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


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


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.


Catalytic properties and crystal structure of quinoprotein aldose sugar dehydrogenase from hyperthermophilic archaeon Pyrobaculum aerophilum

We identified a gene encoding a soluble quinoprotein glucose dehydrogenase homologue in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The enzyme was extremely thermostable, and the activity of the pyrroloquinoline quinone (PQQ)-bound holoenzyme was not lost after incubation at 100 degrees C for 10 min. The crystal structure of the enzyme was determined in both the apoform and as the PQQ-bound holoenzyme. The overall fold of the P. aerophilum enzyme showed significant similarity to that of soluble quinoprotein aldose sugar dehydrogenase (Asd) from E. coli. However, clear topological differences were observed in the two long loops around the PQQ-binding sites of the two enzymes. Structural comparison revealed that the hyperthermostability of the P. aerophilum enzyme is likely attributable to the presence of an extensive aromatic pair network located around a beta-sheet involving N- and C-terminal beta-strands.


A novel cytotoxic protein, Karatoxin, from the dorsal spines of the the redfin velvetfihi, Hypodytes rubripinnis


An amperometric D-amino acid biosensor prepared with a thermostable D-Proline dehydrogenase and a carbon nanotube-ionic liquid gel

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.


Development of a D-amino acids electrochemical sensor based on immobilization of thermostable D-Proline dehydrogenase within agar gel membrane

A novel biosensor for determination of d-amino acids (DAAs) in biological samples by using an electrode based on immobilization of a thermostable d-Proline dehydrogenase (d-Pro DH) within an agar gel membrane was developed. The electrode was simply prepared by spin-coating the agar solution with the d-Pro DH on a glassy carbon (GC) electrode. An electrocatalytic oxidation current of 2,6-dichloroindophenol (DCIP) was observed at -100mV vs. Ag/AgCl with the addition of 5 and 20mmolL(-1)d-proline. The current response and its relative standard deviation were 0.15muA and 7.6% (n=3), respectively, when it was measured in a pH 8.0 phosphate buffer solution containing 10mmolL(-1)d-proline and 0.5mmolL(-1) DCIP at 50 degrees C. The current response of d-proline increased with increase of the temperature of the sample solution up to 70 degrees C. The electrocatalytic response at the d-Pro DH/agar immobilized electrode subsequently maintained for 80 days. Finally, the d-Pro DH/agar immobilized electrode was applied to determination of DAAs in a human urine sample. The determined value of DAAs in the human urine and its R.S.D. were 1.39+/-0.12mmolL(-1) and 8.9% (n=3), respectively.


Structure of l-aspartate oxidase from the hyperthermophilic archaeon Sulfolobus tokodaii.

The crystal structure of the highly thermostable l-aspartate oxidase (LAO) from the hyperthermophilic archaeon Sulfolobus tokodaii was determined at a 2.09 A resolution. The factors contributing to the thermostability of the enzyme were analyzed by comparing its structure to that of Escherichia coli LAO. Like E. coli LAO, the S. tokodaii enzyme consists of three domains: an FAD-binding domain, an alpha+beta capping domain, and a C-terminal three-helix bundle. However, the situation of the linker between the FAD-binding domain and C-terminal three-helix bundle in S. tokodaii LAO is completely different from that in E. coli LAO, where the linker is situated near the FAD-binding domain and has virtually no interaction with the rest of the protein. In S. tokodaii LAO, this linker is situated near the C-terminal three-helix bundle and contains a beta-strand that runs parallel to the C-terminal strand. This results in the formation of an additional beta-sheet, which appears to reduce the flexibility of the C-terminal region. Furthermore, the displacement of the linker enables formation of a 5-residue ion-pair network between the FAD-binding and C-terminal domains, which strengthens the interdomain interactions. These features might be the main factors contributing to the high thermostability of S. tokodaii LAO.

