Protective effect of oleic acid against very long-chain fatty acid-induced apoptosis in peroxisome-deficient CHO cells

Very long-chain fatty acids (VLCFAs) are degraded exclusively in peroxisomes, as evidenced by the accumulation of VLCFAs in patients with certain peroxisomal disorders. Although accumulation of VLCFAs is considered to be associated with health issues, including neuronal degeneration, the mechanisms underlying VLCFAs-induced tissue degeneration remain unclear. Here, we report the toxic effect of VLCFA and protective effect of C18: 1 FA in peroxisome-deficient CHO cells. We examined the cytotoxicity of saturated and monounsaturated VLCFAs with chain-length at C20-C26, and found that longer and saturated VLCFA showed potent cytotoxicity at lower accumulation levels. Furthermore, the extent of VLCFA-induced toxicity was found to be associated with a decrease in cellular C18:1 FA levels. Notably, supplementation with C18:1 FA effectively rescued the cells from VLCFA-induced apoptosis without reducing the cellular VLCFAs levels, implying that peroxisome-deficient cells can survive in the presence of accumulated VLCFA, as long as the cells keep sufficient levels of cellular C18:1 FA. These results suggest a therapeutic potential of C18:1 FA in peroxisome disease and may provide new insights into the pharmacological effect of Lorenzo's oil, a 4:1 mixture of C18:1 and C22:1 FA.

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids


Degradation of glycosylinositol phosphoceramide during plant tissue homogenization


First crystal structure of an NADP+-dependent L-arginine dehydrogenase belonging to the μ-crystallin family

International Journal of Biological Macromolecules


Peroxisomes attenuate cytotoxicity of very long-chain fatty acids

One of the major functions of peroxisomes in mammals is oxidation of very long-chain fatty acids (VLCFAs). Genetic defects in peroxisomal β-oxidation result in the accumulation of VLCFAs and lead to a variety of health problems, such as demyelination of nervous tissues. However, the mechanisms by which VLCFAs cause tissue degeneration have not been fully elucidated. Recently, we found that the addition of small amounts of isopropanol can enhance the solubility of saturated VLCFAs in an aqueous medium. In this study, we characterized the biological effect of extracellular VLCFAs in peroxisome-deficient Chinese hamster ovary (CHO) cells, neural crest-derived pheochromocytoma cells (PC12), and immortalized adult Fischer rat Schwann cells (IFRS1) using this solubilizing technique. C20:0 FA was the most toxic of the C16-C26 FAs tested in all cells. The basis of the toxicity of C20:0 FA was apoptosis and was observed at 5 μM and 30 μM in peroxisome-deficient and wild-type CHO cells, respectively. The sensitivity of wild-type CHO cells to cytotoxic C20:0 FA was enhanced in the presence of a peroxisomal β-oxidation inhibitor. Further, a positive correlation was evident between cell toxicity and the extent of intracellular accumulation of toxic FA. These results suggest that peroxisomes are pivotal in the detoxification of apoptotic VLCFAs by preventing their accumulation.


Nonspecific phospholipase C3 of radish has phospholipase D activity toward glycosylinositol phosphoceramide

Glycosylinositol phosphoceramide (GIPC) is a major sphingolipid in the plasma membranes of plants. Previously, we found an enzyme activity that produces phytoceramide 1-phosphate (PC1P) by hydrolysis of the D position of GIPC in cabbage and named this activity as GIPC-phospholipase D (PLD). Here, we purified GIPC-PLD by sequential chromatography from radish roots. Peptide mass fingerprinting analysis revealed that the potential candidate for GIPC-PLD protein was nonspecific phospholipase C3 (NPC3), which has not been characterized as a PLD. The recombinant NPC3 protein obtained by heterologous expression system in Escherichia coli produced PC1P from GIPC and showed essentially the same enzymatic properties as those we characterized as GIPC-PLD in cabbage, radish and Arabidopsis thaliana. From these results, we conclude that NPC3 is one of the enzymes that degrade GIPC.


Crystal structure of a novel type of ornithine δ-aminotransferase from the hyperthermophilic archaeon Pyrococcus horikoshii

Ornithine δ-aminotransferase (Orn-AT) activity was detected for the enzyme annotated as a γ-aminobutyrate aminotransferase encoded by PH1423 gene from Pyrococcus horikoshii OT-3. Crystal structures of this novel archaeal ω-aminotransferase were determined for the enzyme in complex with pyridoxal 5'-phosphate (PLP), in complex with PLP and l-ornithine (l-Orn), and in complex with N-(5'-phosphopyridoxyl)-l-glutamate (PLP-l-Glu). Although the sequence identity was relatively low (28%), the main-chain coordinates of P. horikoshii Orn-AT monomer showed notable similarity to those of human Orn-AT. However, the residues recognizing the α-amino group of l-Orn differ between the two enzymes. In human Orn-AT, Tyr55 and Tyr85 recognize the α-amino group, whereas the side chains of Thr92* and Asp93*, which arise from a loop in the neighboring subunit, form hydrogen bonds with the α-amino group of the substrate in P. horikoshii enzyme. Site-directed mutagenesis suggested that Asp93* plays critical roles in maintaining high affinity for the substrate. This study provides new insight into the substrate binding of a novel type of Orn-AT. Moreover, the structure of the enzyme with the reaction-intermediate analogue PLP-l-Glu bound provides the first structural evidence for the "Glu switch" mechanism in the dual substrate specificity of Orn-AT.


Characterization of uptake and metabolism of very long-chain fatty acids in peroxisome-deficient CHO cells

Fatty acids (FAs) longer than C20 are classified as very long-chain fatty acids (VLCFAs). Although biosynthesis and degradation of VLCFAs are important for the development and integrity of the myelin sheath, knowledge on the incorporation of extracellular VLCFAs into the cells is limited due to the experimental difficulty of solubilizing them. In this study, we found that a small amount of isopropanol solubilized VLCFAs in aqueous medium by facilitating the formation of the VLCFA/albumin complex. Using this solubilizing technique, we examined the role of the peroxisome in the uptake and metabolism of VLCFAs in Chinese hamster ovary (CHO) cells. When wild-type CHO cells were incubated with saturated VLCFAs (S-VLCFAs), such as C23:0 FA, C24:0 FA, and C26:0 FA, extensive uptake was observed. Most of the incorporated S-VLCFAs were oxidatively degraded without acylation into cellular lipids. In contrast, in peroxisome-deficient CHO cells uptake of S-VLCFAs was marginal and oxidative metabolism was not observed. Extensive uptake and acylation of monounsaturated (MU)-VLCFAs, such as C24:1 FA and C22:1 FA, were observed in both types of CHO cells. However, oxidative metabolism was evident only in wild-type cells. Similar manners of uptake and metabolism of S-VLCFAs and MU-VLCFAs were observed in IFRS1, a Schwan cell-derived cell line. These results indicate that peroxisome-deficient cells limit intracellular S-VLCFAs at a low level by halting uptake, and as a result, peroxisome-deficient cells almost completely lose the clearance ability of S-VLCFAs accumulated outside of the cells.


