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The amino acid sequence of the OCC_10945 gene product from the hyperthermophilic archaeon Thermococcus litoralisDSM5473, originally annotated as γ-aminobutyrate aminotransferase, is highly similar to that of the uncharacterized pyridoxal 5ʹ-phosphate (PLP)-dependent amino acid racemase from Pyrococcus horikoshii. The OCC_10945 enzyme was successfully overexpressed in Escherichia coliby coexpression with a chaperone protein. The purified enzyme demonstrated PLP-dependent amino acid racemase activity primarily toward Met and Leu. Although PLP contributed to enzyme stability, it only loosely bound to this enzyme. Enzyme activity was strongly inhibited by several metal ions, including Co2+and Zn2+, and nonsubstrate amino acids such as l-Arg and l-Lys. These results suggest that the underlying PLP-binding and substrate recognition mechanisms in this enzyme are significantly different from those of the other archaeal and bacterial amino acid racemases. This is the first description of a novel PLP-dependent amino acid racemase with moderate substrate specificity in hyperthermophilic archaea.Graphical AbstractThe GABA-ATs classified into group 4 in Thermococcales order of archaea were identified as PLP-dependent moderate-substrate specificity amino acid racemase.

ABSTRACTThe orientation of the three domains in the bifunctional aspartate kinase-homoserine dehydrogenase (AK-HseDH) homologue found in Thermotoga maritimatotally differs from those observed in previously known AK-HseDHs; the domains line up in the order HseDH, AK, and regulatory domain. In the present study, the enzyme produced in Escherichia coliwas characterized. The enzyme exhibited substantial activities of both AK and HseDH. L-Threonine inhibits AK activity in a cooperative manner, similar to that of Arabidopsis thalianaAK-HseDH. However, the concentration required to inhibit the activity was much lower (K0.5 = 37 μM) than that needed to inhibit the A. thalianaenzyme (K0.5 = 500 μM). In contrast to A. thalianaAK-HseDH, Hse oxidation of the T. maritimaenzyme was almost impervious to inhibition by L-threonine. Amino acid sequence comparison indicates that the distinctive sequence of the regulatory domain in T. maritimaAK-HseDH is likely responsible for the unique sensitivity to L-threonine.Abbreviations: AK: aspartate kinase; HseDH: homoserine dehydrogenase; AK–HseDH: bifunctional aspartate kinase–homoserine dehydrogenase; AsaDH: aspartate–β–semialdehyde dehydrogenase; ACT: aspartate kinases (A), chorismate mutases (C), and prephenate dehydrogenases (TyrA, T).

The hydrophobic effect on electrodeposition of an enzyme CotA was investigated. The CotA was electrodeposited on a gold disc electrode modified with an alkane thiol self-assembled monolayer (SAM). The CotA immobilized hydrophobic SAM gold electrodes exhibited a larger electrochemical activity compared with the hydrophilic SAM and bare electrodes. In particular, large current responses were observed in an ethanethiol or a 1-butanethiol SAM gold electrode. In addition, effect of addition of non-ionic surfactant Triton X on the electrodeposition was checked using the SAM gold electrode. The addition of Triton X had a positive effect on the electrodeposition.

Methylmalonyl-CoA mutase (MCM) requires 5′-deoxyadenosylcobalamin (AdoCbl) as a cofactor and is widely distributed in organisms from bacteria and animals. Although genes encoding putative MCMs are present in many archaea, they are separately encoded in large and small subunits. The large and small subunits of archaeal MCM are similar to the catalytic and AdoCbl-binding domains of human MCM, respectively. In Pyrococcus horikoshiiOT3, putative genes PH1306and PH0275encode the large and small subunits, respectively. Because information on archaeal MCM is extremely restricted, we examined the functional and structural characteristics of P. horikoshiiMCM.Reconstitution experiments using recombinant PH0275 and PH1306 showed that these proteins assemble in equimolar ratios and form of heterotetrameric complexes in the presence of AdoCbl. Subsequent immunoprecipitation experiments using anti-PH0275 and anti-PH1306 antibodies suggested that PH0275 and PH1306 form a complex in P. horikoshiicells in the presence of AdoCbl.

We previously found a very large NAD-dependent glutamate dehydrogenase with approximately 170 kDa subunit from Janthinobacterium lividum(Jl-GDH) and predicted that GDH reaction occurred in the central domain of the subunit. To gain further insights into the role of the central domain, several single point mutations were introduced. The enzyme activity was completely lost in all single mutants of R784A, K810A, K820A, D885A, and S1142A. Because, in sequence alignment analysis, these residues corresponded to the residues responsible for glutamate binding in well-known small GDH with approximately 50 kDa subunit, very large GDH and well-known small GDH may share the same catalytic mechanism. In addition, we demonstrated that C1141, one of the three cysteine residues in the central domain, was responsible for the inhibition of enzyme activity by HgCl2, and HgCl2functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl2was found to function as an activating compound for a wild-type Jl-GDH. This suggests that the mechanism for the activation is entirely different from that for the inhibition.

