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A gene encoding a 2-deoxy-d-ribose-5-phosphate aldolase (DERA) homolog was identified in the hyperthermophilic Archaea Aeropyrum pernix. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified and characterized. The enzyme is an extremely thermostable DERA; its activity was not lost after incubation at 100 degrees C for 10 min. The enzyme has a molecular mass of approximately 93 kDa and consists of four subunits with an identical molecular mass of 24 kDa. This is the first report of the presence of tetrameric DERA. The three-dimensional structure of the enzyme was determined by x-ray analysis. The subunit folds into an alpha/beta-barrel. The asymmetric unit consists of two homologous subunits, and a crystallographic 2-fold axis generates the functional tetramer. The main chain coordinate of the monomer of the A. pernix enzyme is quite similar to that of the E. coli enzyme. There was no significant difference in hydrophobic interactions and the number of ion pairs between the monomeric structures of the two enzymes. However, a significant difference in the quaternary structure was observed. The area of the subunit-subunit interface in the dimer of the A. pernix enzyme is much larger compared with the E. coli enzyme. In addition, the A. pernix enzyme is 10 amino acids longer than the E. coli enzyme in the N-terminal region and has an additional N-terminal helix. The N-terminal helix produces a unique dimer-dimer interface. This promotes the formation of a functional tetramer of the A. pernix enzyme and strengthens the hydrophobic intersubunit interactions. These structural features are considered to be responsible for the extremely high stability of the A. pernix enzyme. This is the first description of the structure of hyperthermophilic DERA and of aldolase from the Archaea domain.

The gene encoding the ATP-dependent glucokinase of hyperthermophilic archaeon Aeropyrum pernix was identified, cloned, and functionally expressed in Escherichia coli. The deduced amino acid sequence showed 40% identity to that of the putative glucokinase from hyperthermophilic archaeon Pyrobacurum aerophilum. The purified recombinant enzyme was a monomer with a molecular mass of 35 kDa. The enzyme retained its full activity on heating at 70 degrees C for 10 min and retained 65% of the activity after 10-min incubation at 100 degrees C. The enzyme exclusively catalyzed the phosphorylation of D-glucose using ATP as a phosphoryl donor. ITP was accepted in addition to ATP. The rate dependence with both glucose and ATP followed Michaelis-Menten kinetics, with apparent K(m) values of 0.054 and 0.50 mM, respectively. The enzyme activity required divalent cations; Mg(2+), which was most effective, could partially be replaced by Mn(2+) or Ca(2+). Phylogenetic analysis revealed that the glucokinase from A. pernix does not belong to the clusters of enzymes found in bacteria and eukarya. This is the first description of the characteristics of an ATP-dependent glucokinase from an archaeon.

Although ATP is the most common phosphoryl group donor for kinases, some kinases from certain hyperthermophilic archaea such as Pyrococcus horikoshii and Thermococcus litoralis use ADP as the phosphoryl donor. Those are ADP-dependent glucokinases (ADPGK) and phosphofructokinases in their glycolytic pathway. Here, we succeeded in gene cloning the ADPGK from P. horikoshii OT3 (phGK) in Escherichia coli,and in easy preparation of the enzyme, crystallization, and the structure determination of the apo enzyme. Recently, the three-dimensional structure of the ADPGK from T. litoralis (tlGK) in a complex with ADP was reported. The overall structure of two homologous enzymes (56.7%) was basically similar: This means that they consisted of large alpha/beta-domains and small domains. However, a marked adjustment of the two domains, which is a 10-A translation and a 20 degrees rotation from the conserved GG sequence located at the center of the hinge, was observed between the apo-phGK and ADP-tlGK structures. The ADP-binding loop (430-439) was disordered in the apo form. It is suggested that a large conformational change takes place during the enzymatic reaction.

Bacillus stearothermophilus H-804 isolated from a hot spring in Beppu, Japan, produced an ammonia-specific NAD synthetase (EC 6.3.1.5). The enzyme specifically used NH3 as an amide donor for the synthesis of NAD as it formed AMP and pyrophosphate from deamide-NAD and ATP. None of the l-amino acids tested, such as l-asparagine or l-glutamine, or other amino compounds such as urea, uric acid, or creatinine was used instead of NH3. Mg2+ was needed for the activity, and the maximum enzyme activity was obtained with 3 mM MgCl2. The molecular mass of the native enzyme was 50 kDa by gel filtration, and SDS-PAGE showed a single protein band at the molecular mass of 25 kDa. The optimum pH and temperature for the activity were from 9.0 to 10.0 and 60 degrees C, respectively. The enzyme was stable at a pH range of 7.5 to 9.0 and up to 60 degrees C. The Km for NH3, ATP, and deamide-NAD were 0.91, 0.052, and 0.028 mM, respectively. The gene encoding the enzyme consisted of an open reading frame of 738 bp and encoded a protein of 246 amino acid residues. The deduced amino acid sequence of the gene had about 32% homology to those of Escherichia coli and Bacillus subtilis NAD synthetases. We caused the NAD synthetase gene to be expressed in E. coli at a high level; the enzyme activity (per liter of medium) produced by the recombinant E. coli was 180-fold that of B. stearothermophilus H-804. The specific assay of ammonia and ATP (up to 25 microM) with this stable NAD synthetase was possible.