Massspectrometric analysis of sphingomyelin with N-alfa-hydroxy fatty acyl residue in mouse tissues


Characterization of novel moderate-substrate specificity amino acid racemase from the hyperthermophilic archaeon Thermococcus litoralis.

The amino acid sequence of the OCC_10945 gene product from the hyperthermophilic archaeon Thermococcus litoralis DSM5473, originally annotated as γ-aminobutyrate aminotransferase, is highly similar to that of the uncharacterized pyridoxal 5'-phosphate (PLP)-dependent amino acid racemase from Pyrococcus horikoshii. The OCC_10945 enzyme was successfully overexpressed in Escherichia coli by 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.

Bioscience, Biotechnology, and Biochemistry


Isolation of glycosylinositol phosphoceramide and phytoceramide 1-phosphate in plants and their chemical stabilities.

Glycosylinositol phosphoceramide (GIPC) is a sphingophospholipid in plants. Recently, we identified that GIPC is hydrolyzed to phytoceramide 1-phosphate (PC1P) by an uncharacterized phospholipase D activity following homogenization of certain plant tissues. We now developed methods for isolation of GIPC and PC1P from plant tissues and characterized their chemical stabilities. Hydrophilic solvents, namely a lower layer of a mixed solvent system consisting of isopropanol/hexane/water (55:20:25, v/v/v) was efficient solvent for extraction and eluent in column chromatography. GIPC was isolated by Sephadex column chromatography followed by TLC. A conventional method, such as the Bligh and Dyer method, was applicable for PC1P extraction. Specifically, PC1P was isolated by TLC following mild alkali treatment of lipid extracts of plants. The yields of GIPC and PC1P in our methods were both around 50-70%. We found that PC1P is tolerant against heat (up to 125 °C), strong acid (up to 10 M HCl), and mild alkali (0.1 M KOH). In contrast, significant degradation of GIPC occurred at 100 °C and 1.0 M HCl treatment, suggesting the instability of the inositol glycan moiety in these conditions. These data will be useful for further biochemical and nutritional studies on these sphingolipids.

Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences


FAD-dependent dehydrogenase complexes and PLP-dependent amino acid racemases from hyperthermophilic archaea

超好熱アーキアのFAD依存性脱水素酵素複合体及びPLP依存性アミノ酸ラセマーゼの研究

We have paid much attention to screen and characterize enzymes from hyperthermophiles for analysis of their metabolic pathways of amino acids and utilization of the enzymes as the functional element for biosensor. In this review, we introduce two kinds of enzymes, FAD-dependent L-proline dehydrogenase complex and PLP-dependent amino acid racemase, from hyperthermophilic archaea. Two different FAD-dependent L-proline dehydrogenase activities were detected in the screening process of stable dye-linked dehydrogenase as specific element for electrochemical biosensor. The first enzyme found in Thermococcus profundus formed α β γ δ structure with not only proline dehydrogenase activity but also NADH dehydrogenase activity. We have succeeded in the construction of DNA sensing system using this enzyme. The second one from Pyrococcus horikoshii formed α4β4 structure and had FAD, FMN, ATP and Fe as cofactors. On the other hand, PLP-dependent amino acid racemase with broad substrate specificity was found in the process of investigation into D-amino acid utilization in the growth of P. horikoshii. Furthermore, we found another amino acid racemase specific for Ala and Ser by functional analysis of the homologs of the amino acid racemase. Because the genes of both dehydrogenases and racemases are widely conserved in the genomes of Pyrococcus and Thermococcus species, these enzymes may play important roles in the amino acid metabolisms.

著者らはこれまで，アミノ酸代謝経路の分析やバイオセンサー素子としての酵素の応用利用のために，超好熱菌由来酵素のスクリーニングや機能解析を進めてきた．この総合論文では，これまでに見出した2種類の酵素，FAD依存性L-プロリン脱水素酵素複合体とPLP依存性アミノ酸ラセマーゼについて紹介する．まず，電気化学的バイオセンサー用の機能性素子として利用可能な安定な色素依存性脱水素酵素をスクリーニングする過程で2種類の異なるL-プロリン脱水素酵素活性を見出した．最初に見出したThermococcus profundus由来の酵素はαβγδ構造の複合体酵素であり，L-プロリン脱水素酵素活性だけでなく，NADH脱水素酵素活性も示した．この酵素を用いたDNAセンシングシステムの構築にも成功している．Pyrococcus horikoshiiから見出した第2の酵素もα4β4構造の複合体酵素であり，補因子としてFAD，FMN，ATP，鉄を有していた．一方で，低基質特異性を示すPLP依存性アミノ酸ラセマーゼはP. horikoshiiの増殖におけるD-アミノ酸資化性を調査する過程で見つかった．さらに著者らは，ホモログ酵素の機能解析によってAlaとSerに特異的なアミノ酸ラセマーゼも見出した．これらの脱水素酵素やラセマーゼは両方とも，Thermococcales目超好熱アーキアに広く保存されており，アミノ酸代謝において重要な役割を担っていると考えられる．


Quantitative Analysis of Glycosylinositol Phosphoceramide and Phytoceramide 1-Phosphate in Vegetables

Previously, we found an unidentified sphingolipid in cabbage, and determined it as phytoceramide 1-phosphate (PC1P). PC1P is found to be produced from glycosylinositol phosphoceramide (GIPC) by the action of phospholipase D (PLD) activity. Although GIPC is abundant sphingolipid, especially in cruciferous vegetables, amount of daily intake, digestibility and nutritional activity of GIPC are not well understood. Here, we investigated amounts of GIPC and PC1P in vegetables. GIPC was found in all vegetables examined (13 kinds) at levels 3-20 mg/100 g (wet weight). On the other hand, PC1P was present in limited vegetables which show higher GIPC-PLD activity, such as inner cabbage leaves (5.2 mg/100 g). Because PC1P is formed during homogenization by activated GIPC-PLD, level of PC1P in boiled cabbage leaves was very low. Although digestibility of GIPC is unknown at present, a portion of dietary GIPC is considered to be converted to PC1P during mastication by plant-derived GIPC-PLD activity in some vegetables.

