein was brought to 65% saturation with ammonium sulfate and the resulting precipitate stored at 277 K. The enzyme activity was found to be 340 units mg -1 using the method of Consalvi et al (1991) and the purity further checked by sodium dodecyl sulfate and native polyacrylamide gel electrophoresis.…”

“…Experimental P. furiosus cells were grown on peptone and yeast extract (Raven, Ladwa, Cossar & Sharp, 1992) and all the purification steps were carried out at 277 K, using a modified procedure based on Consalvi et al (1991). The cells were resuspended in 2 volumes of 20 mM Tris-HC1 at pH 8 and ground in a mortar with sea sand.…”