We fabricated a thermostable quinoprotein aldose sugar dehydrogenase (tPQQ-ASD derived from a hyperthermophilic archaeon Pyrobaculum aerophilum)immobilized electrode. The electrode was prepared by immobilizing agarose gel mixed with the enzyme and carbon nanofiber (CNF) on a carbon paste (CP) electrode containing p-benzoquinone (BQ) as an electron mediator. The electrocatalytic response was clearly observed by the addition of D-glucose at the electrode. The electrode properties such as pH, temperature dependency and substrate selectivity basically followed the enzyme properties. The current response against D-glucose increased with measurement temperatures up to 70°C, and response perturbation caused by dissolved oxygen level was not observed at the electrode. As for the results of long-term stability evaluation, the current response was stable for 30 days when the electrode was stored in HEPES buffer solution (pH 7.0) at 4°C.

A third novel type of dye-linked L-proline dehydrogenase (LPDH) has recently been found in the hyperthermophilic archaeon, Pyrobaculum calidifontis, by Satomura et al.The gene encoding the enzyme homologue was identified in the aerobic hyperthermophilic archaeon, Aeropyrum pernix. The gene was successfully expressed in Escherichia coli, and the product was purified to homogeneity and characterized. The expressed enzyme was highly thermostable LPDH having a molecular mass of about 88 kDa and a homodimeric structure. The preferred substrate for the enzyme was L-proline with 2,6-dichloroindophenol (DCIP) as the electron acceptor. However, the enzyme did not utilize ferricyanide as the electron acceptor, in contrast to all other known LPDHs. The electrochemical determination of L-proline at concentrations from 0 to 0.7 mMwas achieved by using A. pernixLPDH. A phylogenetic analysis revealed A. pernixLPDH to be clustered with the third type of LPDHs, and to be clearly separated from the clusters of previously known heterooligomeric LPDHs.

The kinetics of a very large NAD-dependent glutamate dehydrogenase from Janthinobacterium lividumshowed positive cooperativity toward α-ketoglutarate and NADH, and the Michaelis-Menten type toward ammonium chloride in the absence of the catalytic activator, L-aspartate. An increase in the maximum activity accompanied the decrease in the S0.5values for α-ketoglutarate and NADH with the addition of L-aspartate, and the kinetic response for α-ketoglutarate changed completely to a typical Michaelis-Menten type in the presence of 10 mML-aspartate.

A chemoattractant lectin from the dorsal spines of the redfin velvetfish, Hypodytes rubripinnis, was isolated using a combination of affinity chromatography techniques. The glycoprotein, with a molecular mass of 110 kDa, is named Karatoxin. Karatoxin caused agglutination of rabbit erythrocytes. This agglutination was effectively inhibited by d-mannose. In addition, Karatoxin exhibited not only mitogenic activity in the presence of murine splenocytes, but also chemotactic activity in the presence of guinea-pig neutrophils and macrophages. Thus, Karatoxin appears to be a novel chemoattractant lectin. These results suggest that the redfin velvetfish Hypodytes rubripinnismay be a novel source of biologically active substances.

Carbon nanotube (CNT) gel, which is composed of a mixture of single-wall CNT, an ionic liquid, and a thermostable D-proline dehydrogenase (D-Pro DH) immobilized electrode was utilized for the determination of D-amino acids (DAAs) in food samples. When a critical comparison with CNT, Ketjen Black (KB), and carbon powder (CP) was also carried out, the CNT/D-Pro DH immobilized electrode showed the highest sensitivity and the lowest detection limit of D-proline. In addition, the CNT/D-Pro DH immobilized electrode was applied to detection of DAAs in rice wine and vinegar samples. The concentrations of DAAs in rice wine and vinegar samples were 0.0210 ± 0.0001 and 0.55 ± 0.05 mmol L−1, respectively.

A gene encoding an ADP-dependent phosphofructokinase homologue has been identified in the hyperthermophilic archaeon Methanococcus jannaschiivia 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 Kmvalues for d-fructose 6-phosphate and d-glucose were calculated to be 0.010 and 1.6 mm, respectively. The Kmvalues for ADP were 0.032 and 0.63 mmwhen 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-linkedd-proline dehydrogenase was found in the crude extract of a hyperthermophilic archaeon, Pyrobaculum islandicumJCM 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 °C for 10 min and after incubation in the range of pH 4.0–10.0 at 50 °C for 10 min. The enzyme-catalyzed dehydrogenation of several d-amino acids was carried out using 2,6-dichloroindophenol as an electron acceptor, andd-proline was the most preferred substrate among thed-amino acids. The Michaelis constants ford-proline and 2,6-dichloroindophenol were determined to be 4.2 and 0.14 mm, respectively. Δ1-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. islandicumenzyme showed the highest similarity (38%) with that of the probable oxidoreductase in Sulfolobus solfataricus, but low similarity with those ofd-alanine dehydrogenases from the mesophiles so far reported. This shows that the membrane-bound d-proline dehydrogenase from Pb. islandicumis a novel FAD-dependent amino acid dehydrogenase.