Journal of Nutritional Science and Vitaminology


Glycosylinositol phosphoceramide-specific phospholipase D activity catalyzes transphosphatidylation

Glycosylinositol phosphoceramide (GIPC) is the most abundant sphingolipid in plants and fungi. Recently, we detected GIPC-specific phospholipase D (GIPC-PLD) activity in plants. Here, we found that GIPC-PLD activity in young cabbage leaves catalyzes transphosphatidylation. The available alcohol for this reaction is a primary alcohol with a chain length below C4. Neither secondary alcohol, tertiary alcohol, choline, serine nor glycerol serves as an acceptor for transphosphatidylation of GIPC-PLD. We also found that cabbage GIPC-PLD prefers GIPC containing two sugars. Neither inositol phosphoceramide, mannosylinositol phosphoceramide nor GIPC with three sugar chains served as substrate. GIPC-PLD will become a useful catalyst for modification of polar head group of sphingophospholipid.


A novel PLP-dependent Alanine/Serine racemase from the hyperthermophilic archaeon Pyrococcus horikoshii OT-3

We recently identified and characterized a novel broad substrate specificity amino acidracemase (BAR) from the hyperthermophilic archaeon Pyrococcus horikoshii OT-3.Three genes, PH0782, PH1423, and PH1501, encoding homologs exhibiting about45% sequence identity with BAR were present in the P. horikoshii genome. In this study,we detected pyridoxal 50-phosphate (PLP)-dependent amino acid racemase activity inthe protein encoded by PH0782. The enzyme showed activity toward Ala, Ser, Thr,and Val, but the catalytic efficiency with Thr or Val was much lower than with Ala orSer. The enzyme was therefore designated Ala/Ser racemase (ASR). Like BAR, ASRwas highly stable at high temperatures and over a wide range of pHs, though itshexameric structure differed from the dimeric structure of BAR. No activity was detectedin K291A or D234A ASR mutants. This suggests that, as in Ile 2-epimerase (ILEP) fromLactobacillus buchneri JCM1115, these residues are involved in Schiff base formationand substrate interaction, respectively. Unlike BAR, enhanced ASR activity was notdetected in P. horikoshii cells cultivated in the presence of D-Ala or D-Ser. This is thefirst description of a PLP-dependent fold type I ASR in archaea.


First characterization of an archaeal amino acid racemase with broad substrate specificity from the hyperthermophile Pyrococcus horikoshii OT-3 induced by D-amino acids

A novel amino acid racemase with broad substrate specificity (BAR) was recently isolated from the hyperthermophilic archaeon Pyrococcus horikoshii OT-3. Characterization of this enzyme has been difficult, however, because the recombinant enzyme is produced mainly as an inclusion body in Escherichia coli. In this study, expression of the recombinant protein into the soluble fraction was markedly improved by co-expression with chaperone molecules. The purified enzyme retained its full activity after incubation at 80°C for at least 2h in buffer (pH 710), making this enzyme the most thermostable amino acid racemase so far known. Besides the nine amino acids containing hydrophobic and aromatic amino acids previously reported (Kawakami etal., Amino Acids, 47, 15791587, 2015), the enzyme exhibited substantial activity toward Thr (about 42% of relative activity toward Phe) and showed no activity toward Arg, His, Gln, and Asn. The substrate specificity of this enzyme thus differs markedly from those of other known amino acid racemases. In particular, the high reaction rate with Trp and Tyr, in addition to Leu, Met and Phe as substrates is a noteworthy feature of this enzyme. The high reactivity toward Trp and Tyr, as well as extremely high thermostability, is likely a major advantage of using BAR for biochemical conversion of these aromatic amino acids.

Journal of Bioscience and Bioengineering


Screening and analysis of edible seaweeds in the ability to adsorb Shiga toxin.


First characterization of extremely halophilic 2-deoxy-D-ribose-5-phosphate aldolase

2-Deoxy-d-ribose-5-phosphate aldolase (DERA) catalyzes the aldol reaction between two aldehydes and is thought to be a potential biocatalyst for the production of a variety of stereo-specific materials. A gene encoding DERA from the extreme halophilic archaeon, Haloarcula japonica, was overexpressed in Escherichia coli. The gene product was successfully purified, using procedures based on the protein's halophilicity, and characterized. The expressed enzyme was stable in a buffer containing 2 M NaCl and exhibited high thermostability, retaining more than 90% of its activity after heating at 70 °C for 10 min. The enzyme was also tolerant to high concentrations of organic solvents, such as acetonitrile and dimethylsulfoxide. Moreover, H. japonica DERA was highly resistant to a high concentration of acetaldehyde and retained about 35% of its initial activity after 5-h' exposure to 300 mM acetaldehyde at 25 °C, the conditions under which E. coli DERA is completely inactivated. The enzyme exhibited much higher activity at 25 °C than the previously characterized hyperthermophilic DERAs (Sakuraba et al., 2007). Our results suggest that the extremely halophilic DERA has high potential to serve as a biocatalyst in organic syntheses. This is the first description of the biochemical characterization of a halophilic DERA.


Identification of a novel amino acid racemase from a hyperthermophilic archaeon Pyrococcus horikoshii OT-3 induced by D-amino acids

To date, there have been few reports analyzing the amino acid requirement for growth of hyperthermophilic archaea. We here found that the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 requires Thr, Leu, Val, Phe, Tyr, Trp, His and Arg in the medium for growth, and shows slow growth in medium lacking Met or Ile. This largely corresponds to the presence, or absence, of genes related to amino acid biosynthesis in its genome, though there are exceptions. The amino acid requirements were dramatically lost by addition of D-isomers of Met, Leu, Val, allo-Ile, Phe, Tyr, Trp and Arg. Tracer analysis using (14)C-labeled D-Trp showed that D-Trp in the medium was used as a protein component in the cells, suggesting the presence of D-amino acid metabolic enzymes. Pyridoxal 5'-phosphate (PLP)-dependent racemase activity toward Met, Leu and Phe was detected in crude extract of P. horikoshii and was enhanced in cells grown in the medium supplemented with D-amino acids, especially D-allo-Ile. The gene encoding the racemase was narrowed down to one open reading frame on the basis of enzyme purification from P. horikoshii cells, and the recombinant enzyme exhibited PLP-dependent racemase activity toward several amino acids, including Met, Leu and Phe, but not Pro, Asp or Glu. This is the first report showing the presence in a hyperthermophilic archaeon of a PLP-dependent amino acid racemase with broad substrate specificity that is likely responsible for utilization of D-amino acids for growth.


