Growth and gas production for hyperthermophilic archaebacterium, <i>Pyrococcus furiosus</i>

Malik, Bisma; Su, Wanting; Wald, H. L.; Blumentals, I. I.; Kelly, Robert M.

doi:10.1002/bit.260340805

Cited by 40 publications

(22 citation statements)

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“…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 elemental sulfur to HIS as a form of detoxification, since H2 is a growth inhibitor [6].In P. ,furio.sus we found a high content of an extremely thermostable glutamate dehydrogenase that represents about 20% of the total protein expressed in the microorganism. This paper reports the purification and some general properties of glutamate dehydrogenase from P. furiosus as a first step to clarify the metabolic role of this overexpressed enzyme in the survival of the microorganism in marine superheated sources, the natural habitat of P. furiosus.…”

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Extremely thermostable glutamate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus furiosus

Consalvi

Chiaraluce

Politi

et al. 1991

European Journal of Biochemistry

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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...

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Extremely thermostable glutamate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus furiosus

Consalvi

Chiaraluce

Politi

et al. 1991

European Journal of Biochemistry

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“…The two Hyhs are very similar in their molecular and enzymatic properties, except that Hyh-II is approximately an order of magnitude less active than Hyh-I. Because P. furiosus is known to evolve H 2 as a metabolite (6,29,42), the physiological role of the two Hyhs was suggested to be the disposal of excess reducing equivalents in the form of H 2 (25).…”

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Distinct Physiological Roles of the Three [NiFe]-Hydrogenase Orthologs in the Hyperthermophilic Archaeon Thermococcus kodakarensis

et al. 2011

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Hydrogenases catalyze the reversible oxidation of molecular hydrogen (H 2 ) and play a key role in the energy metabolism of microorganisms in anaerobic environments. The hyperthermophilic archaeon Thermococcus kodakarensis KOD1, which assimilates organic carbon coupled with the reduction of elemental sulfur (S 0 ) or H 2 generation, harbors three gene operons encoding [NiFe]-hydrogenase orthologs, namely, Hyh, Mbh, and Mbx. In order to elucidate their functions in vivo, a gene disruption mutant for each [NiFe]-hydrogenase ortholog was constructed. The Hyh-deficient mutant (PHY1) grew well under both H 2 S-and H 2 -evolving conditions. H 2 S generation in PHY1 was equivalent to that of the host strain, and H 2 generation was higher in PHY1, suggesting that Hyh functions in the direction of H 2 uptake in T. kodakarensis under these conditions. Analyses of culture metabolites suggested that significant amounts of NADPH produced by Hyh are used for alanine production through glutamate dehydrogenase and alanine aminotransferase. On the other hand, the Mbh-deficient mutant (MHD1) showed no growth under H 2 -evolving conditions. This fact, as well as the impaired H 2 generation activity in MHD1, indicated that Mbh is mainly responsible for H 2 evolution. The copresence of Hyh and Mbh raised the possibility of intraspecies H 2 transfer (i.e., H 2 evolved by Mbh is reoxidized by Hyh) in this archaeon. In contrast, the Mbx-deficient mutant (MXD1) showed a decreased growth rate only under H 2 S-evolving conditions and exhibited a lower H 2 S generation activity, indicating the involvement of Mbx in the S 0 reduction process. This study provides important genetic evidence for understanding the physiological roles of hydrogenase orthologs in the Thermococcales.

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“…This obligate anaerobe grows optimally at 100'C by the fermentation of carbohydrates and peptides in which excess reductant, generated mainly in the form of reduced ferredoxin (2,5,30), is disposed of either as H2, or if S is added to the growth medium, as H2S (18). Both simple and complex sulfidic compounds serve as substrates for H2S production (9,29,49). Although S0 reduction was originally thought to be a mechanism of detoxifying inhibitory H2 (18), a more recent study showed that S0 reduction plays a role in energy conservation (42).…”

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Sulfide dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus: a new multifunctional enzyme involved in the reduction of elemental sulfur

1994

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Pyrococcus furiosus is an anaerobic archaeon that grows optimally at 1000C by the fermentation of carbohydrates yielding acetate, C02, and H2 as the primary products. If elemental sulfur (SO) or polysulfide is added to the growth medium, H2S is also produced. The cytoplasmic hydrogenase of P. furiosus, which is responsible for H2 production with ferredoxin as the electron donor, has been shown to also catalyze the reduction of polysulfide to H2S (K. Ma, R. N. Schicho (33). The majority are strict anaerobes, and most are obligately dependent upon the reduction of elemental sulfur (S) to H2S for optimal growth. Molecular H2 or organic compounds serve as electron donors for the apparent respiration of So. Some of the heterotrophic species are able to grow in the absence of S by fermentation-type metabolisms that result in the production of H2. In such cases, the addition of So to the growth medium leads to H2S production and growth stimulation. Since the hyperthermophiles are the most slowly evolving of known life forms, it has been suggested that S0 respiration may represent one of the earliest mechanisms of energy conservation from an evolutionary perspective (40, 47).The mechanisms by which hyperthermophilic organisms reduce So to H2S and the natures of the enzymes involved are far from clear. The situation is complicated by the fact that the abiotic reduction of S0 can also occur at the growth temperatures of these organisms (9). Also, because of the low solubility of S in aqueous media, golysulfide is thought to be the true substrate for microbial S reduction (9,40,41 nism of H2S production from S0 has been investigated in only one autotrophic hyperthermophile, Pyrodictium brockii, an obligate S-reducing species which grows optimally at 1050C. This organism was shown to have a primitive membrane-bound electron transport chain for coupling H2 oxidation and S0 reduction (36). The chain contained hydrogenase, a cytochrome, and a novel quinone, but the So-reducing entity was not purified. In fact, there have been few reports on S5 reduction by mesophilic organisms. For example, Zophel and coworkers screened several different So-reducing bacteria for sulfur oxidoreductase activity (50) and the enzyme responsible in "Spirillum" strain 5175 (44) was postulated to be an iron-sulfur protein possibly associated with a c-type cytochrome (51). However, only one S0-reducing enzyme has been purified and characterized from a mesophile, that from Wolinella succinogenes (20,21,43), an organism which grows with formate as the electron donor and S0 as the electron acceptor (27). From sequencing analysis, it was postulated that its polysulfide reductase is composed of three subunits and is a molybdopterin-containing iron-sulfur protein (21).We are investigating the So-reducing activities of heterotrophic hyperthermophiles such as P. furiosus (18). This obligate anaerobe grows optimally at 100'C by the fermentation of carbohydrates and peptides in which excess reductant, generated mainly in the form of reduced ferredoxin (2,5,30)...

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Growth and gas production for hyperthermophilic archaebacterium, Pyrococcus furiosus

Cited by 40 publications

References 10 publications

Extremely thermostable glutamate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus furiosus

Extremely thermostable glutamate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus furiosus

Distinct Physiological Roles of the Three [NiFe]-Hydrogenase Orthologs in the Hyperthermophilic Archaeon Thermococcus kodakarensis

Sulfide dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus: a new multifunctional enzyme involved in the reduction of elemental sulfur

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