A gene encoding the L-aspartate oxidase homologue was identified via genome sequencing in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3. We succeeded in expressing the encoding gene in Escherichia coli and purified the product to homogeneity. Characterization of the protein revealed that it is the most thermostable L-aspartate oxidase detected so far. In addition to the oxidase activity, the enzyme catalyzed L-aspartate dehydrogenation in the presence of an artificial electron acceptor such as phenazine methosulfate, 2,6-dichlorophenol-indophenol, and ferricyanide. L-Aspartate oxidase is known to function as the first enzyme in the de novo NAD biosynthetic pathway in prokaryotes. By a similarity search in public databases, the genes that encode the homologue of all other enzymes involved in the pathway were identified in the P. horikoshii OT-3 genome. This suggests that P. horikoshii OT-3 may use the de novo NAD biosynthetic pathway under anaerobic conditions.

Glutamate dehydrogenase from an aerobic hyperthermophilic archaeon, Aeropyrum pernix K1, was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol (PEG) 400 as the precipitant. The crystals belong to the hexagonal space group P6(3), with unit-cell parameters a = b = 98.9, c = 394.8 A, alpha = beta = 90, gamma = 120 degrees. The asymmetric unit contained one hexamer of the enzyme, giving a crystal volume per enzyme mass (V(M)) of 1.98 A(3) Da(-1) and a solvent content of 37.3%. The X-ray diffraction data were collected to a resolution of 3.0 A at the BL6B beamline in the Photon Factory with an overall R(sym) of 13.8% and a completeness of 87.1%.

Leucine dehydrogenase is an octameric enzyme which belongs to the superfamily of amino-acid dehydrogenases and catalyses the reversible oxidative deamination of leucine to 2-ketoisocaproate, with the corresponding reduction of the cofactor NAD(+). Catalysis by this enzyme is thought to involve a large-scale motion of the enzyme's two domains between an 'open' and 'closed' form, with the latter representing a conformation of the enzyme in which the partners involved in the hydride-transfer reaction are appropriately positioned for catalysis. Whilst a structure for the open form of the enzyme has been determined, the nature of the closed form has yet to be observed. In order to trap a closed form, crystals of the complexes of leucine dehydrogenase from Thermoactinomyces intermedius with 2-ketoisocaproate and with 2-ketoisocaproate and NAD(+) have been obtained by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The crystals of the binary complex with 2-ketoisocaproate belong to space group P2(1)2(1)2(1), with approximate unit-cell parameters a = 106, b = 118, c = 320 A and an octamer in the asymmetric unit, corresponding to a V(M) of 3.1 A(3) Da(-1). The crystals of the non-productive ternary complex belong to space group P6(1) or P6(5), with approximate unit-cell parameters a = b = 117, c = 502 A and an octamer in the asymmetric unit, corresponding to a V(M) of 3.0 A(3) Da(-1). These crystals diffract X-rays on a synchrotron-radiation source to at least 2.8 and 3.3 A resolution, respectively, and are suitable for a full structure determination.

The thermostability of Flavobacterium meningosepticum glycerol kinase was increased by the change from Ser329 to Asp [Protein Eng., 14, 663-667 (2001)]. Based on a three-dimensional structure model of the mutant, we have postulated that a new charged-neutral hydrogen bond was formed between Asp329 and Ser414, and the formation of the hydrogen bond contributed to the stabilization of the tertiary structure and increased thermostability of the mutant enzyme. If the postulation is the case, FGK thermostabilization would be possible similarly by the single amino acid substitution from Ser414 to another amino acid which could form the hydrogen bond with Ser329. We did a single amino acid substitution of the wild-type enzyme from Ser414 to Asn. As we expected, S414N showed comparable thermostability to that of S329D. On the other hand, a difference in kinetic properties for ATP between S414N and S329D was observed.