Identification of catalytic residues of a very large NAD-glutamate dehydrogenase from Janthinobacterium lividum by site-directed mutagenesis

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 HgCl2 functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl2 was 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.

Bioscience, Biotechnology, and Biochemistry


Comparative analysis of the catalytic components in the archaeal dye-linked L-proline dehydrogenase complexes.

Two types of hetero-oligomeric dye-linked L-proline dehydrogenases (44 and types) are expressed in the hyperthermophilic archaea belonging to Thermococcales. In both enzymes, the subunit (PDH) is responsible for catalyzing L-proline dehydrogenation. The genes encoding the two enzyme types form respective clusters that are completely conserved among Pyrococcus and Thermococcus strains. To compare the enzymatic properties of PDHs from 44- and -type enzyme complexes, eight PDHs (four of each type) from Pyrococcus furiosus DSM3638, Pyrococcus horikoshii OT-3, Thermococcus kodakaraensis KOD1 JCM12380 and Thermococcus profundus DSM9503 were expressed in Escherichia coli cells and purified to homogeneity using one-step Ni-chelating chromatography. The 44-type PDHs showed greater thermostability than most of the -type PDHs: the former retained more than 80 % of their activity after heating at 70 °C for 20 min, while the latter showed different thermostabilities under the same conditions. In addition, the 44-type PDHs utilized ferricyanide as the most preferable electron acceptor, whereas -type PDHs preferred 2, 6-dichloroindophenol, with one exception. These results indicate that the differences in the enzymatic properties of the PDHs likely reflect whether they were from an - or 44-type complex, though the wider divergence observed within -type PDHs based on the phylogenetic analysis may also be responsible for their inconsistent enzymatic properties. By contrast, differences in the kinetic parameters among the PDHs did not reflect the complex type. Interestingly, the k cat value for free 44-type PDH from P. horikoshii was much larger than the value for the same subunit within the 44-complex. This indicates that the isolated PDH could be a useful element for an electrochemical system for detection of L-proline.


Crystal structure of novel dye-linked L-proline dehydrogenase from hyperthermophilic archaeon Aeropyrum pernix.

Two types of dye-linked L-proline dehydrogenase (PDH1, α4β4-type hetero-octamer, and PDH2, αβγδ-type heterotetramer) have been identified so far in hyperthermophilic archaea. Here, we report the crystal structure of a third type of L-proline dehydrogenase, found in the aerobic hyperthermophilic archaeon Aeropyrum pernix, whose structure (homodimer) is much simpler than those of previously studied L-proline dehydrogenases. The structure was determined at a resolution of 1.92 Å. The asymmetric unit contained one subunit, and a crystallographic 2-fold axis generated the functional dimer. The overall fold of the subunit showed similarity to that of the PDH1 β-subunit, which is responsible for catalyzing L-proline dehydrogenation. However, the situation at the subunit-subunit interface of the A. pernix enzyme was totally different from that in PDH1. The presence of additional surface elements in the A. pernix enzyme contributes to a unique dimer association. Moreover, the C-terminal Leu(428), which is provided by a tail extending from the FAD-binding domain, shielded the active site, and an L-proline molecule was entrapped within the active site cavity. The K(m) value of a Leu(428) deletion mutant for L-proline was about 800 times larger than the K(m) value of the wild-type enzyme, although the k(cat) values did not differ much between the two enzymes. This suggests the C-terminal Leu(428) is not directly involved in catalysis, but it is essential for maintaining a high affinity for the substrate. This is the first description of an LPDH structure with L-proline bound, and it provides new insight into the substrate binding of LPDH.


Expression, purification, crystallization and preliminary X-ray diffraction analysis of a galactose 1-phosphate uridylyltransferase from the hyperthermophilic archaeon Pyrobaculum aerophilum.

A galactose 1-phosphate uridylyltransferase from the hyperthermophilic archaeon Pyrobaculum aerophilum was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 8000 as the precipitant. The crystals belonged to the tetragonal space group P4(1), with unit-cell parameters a = b = 73.3, c = 126.1 Å, and diffracted to 2.73 Å resolution on beamline BL5A at the Photon Factory. The overall R(merge) was 7.3% and the data completeness was 99.8%.

Acta Crystallographica. Section F, Structural Biology and Crystallization Communications


L-proline dehydrogenases in hyperthermophilic archaea: distribution, function, structure, and application.

Dye-linked L-proline dehydrogenase (ProDH) catalyzes the oxidation of L-proline to ∆(1)-pyrroline-5-carboxylate (P5C) in the presence of artificial electron acceptors. The enzyme is known to be widely distributed in bacteria and eukarya, together with nicotinamide adenine dinucleotide (phosphate)-dependent P5C dehydrogenase, and to function in the metabolism of L-proline to L-glutamate. In addition, over the course of the last decade, three other types of ProDH with molecular compositions completely different from previously known ones have been identified in hyperthermophilic archaea. The first is a heterotetrameric αβγδ-type ProDH, which exhibits both ProDH and reduced nicotinamide adenine dinucleotide dehydrogenase activity and includes two electron transfer proteins. The second is a heterooctameric α(4)β(4)-type ProDH, which uses flavin adenine dinucleotide, flavin mononucleotide, adenosine triphosphate, and Fe as cofactors and creates a new electron transfer pathway. The third is a recently identified homodimeric ProDH, which exhibits the greatest thermostability among these archaeal ProDHs. This minireview focuses on the functional and structural properties of these three types of archaeal ProDH and their distribution in archaea. In addition, we will describe the specific application of hyperthermostable ProDH for use in a biosensor and for DNA sensing.


Crystallization and preliminary X-ray analysis of a dye-linked D-lactate dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix.

A dye-linked D-lactate dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol 8000 as the precipitant. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 63.4, b = 119.4, c = 70.2 Å, β = 112.0°, and diffracted to 2.0 Å resolution on the BL26B1 beamline at SPring-8. The overall R(merge) was 4.5% and the completeness was 99.8%.


Electrochemical behavior of dye-linked L-proline dehydrogenase on glassy carbon electrodes modified by multi-walled carbon nanotubes.