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics


Molecular and functional characterization of D-3-phosphoglycerate dehydrogenase in the serine biosynthetic pathway of the hyperthermophilic archaeon Sulfolobus tokodaii

A gene (ST1218) encoding a D-3-phosphoglycerate dehydrogenase (PGDH; EC 1.1.1.95) homolog was found in the genome of Sulfolobus tokodaii strain 7 by screening a database of enzymes likely to contribute to l-serine biosynthesis in hyperthermophilic archaea. After expressing the gene in Escherichia coli, the PGDH activity of the recombinant enzyme was assessed. Homogeneous PGDH was obtained using conventional chromatography steps, though during the purification an unexpected decline in enzyme activity was observed if the enzyme was stored in plastic tubes, but not in glass ones. The purified enzyme was a homodimer with a subunit molecular mass of about 35kDa and was highly thermostable. It preferably acted as an NAD-dependent D-3-phosphoglycerate (3PGA) dehydrogenase. Although NADP had no activity as the electron acceptor, both NADPH and NADH acted as electron donors. Kinetic analyses indicated that the enzyme reaction proceeds via a Theorell-Chance Bi-Bi mechanism. Unlike E. coli PGDH, the S. tokodaii enzyme was not inhibited by l-serine. In addition, both the NAD-dependent 3PGA oxidation and the reverse reaction were enhanced by phosphate and sulfate ions, while NADPH-dependent 3-phosphohydroxypyruvate (PHP) reduction was inhibited. Thus S. tokodaii PGDH appears to be subject to a novel regulatory mechanism not seen elsewhere. A database analysis showed that ST1218 gene forms a cluster with ST1217 gene, and a functional analysis of the ST1217 product expressed in E. coli revealed that it possesses l-glutamate-PHP aminotransferase activity. Taken together, our findings represent the first example of a phosphorylated serine pathway in a hyperthermophilic archaeon.


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.


Crystal structure of archaeal highly thermostable L-aspartate dehydrogenase-NAD-citrate ternary complex

The crystal structure of the highly thermostable L-aspartate dehydrogenase (L-aspDH; EC 1.4.1.21) from the hyperthermophilic archaeon Archaeoglobus fulgidus was determined in the presence of NAD and a substrate analog, citrate. The dimeric structure of A. fulgidus L-aspDH was refined at a resolution of 1.9 A with a crystallographic R-factor of 21.7% (R(free) = 22.6%). The structure indicates that each subunit consists of two domains separated by a deep cleft containing an active site. Structural comparison of the A. fulgidus L-aspDH/NAD/citrate ternary complex and the Thermotoga maritima L-aspDH/NAD binary complex showed that A. fulgidus L-aspDH assumes a closed conformation and that a large movement of the two loops takes place during substrate binding. Like T. maritima L-aspDH, the A. fulgidus enzyme is highly thermostable. But whereas a large number of inter- and intrasubunit ion pairs are responsible for the stability of A. fulgidus L-aspDH, a large number of inter- and intrasubunit aromatic pairs stabilize the T. maritima enzyme. Thus stabilization of these two L-aspDHs appears to be achieved in different ways. This is the first detailed description of substrate and coenzyme binding to L-aspDH and of the molecular basis of the high thermostability of a hyperthermophilic L-aspDH.


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.


An enzymatic cycling assay for nicotinic acid adenine dinucleotide phosphate using NAD synthetase

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to mobilize Ca(2+) from intracellular stores in a wide variety of organisms, ranging from plants to humans. We have developed a novel enzyme cycling assay for NAADP that involves coupled reactions catalyzed by four enzymes. In this system, NAADP is first converted into nicotinic acid adenine dinucleotide (NAAD) by alkaline phosphatase, after which the NAAD is converted to NAD, AMP, and PPi by NAD synthetase (NADS) in the presence of ATP and ammonia. The NAD is then amplified using an enzyme cycling system driven by glucose dehydrogenase and diaphorase. The resultant formation of formazan dye is measured spectrophotometrically based on the increase in absorbance at 450 nm. Using this method, NAADP (20-400 nM) was assayed, and a highly linear correlation was obtained between the NAADP concentration and the increase in absorbance at 450 nm. The cycling rate was approximately 95 cycles/min. In addition, the within-run coefficients of variation (CVs) for 25, 50, and 100 nM NAADP solutions were 9.33, 4.86, and 3.13%, respectively. Interference by NAD analogs (e.g., NAAD, NADP) in the sample was eliminated prior to running the assay by treating the sample with NADS and NAD nucleosidase (NADase). In sum, our findings indicate this enzyme cycling assay to be readily applicable for determination for NAADP in a variety of biological samples and to be particularly appropriate for use with an autoanalyzer.