A gene encoding an ADP-dependent phosphofructokinase homologue has been identified in the hyperthermophilic archaeon Methanococcus jannaschii via genome sequencing. The gene encoded a protein of 462 amino acids with a molecular weight of 53,361. The deduced amino acid sequence of the gene showed 52 and 29% identities to the ADP-dependent phosphofructokinase and glucokinase from Pyrococcus furiosus, respectively. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified and characterized. To our surprise, the enzyme showed high ADP-dependent activities for both glucokinase and phosphofructokinase. A native molecular mass was estimated to be 55 kDa, and this indicates the enzyme is monomeric. The reaction rate for the phosphorylation of D-glucose was almost 3 times that for D-fructose 6-phosphate. The K(m) values for D-fructose 6-phosphate and D-glucose were calculated to be 0.010 and 1.6 mm, respectively. The K(m) values for ADP were 0.032 and 0.63 mm when D-glucose and D-fructose 6-phosphate were used as a phosphoryl group acceptor, respectively. The gene encoding the enzyme is proposed to be an ancestral gene of an ADP-dependent phosphofructokinase and glucokinase. A gene duplication event might lead to the two enzymatic activities.

The activity of dye-linked d-proline dehydrogenase was found in the crude extract of a hyperthermophilic archaeon, Pyrobaculum islandicum JCM 9189. The dye-linked d-proline dehydrogenase was a membrane associated enzyme and was solubilized from the membrane fractions by treatment with Tween 20. The solubilized enzyme was purified 34-fold in the presence of 0.1% Tween 20 by four sequential chromatographies. The enzyme has a molecular mass of about 145 kDa and consisted of homotetrameric subunits with a molecular mass of about 42 kDa. The N-terminal amino acid sequence of the subunit was MKVAIVGGGIIGLFTAYHLRQQGADVVI. The enzyme retained its full activity both after incubation at 80 degrees C for 10 min and after incubation in the range of pH 4.0-10.0 at 50 degrees C for 10 min. The enzyme-catalyzed dehydrogenation of several d-amino acids was carried out using 2,6-dichloroindophenol as an electron acceptor, and d-proline was the most preferred substrate among the d-amino acids. The Michaelis constants for d-proline and 2,6-dichloroindophenol were determined to be 4.2 and 0.14 mm, respectively. Delta(1)-Pyrroline-2-carboxylate was identified as the reaction product from d-proline by thin layer chromatography. The prosthetic group of the enzyme was identified to be FAD by high-performance liquid chromatography. The gene encoding the enzyme was cloned and expressed in Escherichia coli. The nucleotide sequence of the dye-linked d-proline dehydrogenase gene was determined and encoded a peptide of 363 amino acids with a calculated molecular weight of 40,341. The amino acid sequence of the Pb. islandicum enzyme showed the highest similarity (38%) with that of the probable oxidoreductase in Sulfolobus solfataricus, but low similarity with those of d-alanine dehydrogenases from the mesophiles so far reported. This shows that the membrane-bound d-proline dehydrogenase from Pb. islandicum is a novel FAD-dependent amino acid dehydrogenase.

The temperature dependence of the steady-state kinetic parameters for a glutamate dehydrogenase from Aeropyrum pernix K1 was investigated. The enzyme showed a biphasic kinetic characteristic for L-glutamate and a monophasic one for NADP at 50-90 degrees C. At low concentrations of L-glutamate the Km decreased from 2.02 to 0.56 mM and the catalytic efficiency (Vmax/Km) markedly increased (4-150 micromol x mg(-1) x mM(-1)) along with the increase of temperature from 50 to 90 degrees C. At high concentrations of the substrate the Km was fairly high and approximately constant (around 225 mM), and the catalytic efficiency was low and its temperature-dependent change was small. The Km (0.039 mM) for NADP did not change with the increase of temperature. In the reductive amination, the Kms for 2-oxoglutarate (1.81 and 9.37 mM at low and high levels of ammonia, respectively) were independent on temperature, but the Kms for ammonia and NADPH rose from 86 to 185 mM and 0.050 to 0.175 mM, respectively.

Hyperthermophiles, a group of microorganisms whose optimum growth temperatures are above 80 degrees C, have been isolated mainly from marine and continental volcanic environments. They are viewed as potential sources of extraordinarily stable biomolecules with applications in novel industrial processes. Most hyperthermophiles belong to the domain Archaea, the third domain of life, and are considered to be the most ancient of all extant life forms. Recent studies have revealed unusual energy metabolic processes in hyperthermophilic archaea, e.g. a modified Embden-Meyerhof pathway, that have not been observed so far in organisms belonging to the Bacteria and Eucarya domains. Several novel enzymes--ADP-dependent glucokinase, ADP-dependent phosphofruktokinase, glyceraldehyde-3-phosphate ferredoxin oxidoreductase, phosphoenolpyruvate synthase, pyruvate: ferredoxin oxidoreductase, and ADP-forming acetyl-CoA synthetase--have been found to be involved in the modified Embden-Meyerhof pathway of the hyperthermophilic archaeon Pyrococcus furiosus. In addition, a novel regulation site for energy metabolism and a unique mode of ATP regeneration have been postulated to exist in the pathway of P. furiosus. The metabolic design observed in this microorganism might reflect the situation at an early stage of evolution. This review focuses mainly on the unique energy metabolism and related enzymes of P. furiosus that have recently been described.