A glassy carbon electrode (GC) was modified by multi-walled carbon nanotubes (MWCNTs). The modified electrode showed a pair of redox peaks that resulted from the oxygen-containing functional groups on the nanotube surface. A recombinant thermostable dye-linked L-proline dehydrogenase (L-proDH) from hyperthermophilic archaeon (Thermococcus profundus) was further immobilized by physical adsorption. The modified electrode (GC/MWCNTs/L-proDH) exhibited an electrocatalytic signal for L-proline compared to bare GC, GC/L-proDH and GC/MWCNTs electrodes, which suggested that the presence of MWCNTs efficiently enhances electron transfer between the active site of enzyme and electrode surface. The immobilized L-proDH showed a typical Michaelis-Menten catalytic response with lower apparent constant.


The unique kinetic behavior of the very large NAD-dependent glutamate dehydrogenase from Janthinobacterium lividum.

The kinetics of a very large NAD-dependent glutamate dehydrogenase from Janthinobacterium lividum showed positive cooperativity toward alpha-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 S(0.5) values for alpha-ketoglutarate and NADH with the addition of L-aspartate, and the kinetic response for alpha-ketoglutarate changed completely to a typical Michaelis-Menten type in the presence of 10 mM L-aspartate.


Refolding, characterization and crystal structure of (S)-malate dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix.

Tartrate oxidation activity was found in the crude extract of an aerobic hyperthermophilic archaeon Aeropyrum pernix, and the enzyme was identified as (S)-malate dehydrogenase (MDH), which, when produced in Escherichia coli, was mainly obtained as an inactive inclusion body. The inclusion body was dissolved in 6 M guanidine-HCl and gradually refolded to the active enzyme through dilution of the denaturant. The purified recombinant enzyme consisted of four identical subunits with a molecular mass of about 110 kDa. NADP was preferred as a coenzyme over NAD for (S)-malate oxidation and, unlike MDHs from other sources, this enzyme readily catalyzed the oxidation of (2S,3S)-tartrate and (2S,3R)-tartrate. The tartrate oxidation activity was also observed in MDHs from the hyperthermophilic archaea Methanocaldococcus jannaschii and Archaeoglobus fulgidus, suggesting these hyperthermophilic MDHs loosely bind their substrates. The refolded A. pernix MDH was also crystallized, and the structure was determined at a resolution of 2.9 A. Its overall structure was similar to those of the M. jannaschii, Chloroflexus aurantiacus, Chlorobium vibrioforme and Cryptosporidium parvum [lactate dehydrogenase-like] MDHs with root-mean-square-deviation values between 1.4 and 2.1 A. Consistent with earlier reports, Ala at position 53 was responsible for coenzyme specificity, and the next residue, Arg, was important for NADP binding. Structural comparison revealed that the hyperthermostability of the A. pernix MDH is likely attributable to its smaller cavity volume and larger numbers of ion pairs and ion-pair networks, but the molecular strategy for thermostability may be specific for each enzyme.

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics


Structure of a D-tagatose 3-epimerase-related protein from the hyperthermophilic bacterium Thermotoga maritima.

The crystal structure of a D-tagatose 3-epimerase-related protein (TM0416p) encoded by the hypothetical open reading frame TM0416 in the genome of the hyperthermophilic bacterium Thermotoga maritima was determined at a resolution of 2.2 A. The asymmetric unit contained two homologous subunits and a dimer was generated by twofold symmetry. The main-chain coordinates of the enzyme monomer proved to be similar to those of D-tagatose 3-epimerase from Pseudomonas cichorii and D-psicose 3-epimerase from Agrobacterium tumefaciens; however, TM0416p exhibited a unique solvent-accessible substrate-binding pocket that reflected the absence of an alpha-helix that covers the active-site cleft in the two aforementioned ketohexose 3-epimerases. In addition, the residues responsible for creating a hydrophobic environment around the substrate in TM0416p differ entirely from those in the other two enzymes. Collectively, these findings suggest that the substrate specificity of TM0416p is likely to differ substantially from those of other D-tagatose 3-epimerase family enzymes.


A novel flavin adenine dinucleotide (FAD) containing d-lactate dehydrogenase from the thermoacidophilic crenarchaeota Sulfolobus tokodaii strain 7: purification, characterization and expression in Escherichia coli.

Dye-linked D-lactate dehydrogenase activity was found in the crude extract of a continental thermoacidophilic crenarchaeota, Sulfolobus tokodaii strain 7, and was purified 375-fold through four sequential chromatography steps. With a molecular mass of about 93 kDa, this enzyme was a homodimer comprised of identical subunits with molecular masses of about 48 kDa. The enzyme retained its full activity after incubation at 80 degrees C for 10 min and after incubation at pHs ranging from 6.5 to 10.0 for 30 min at 50 degrees C. The preferred substrate for this enzyme was D-lactate, with 2,6-dichloroindophenol serving as the electron acceptor. Using high-performance liquid chromatography (HPLC), the enzyme's prosthetic group was determined to be flavin adenine dinucleotide (FAD). Its N-terminal amino acid sequence was MLEGIEYSQGEEREDFVGFKIKPKI. Using that sequence and previously reported genome information, the gene encoding the enzyme (ST0649) was identified. It was subsequently cloned and expressed in Escherichia coli and found to encode a polypeptide of 440 amino acids with a calculated molecular weight of 49,715. The amino acid sequence of this dye-linked D-lactate dehydrogenase showed higher homology (39% identity) with that of a glycolate oxidase subunit homologue from Archaeoglobus fulgidus, but less similarity (32% identity) to D-lactate dehydrogenase from A. fulgidus. Taken together, our findings indicate that the dye-linked D-lactate dehydrogenase from S. tokodaii is a novel type of FAD containing D-lactate dehydrogenase.

Journal of Bioscience and Bioengineering


Sequential aldol condensation catalyzed by hyperthermophilic 2-deoxy-D-ribose-5-phosphate aldolase.

Genes encoding 2-deoxy-d-ribose-5-phosphate aldolase (DERA) homologues from two hyperthermophiles, the archaeon Pyrobaculum aerophilum and the bacterium Thermotoga maritima, were expressed individually in Escherichia coli, after which the structures and activities of the enzymes produced were characterized and compared with those of E. coli DERA. To our surprise, the two hyperthermophilic DERAs showed much greater catalysis of sequential aldol condensation using three acetaldehydes as substrates than the E. coli enzyme, even at a low temperature (25 degrees C), although both enzymes showed much less 2-deoxy-d-ribose-5-phosphate synthetic activity. Both the enzymes were highly resistant to high concentrations of acetaldehyde and retained about 50% of their initial activities after a 20-h exposure to 300 mM acetaldehyde at 25 degrees C, whereas the E. coli DERA was almost completely inactivated after a 2-h exposure under the same conditions. The structure of the P. aerophilum DERA was determined by X-ray crystallography to a resolution of 2.0 A. The main chain coordinate of the P. aerophilum enzyme monomer was quite similar to those of the T. maritima and E. coli enzymes, whose crystal structures have already been solved. However, the quaternary structure of the hyperthermophilic enzymes was totally different from that of the E. coli DERA. The areas of the subunit-subunit interface in the dimer of the hyperthermophilic enzymes are much larger than that of the E. coli enzyme. This promotes the formation of the unique dimeric structure and strengthens the hydrophobic intersubunit interactions. These structural features are considered responsible for the extremely high stability of the hyperthermophilic DERAs.