Analytical Biochemistry: Methods in the Biological Sciences


Comparative analysis of the protein folding activities of two chaperonin subunits of Thermococcus strain KS-1; the effects of beryllium fluoride

We conducted a comparative analysis of the effects of beryllium fluoride (BeFx) on protein folding mediated by the alpha- and beta-subunit homooligomers (alpha16mer or beta16mer) from the hyperthermophilic archaeum Thermococcus strain KS-1. BeFx inhibited the ATPase activities of both alpha16mer and beta16mer with equal efficiency. This indicated that BeFx replaces the gamma-phosphate of chaperonin-bound ATP, thereby forming a stable chaperonin-ADP-BeFx complex. In the presence of ATP and BeFx, both of the two chaperonin subunits mediated green fluorescent protein (GFP) folding. Gel filtration chromatography revealed that the refolded GFP was retained by both chaperonins. Protease digestion and electron microscopic analyses showed that both chaperonin-ADP-BeFx complexes of the two subunits adopted a symmetric closed conformation with the built-in lids of both rings closed and that protein folding took place in their central cavities. These data indicated that basic protein folding mechanisms of alpha16mer and beta16mer are likely similar although there were some apparent differences. While beta16mer-mediated GFP refolding in the presence of ATP-BeFx that proceeded more rapidly than in the presence of ATP alone and reached a twofold higher plateau than that achieved with AMP-PNP, the folding mediated by the alpha16mer that proceeded with much lower yields. A mutant of alpha16mer, trapalpha, which traps the unfolded and partially folded substrate protein, did not affect the ATP-BeFx-dependent GFP folding by beta16mer but it suppressed that mediated by alpha16mer to the level of spontaneous folding. These results suggested that beta16mer differed from the alpha16mer in nucleotide binding affinity or the rate of nucleotide hydrolysis.


Structure of a hyperthermophilic archaeal homing endonuclease, I-Tsp061I: contribution of cross-domain polar networks to thermostability

A novel LAGLIDADG-type homing endonuclease (HEase), I-Tsp061I, from the hyperthermophilic archaeon Thermoproteus sp. IC-061 16 S rRNA gene (rDNA) intron was characterized with respect to its structure, catalytic properties and thermostability. It was found that I-Tsp061I is a HEase isoschizomer of the previously described I-PogI and exhibits the highest thermostability among the known LAGLIDADG-type HEases. Determination of the crystal structure of I-Tsp061I at 2.1 A resolution using the multiple isomorphous replacement and anomalous scattering method revealed that the overall fold is similar to that of other known LAGLIDADG-type HEases, despite little sequence similarity between I-Tsp061I and those HEases. However, I-Tsp061I contains important cross-domain polar networks, unlike its mesophilic counterparts. Notably, the polar network Tyr6-Asp104-His180-107O-HOH12-104O-Asn177 exists across the two packed alpha-helices containing both the LAGLIDADG catalytic motif and the GxxxG hydrophobic helix bundle motif. Another important structural feature is the salt-bridge network Asp29-Arg31-Glu182 across N and C-terminal domain interface, which appears to contribute to the stability of the domain/domain packing. On the basis of these structural analyses and extensive mutational studies, we conclude that such cross-domain polar networks play key roles in stabilizing the catalytic center and domain packing, and underlie the hyperthermostability of I-Tsp061I.


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.

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics


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


Bilirubin oxidase activity of Bacillus subtilis CotA

The spore coat protein CotA from Bacillus subtilis was previously identified as a laccase. We have now found that CotA also shows strong bilirubin oxidase activity and markedly higher affinity for bilirubin than conventional bilirubin oxidase. This is the first characterization of bilirubin oxidase activity in a bacterial protein.


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.


The first crystal structure of an archaeal helical repeat protein

The crystal structure of ST1625p, a protein encoded by a hypothetical open reading frame ST1625 in the genome of the hyperthermophilic archaeon Sulfolobus tokodaii, was determined at 2.2 A resolution. The only sequence similarity exhibited by the amino-acid sequence of ST1625p was a 33% identity with the sequence of SSO0983p from S. solfataricus. The 19 kDa monomeric protein was observed to consist of a right-handed superhelix assembled from a tandem repeat of ten alpha-helices. A structural homology search using the DALI and MATRAS algorithms indicates that this protein can be classified as a helical repeat protein.

Acta Crystallographica. Section F, Structural Biology and Crystallization Communications


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.