The temperature dependence of the steady-state kinetic parameters for a glutamate dehydrogenase from Aeropyrum pernixK1 was investigated. The enzyme showed a biphasic kinetic characteristic for L-glutamate and a monophasic one for NADP at 50–90°C. At low concentrations of L-glutamate the Kmdecreased from 2.02 to 0.56 mMand the catalytic efficiency (Vmax/Km) markedly increased (4–150 μmol•mg−1•mM−1) along with the increase of temperature from 50 to 90°C. At high concentrations of the substrate the Kmwas 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 mMat low and high levels of ammonia, respectively) were independent on temperature, but the Kms for ammonia and NADPH rose from 86 to 185 mMand 0.050 to 0.175 mM, respectively.

The thermostability of Flavobacterium meningosepticumglycerol 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.

Bacillus stearothermophilusH-804 isolated from a hot spring in Beppu, Japan, produced an ammonia-specific NAD synthetase (EC 6.3.1.5). The enzyme specifically used NH3as 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 mMMgCl2. 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°C, respectively. The enzyme was stable at a pH range of 7.5 to 9.0 and up to 60°C. The Kmfor 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 coliand Bacillus subtilisNAD synthetases. We caused the NAD synthetase gene to be expressed in E. coliat a high level; the enzyme activity (per liter of medium) produced by the recombinant E. coliwas 180-fold that of B. stearothermophilusH-804. The specific assay of ammonia and ATP (up to 25 μM) with this stable NAD synthetase was possible.

The thermostability enhancement of Flavobacterium meningosepticum glycerol kinase (FGK) by random mutagenesis in the subunit interface region was investigated. A single Escherichia coli transformant, which produced a more thermostable glycerol kinase than the parent enzyme, was obtained. The nucleotide sequence of the gene of the mutant enzyme (FGK2615) was determined, and the four amino acid replacements were identified as Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys. Although the properties of FGK2615 were fundamentally similar to those of the parent enzyme, the thermostability and Km for ATP had changed. The thermostability of FGK2615 was apparently increased; the temperature at which the enzyme activity is inactivated by 50% for a 30-min incubation of FGK2615 was determined to be 72.1°C which was 3.1°C higher than that of the parent FGK. Four additional mutants each having a single amino acid replacement (Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys) were prepared and their thermostability and Km for substrates were evaluated. The effect of the substitution of Ser329 to Asp is discussed.

In marine fish liver, degradative enzymes able to convert purines to urate have been shown to be located in the cytosol and degradative enzymes able to convert urate to urea and glyoxylate in the peroxisomes. The end products of purine degradation are urea and glyoxylate in fish. Glyoxylate may be converted to glycine by alanine:glyoxylate aminotransferase for the reutilization of purine carbons. The present report describes that hepatic alanine:glyoxylate aminotransferase is located both in the peroxisomes and in the mitochondria in fresh water fish, showing that the intracellular localization of the enzyme differs between fresh water fish and marine fish. This is the first report on the presence of alanine:glyoxylate aminotransferase in fish peroxisomes.

Alcohol:NAD+oxidoreductase was found in the peroxisomes of animal liver for the first time as follows. The distribution of alcohol:NAD+oxidoreductase activity with nonanol as substrate in the light mitochondrial fraction (peroxisome-enriched fraction) of rat liver was examined by centrifugation in a sucrose density gradient. Most of the enzyme activity was localized in the mitochondria, with some activity in the peroxisomes. The administration of clofibrate, a peroxisome proliferator, to rats resulted in a marked increase of the enzyme activity in the peroxisomes, but not in the mitochondria. The enzyme was found to be located in the matrix of the peroxisomes. The evidence was obtained that the enzyme differed from alcohol dehydrogenases and alcohol oxidizing systems found previously. The enzyme activity was not affected by pyrazole, an inhibitor of alcohol dehydrogenase and sodium azide, an inhibitor of catalase. The enzyme was NAD+-dependent and oxidized straight chain aliphatic alcohols with a variety of carbon chains (C2-C18), showing the maximum on nonanol. Kmvalues toward these aliphatic alcohols decreased with increasing chain length. The major reaction product was identified as the carboxylic acid by using high performance liquid chromatography.

A gene encoding a UDP-glucose dehydrogenase homologue was identified in the hyperthermophilic archaeon, Pyrobaculum islandicum. This gene was expressed in Escherichia coli, and the product was purified and characterized. The expressed enzyme is the most thermostable UDP-glucose dehydrogenase so far described, with a half-life of 10 min at 90 °C. The enzyme retained its full activity after incubating in a pH range of 5.0–10.0 for 10 min at 80 °C. The temperature dependence of the kinetic parameters for this enzyme was examined at 37–70 °C. A decrease in Kms for UDP-glucose and NAD was observed with decreasing temperature. This resulted in the enzyme still retaining high catalytic efficiency (Vmax⁄Km) for the substrate and cofactor, even at 37 °C. These characteristics make the enzyme potentially useful for its application at a much lower temperature such as 37 °C than the optimum growth temperature of 100 °C for P. islandicum.