Gene cloning and characterization of the very large NAD-dependent L-glutamate dehydrogenase from the psychrophile Janthinobacterium lividum, isolated from cold soil

NAD-dependent l-glutamate dehydrogenase (NAD-GDH) activity was detected in cell extract from the psychrophile Janthinobacterium lividum UTB1302, which was isolated from cold soil and purified to homogeneity. The native enzyme (1,065 kDa, determined by gel filtration) is a homohexamer composed of 170-kDa subunits (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Consistent with these findings, gene cloning and sequencing enabled deduction of the amino acid sequence of the subunit, which proved to be comprised of 1,575 amino acids with a combined molecular mass of 169,360 Da. The enzyme from this psychrophile thus appears to belong to the GDH family characterized by very large subunits, like those expressed by Streptomyces clavuligerus and Pseudomonas aeruginosa (about 180 kDa). The entire amino acid sequence of the J. lividum enzyme showed about 40% identity with the sequences from S. clavuligerus and P. aeruginosa enzymes, but the central domains showed higher homology (about 65%). Within the central domain, the residues related to substrate and NAD binding were highly conserved, suggesting that this is the enzyme's catalytic domain. In the presence of NAD, but not in the presence of NADP, this GDH exclusively catalyzed the oxidative deamination of l-glutamate. The stereospecificity of the hydride transfer to NAD was pro-S, which is the same as that of the other known GDHs. Surprisingly, NAD-GDH activity was markedly enhanced by the addition of various amino acids, such as l-aspartate (1,735%) and l-arginine (936%), which strongly suggests that the N- and/or C-terminal domains play regulatory roles and are involved in the activation of the enzyme by these amino acids.


Gene expression and characterization of 2-keto-3-deoxygluconate kinase, a key enzyme in the modified Entner-Doudoroff pathway of the aerobic and acidophilic hyperthermophile Sulfolobus tokodaii.

2-Keto-3-deoxygluconate kinase (KDGK) catalyzes the ATP-dependent phosphorylation of 2-keto-3-deoxygluconate, a key intermediate in the modified (semi-phosphorylative) Entner-Doudoroff (ED) glucose metabolic pathway. We identified the gene (ORF ID: ST2478) encoding KDGK in the hyperthermophilic archaeon Sulfolobus tokodaii based on the structure of a gene cluster in a genomic database and functionally expressed it in Escherichia coli. The expressed protein was purified from crude extract by heat treatment and two conventional column chromatography steps, and the partial amino acid sequence in the N-terminal region of the purified enzyme (MAKLIT) was identical to that obtained from the gene sequence. The purified enzyme was extremely thermostable and retained full activity after heating at 80 degrees C for 1 h. The enzyme utilized ATP or GTP, but not ADP or AMP, as a phosphoryl donor and 2-keto-3-deoxy-D-gluconate or 2-keto-D-gluconate as a phosphoryl acceptor. Divalent cations including Mg(2+), Co(2+), Ni(2+), Zn(2+) or Mn(2+) were required for activity, and the apparent Km values for KDG and ATP at 50 degrees C were 0.027 mM and 0.057 mM, respectively. The presence of KDGK means that the hyperthermophilic archaeon S. tokodaii metabolizes glucose via both modified (semi-phosphorylative) and non-phosphorylative ED pathways.


The first archaeal l-aspartate dehydrogenase from the hyperthermophile Archaeoglobus fulgidus: gene cloning and enzymological characterization

A gene encoding an L-aspartate dehydrogenase (EC 1.4.1.21) homologue was identified in the anaerobic hyperthermophilic archaeon Archaeoglobus fulgidus. After expression in Escherichia coli, the gene product was purified to homogeneity, yielding a homodimeric protein with a molecular mass of about 48 kDa. Characterization revealed the enzyme to be a highly thermostable L-aspartate dehydrogenase, showing little loss of activity following incubation for 1 h at up to 80 degrees C. The optimum temperature for L-aspartate dehydrogenation was about 80 degrees C. The enzyme specifically utilized L-aspartate as the electron donor, while either NAD or NADP could serve as the electron acceptor. The Km values for L-aspartate were 0.19 and 4.3 mM when NAD or NADP, respectively, served as the electron acceptor. The Km values for NAD and NADP were 0.11 and 0.32 mM, respectively. For reductive amination, the Km values for oxaloacetate, NADH and ammonia were 1.2, 0.014 and 167 mM, respectively. The enzyme showed pro-R (A-type) stereospecificity for hydrogen transfer from the C4 position of the nicotinamide moiety of NADH. This is the first report of an archaeal L-aspartate dehydrogenase. Within the archaeal domain, homologues of this enzyme occurred in many Methanogenic species, but not in Thermococcales or Sulfolobales species.


L-Proline sensor based on layer-by-layer immobilization of thermostable dye-linked L-proline dehydrogenase and polymerized mediator

L-Proline sensor has been fabricated through multilayer assembly of recombinant dye-linked L-proline dehydrogenase (L-proDH) originally from hyperthermophilic archaeon (Therococcus profundus) and polymerized mediator, poly(allylamine) ferrocene (PAA-Fc), on a gold electrode by electrostatic layer-by-layer alternate adsorption method. The characteristic redox peaks of ferrocene groups were obvious exhibited in cyclic voltammograms, and both anodic and cathodic peaks increased with PAA-Fc/L-proDH bilayer number which confirmed the formation of a multilayer structure. Further experiments showed that an electrode fabricated in this way catalyzed the oxidation of L-proline. It suggested that polymerized mediator could transfer electrons between electrode surface and immobilized L-proDH. Ampeormetric experiments showed that the current response to L-proline increased with PAA-Fc/L-proDH bilayer number. The stability of the sensor was measured, and it kept a 40% relative response after 30 days storage in buffer under 4 °C.

Science and Technology of Advanced Materials


Intersubunit Interaction Induced by Subunit Rearrangement Is Essential for the Catalytic Activity of the Hyperthermophilic Glutamate Dehydrogenase from Pyrobaculum islandicum.

The specific activity of recombinant Pyrobaculum islandicum glutamate dehydrogenase (pis-GDH) expressed in Escherichia coli is much lower than that of the native enzyme. However, when the recombinant enzyme is heated at 90 degrees C or exposed to 5 M urea, the activity increases to a level comparable to that of the native enzyme. Small-angle X-ray scattering measurements revealed that the radius of gyration (R(g,z)) of the hexameric recombinant enzyme was reduced to 47 A from 55 A by either heat or urea, and that the final structure of the active enzyme is the same irrespective of the mechanism of activation. Activation was accompanied by a shift in the peaks of the Kratky plot, though the molecular mass of the enzyme was unchanged. The activation-induced decline in R(g,z) followed first-order kinetics, indicating that activation of the enzyme involved a transition between two states, which was confirmed by singular-value decomposition analysis. When the low-resolution structure of the recombinant enzyme was restored using ab initio modeling, we found it to possess no point symmetry, whereas the heat-activated enzyme possessed 32-point symmetry. In addition, a marked increase in the fluorescence emission was observed with addition of ANS to the inactive recombinant enzyme but not the active forms, indicating that upon activation hydrophobic residues on the surface of the recombinant protein moved to the interior. Taken together, these data strongly suggest that subunit rearrangement, i.e., a change in the quaternary structure of the hexameric recombinant pis-GDH, is essential for activation of the enzyme.


Crystal Structure of a Novel FAD-, FMN-, and ATP-containing L-Proline Dehydrogenase Complex from Pyrococcus horikoshii

Two novel types of dye-linked L-proline dehydrogenase complex (PDH1 and PDH2) were found in a hyperthermophilic archaeon, Pyrococcus horikoshii OT3. Here we report the first crystal structure of PDH1, which is a heterooctameric complex (alphabeta)4 containing three different cofactors: FAD, FMN, and ATP. The structure was determined by x-ray crystallography to a resolution of 2.86 angstroms. The structure of the beta subunit, which is an L-proline dehydrogenase catalytic component containing FAD as a cofactor, was similar to that of monomeric sarcosine oxidase. On the other hand, the alpha subunit possessed a unique structure composed of a classical dinucleotide fold domain with ATP, a central domain, an N-terminal domain, and a Cys-clustered domain. Serving as a third cofactor, FMN was located at the interface between the alpha and beta subunits in a novel configuration. The observed structure suggests that FAD and FMN are incorporated into an electron transfer system, with electrons passing from the former to the latter. The function of ATP is unknown, but it may play a regulatory role. Although the structure of the alpha subunit differs from that of the beta subunit, except for the presence of an analogous dinucleotide domain with a different cofactor, the structural characteristics of PDH1 suggest that each represents a divergent enzyme that arose from a common ancestral flavoenzyme and that they eventually formed a complex to gain a new function. The structural characteristics described here reveal the PDH1 complex to be a unique diflavin dehydrogenase containing a novel electron transfer system.


L-Threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 --- gene cloning and enzymatic characterization

A gene encoding the L-threonine dehydrogenase homologue has been identified in a hyperthermophlic archaeon Pyrococcus horikoshii OT3 via genome sequencing. The gene was cloned and expressed in Escherichia coli. The purified enzyme from the recombinant E. coli was extremely thermostable; the activity was not lost after incubation at 100 degrees C for 20 min. The enzyme (molecular mass: 192 kDa) is composed of a tetrameric structure with a type of subunit (41 kDa). The enzyme is specific for NAD and utilizes L-threonine, L-serine and DL-threo-3-phenylserine as the substrate. The enzyme required divalent cations such as Zn(2+), Mn(2+) and Co(2+) for the activity, and contained one zinc ion/subunit. The K(m) values for L-threonine and NAD at 50 degrees C were 0.20 mM and 0.024 mM, respectively. Kinetic analyses indicated that the L-threonine oxidation reaction proceeds via a random mechanism with regard to the binding of L-threonine and NAD. The enzyme showed pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NADH. This is the first description of the characteristics of an L-threonine dehydrogenase from the archaea domain.


A second novel dye-linked L-proline dehydrogenase complex is present in the hyperthermophilic archaeon Pyrococcus horikoshii OT-3

Two distinguishable activity bands for dye-linked l-proline dehydrogenase (PDH1 and PDH2) were detected when crude extract of the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 was run on a polyacrylamide gel. After purification, PDH1 was found to be composed of two different subunits with molecular masses of 56 and 43 kDa, whereas PDH2 was composed of four different subunits with molecular masses of 52, 46, 20 and 8 kDa. The native molecular masses of PDH1 and PDH2 were 440 and 101 kDa, respectively, indicating that PDH1 has an alpha4beta4 structure, while PDH2 has an alphabetagammadelta structure. PDH2 was found to be similar to the dye-linked l-proline dehydrogenase complex from Thermococcus profundus, but PDH1 is a different type of enzyme. After production of the enzyme in Escherichia coli, high-performance liquid chromatography showed the PDH1 complex to contain the flavins FMN and FAD as well as ATP. Gene expression and biochemical analyses of each subunit revealed that the beta subunit bound FAD and exhibited proline dehydrogenase activity, while the alpha subunit bound ATP, but unlike the corresponding subunit in the T. profundus enzyme, it exhibited neither proline dehydrogenase nor NADH dehydrogenase activity. FMN was not bound to either subunit, suggesting it is situated at the interface between the alpha and beta subunits. A comparison of the amino-acid sequences showed that the ADP-binding motif in the alpha subunit of PDH1 clearly differs from that in the alpha subunit of PDH2. It thus appears that a second novel dye-linked l-proline dehydrogenase complex is produced in P. horikoshii.


First Archaeal Inorganic Polyphosphate/ATP-Dependent NAD Kinase, from Hyperthermophilic Archaeon Pyrococcus horikoshii --- Cloning, Expression, and Characterization

The gene (PH1074) encoding the NAD kinase of the hyperthermophilic archaeon Pyrococcus horikoshii was identified in the genome database, cloned, and functionally expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment at 90 degrees C for 20 min and one successive HiTrap affinity chromatography step. The purified enzyme was easily precipitated by dialysis against phosphate buffer without NaCl and imidazole and was usually stored in buffer containing 0.5 M NaCl and 0.5 M imidazole to avoid precipitation. The molecular mass of the active enzyme was determined to be 145 kDa by a gel filtration method, and the enzyme was composed of a tetramer of 37-kDa subunits. The archaeal enzyme utilized several nucleoside triphosphates, such as GTP, CTP, UTP, and ITP, as well as ATP and inorganic polyphosphates [poly(P)] as phosphoryl donors for NAD phosphorylation. The enzyme utilized poly(P)27 (the average length of the phosphoryl chain was 27) as the most active inorganic polyphosphate for NAD phosphorylation. Thus, this enzyme is categorized as an inorganic polyphosphate/ATP-dependent NAD kinase. The enzyme was the most thermostable NAD kinase found to date: its activity was not lost by incubation at 95 degrees C for 10 min. The enzyme showed classical Michaelis-Menten-type kinetics for NAD and ATP, but not for poly(P)27. The Km values for NAD were determined to be 0.30 and 0.40 mM when poly(P)27 and ATP, respectively, were used as the phosphoryl donors. The Km value for ATP was 0.29 mM, and the concentration of poly(P)27 which gave half of the maximum enzyme activity was 0.59 mM. The enzyme required several metal cations, such as Mg2+, Mn2+, or Ni2+, for its activity. The deduced amino acid sequence showed a low level of identity to those of E. coli ATP-dependent NAD kinase (31%) and the inorganic polyphosphate/ATP-dependent NAD kinase of Mycobacterium tuberculosis (29%). This is the first description of the characteristics of a poly(P)/ATP-dependent NAD kinase from a hyperthermophilic archaeon.


Oxidative stress response in an anaerobic hyperthermophilic archaeon --- presence of a functional peroxiredoxin in Pyrococcus horikoshii

嫌気性超好熱古細菌Pyrococcus horikoshiiに僅かな酸素ストレスを与えると，顕著に誘導されるタンパク質を見出し，これがペルオキシレドキシンであることを明らかにした．嫌気性菌が酸素ストレス応答タンパク質を持っており，これが菌体の酸素耐性に関与する可能性を示した．


Novel Archaeal Alanine:Glyoxylate Aminotransferase from Thermococcus litoralis

超好熱菌に初めてアラニングリオキシル酸アミノトランスフェラーゼを見出した．一次構造解析により系統樹を作製した結果，既知のアラニングリオキシル酸アミノトランスフェラーゼとは全く異なるグループに属することが判明した．本酵素は，芳香族アミノ酸トランスフェラーゼに近縁であるが，反応はアラニンとグリオキシル酸に特異的であった．本菌が本酵素やグリオキシル酸レダクターゼを含むグリオキシル酸代謝経路を持つ事を提唱した．


Gene and primary structures of dye-linked L-proline dehydrogenase from the hyperthermophilic archaeon Thermococcus profundus show the presence of a novel heterotetrameric amino acid dehydrogenase complex

超好熱アーキアThermococcus profundusに見出した新規色素依存性L-プロリン脱水素酵素の遺伝子クローニングに成功し，本酵素がユニークなヘテロテトラマー構造をとること，これらをコードする4種の遺伝子がオペロンを形成することを明らかにした．また，この異種サブユニットの共発現系の構築に成功した．本成果および関連研究により，第5回酵素応用シンポジウム研究奨励賞受賞．平成16年度医科学応用研究財団奨励金受賞．成果は日経バイオテク2004/6/21, pp19-20に掲載された．


The First Crystal Structure of Archaeal Aldolase: Unique Tetrameric Structure of 2-Deoxy-D-ribose-5-phosphate Aldolase from Hyperthermophilic Archaeon Aeropyrum pernix

古細菌のアルドラーゼに関して，初めてX線結晶構造解析に成功した．解析したデオキシリボース5-リン酸アルドラーゼは抗がん剤や抗ウイルス剤の合成中間体の生産に利用できるが，既知の酵素は不安定で産業への実用化が難しい．本酵素は非常に安定性が高く，実用面での有用性を示した．また，その安定性が本酵素のユニークな4次構造に由来することを提唱した．本研究により，平成15年度バイオインダストリー協会「グラント」奨励金受賞．


ADP-Dependent Glucokinase/Phosphofructokinase, a Novel Bifunctional Enzyme from the Hyperthermophilic Archaeon Methanococcus jannaschii

超好熱アーキアMethanococcus jannashiiに見出したグルコキナーゼおよびホスホフルクトキナーゼの両活性を持つ新規酵素について報告している．この酵素がグルコキナーゼおよびホスホフルクトキナーゼの起源であることを提唱した．本酵素の発見により，メタン菌による糖からのメタン生産が理論上可能であることを示した．生化学分野のトップジャーナルの1つであるJ. Biol. Chem.において，特に重要な発見を紹介するAccelerated Publicationに掲載された．本研究により平成14年度ビタミン学会奨励賞受賞．


Dye-linked D-Proline Dehydrogenase from Hyperthermophilic Archaeon Pyrobaculum islandicum Is a Novel FAD-dependent Amino Acid Dehydrogenase

超好熱アーキアPyrobaculum islandicumに初めて見出した新規色素依存性D-プロリン脱水素酵素に関して報告している．本酵素は従来報告されているD-アミノ酸脱水素酵素とは大きく構造や性質が異なることを明らかにした．D-アミノ酸の合成およびD-プロリンの定量用バイオセンサー素子としての有用性が期待できる．


L-Aspartate oxidase is present in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3: characteristics and role in the de novo biosynthesis of nicotinamide adenine dinucleotide proposed by genome sequencing

超好熱アーキアPyrococcus horikoshiiに嫌気性菌で初めて見出したL-アスパラギン酸オキシダーゼについて報告している．本酵素が嫌気条件下でも機能する色素依存性L-アスパラギン酸脱水素酵素反応も触媒することを見出し，NAD生合成系の初発酵素として機能していることを提唱した．本酵素に特徴的な脱水素酵素反応によるL-アスパラギン酸定量への応用が期待できる．本研究により平成14年度ビタミン学会奨励賞受賞．


Purification, Characterization, and Application of a Novel Dye-Linked L-Proline Dehydrogenase from a Hyperthermophilic Archaeon, Thermococcus profundus

超好熱アーキアThermococcus profundusに見出した新規色素依存性L-プロリン脱水素酵素について報告している．本酵素は新規色素依存性酵素の中では非常に安定性が高いことを明らかにした．果物やハチミツの品質管理マーカーであるL-プロリンの定量用バイオセンサー素子としての有用性を明らかにした．