Crystal Structure of the NAD Biosynthetic Enzyme Quinolinate Synthase

A gene encoding a quinolinate synthase has been identified in the hyperthermophilic archaeon Pyrococcus horikoshii via genome sequencing. The gene was overexpressed in Escherichia coli, and the crystal structure of the produced enzyme was determined to 2.0 A resolution in the presence of malate, a substrate analogue. The overall structure exhibits a unique triangular architecture composed of a 3-fold repeat of three-layer (alphabetaalpha) sandwich folding. Although some aspects of the fold homologous to the each domain have been observed previously, the overall structure of quinolinate synthase shows no similarity to any known protein structure. The three analogous domains are related to a pseudo-3-fold symmetry. The active site is located at the interface of the three domains and is centered on the pseudo-3-fold axis. The malate molecule is tightly held near the bottom of the active site cavity. The model of the catalytic state during the first condensation step of the quinolinate synthase reaction indicates that the elimination of inorganic phosphate from dihydroxyacetone phosphate may precede the condensation reaction.


A new enzymatic cycling method for ammonia assay using NAD synthetase

好熱菌由来のアンモニア依存性NADキナーゼとグルコースデヒドロゲナーゼおよびジアホラーゼを利用した，新規酵素サイクリングアッセイ法を開発した．従来の方法に比べ，この方法では生体成分内の微量のアンモニアを簡便かつ正確に定量できることを示した．


The first archaeal agmatinase from anaerobic hyperthermophilic archaeon Pyrococcus horikoshii --- cloning, expression, and characterization

超好熱古細菌Pyrococcus horikoshiiに古細菌で初めてアグマチナーゼを見出し，遺伝子クローニング，大量発現系の構築，および性質決定を行った．超好熱菌のポリアミン代謝に本酵素が機能することを提唱した．

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics


The first crystal structure of hyperthremostable NAD-dependent glutamate dehydrogenase from Pyrobaculum islandicum

超好熱古細菌の2大キングダムの1つであるクレンアーキオータ由来のNAD依存性グルタミン酸脱水素酵素について，初めてX線結晶構造解析に成功した．これまでグルタミン酸脱水素酵素の高い熱安定性が，イオンペアネットワークの増加に由来するとされてきた定説に対し，サブユニット間の疎水性相互作用の重要性を指摘した．また，補酵素特異性と立体構造の相関を明らかにした．


Crystallization and preliminary X-ray diffraction analysis of homing endonuclease I-Tsp061I

超好熱菌由来新規ホーミングエンドヌクレアーゼを見出し，その結晶化に初めて成功した．また，X線結晶構造の初期解析を行った．

Acta Crystallographica. Section D, Biological Crystallography


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．


Crystallization and preliminary X-ray diffraction analysis of the hyperthermostable NAD-dependent glutamate dehydrogenase from Pyrobaculum islandicum

超好熱古細菌の2大キングダムの1つであるクレンアーキオータ由来のグルタミン酸脱水素酵素について，その結晶化に初めて成功した．また，X線結晶構造の初期解析を行った．


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．


Expression of foreign proteins in Escherichia coli by fusing with an archaeal FK506 binding protein

超好熱菌由来のロダネースの発現に関して，同じく超好熱菌由来のシャペロンと融合させることにより，大腸菌における組換え体タンパク質の生産性が飛躍的に向上することを明らかにした．


Highly stable L-lisine 6-dehydrogenase from the thermophile Geobacillus stearothermophilus isolated from a Japanese hot spring --- characterization, gene cloning and sequensing, and expression

これまでほとんど研究例の無いリジンデヒドロゲナーゼを好熱菌に初めて見出し，その遺伝子クローニングに成功し，一次構造を明らかにした．好熱菌由来の酵素は非常に安定であり，リジンの定量に有用性が高いことを示した．


Purification and characterization of leucine dehydrogenase from an alkaliphilic halophile, Natronobacterium magadii MS-3

Journal of Molecular Catalysis B: Enzymatic


Novel halophilic 2-aminobutyrate dehydrogenase from Halobacterium saccahrovorum DSM 1137


Purification and characterization of alanine dehydrogenase from a marine bacterium, Vibro proteolyticus


The Sulfolobus tokodaii gene ST1704 codes highly thermostable glucose dehydrogenase

超好熱菌由来のグルコース脱水素酵素の遺伝子クローニング，大量発現系の構築に成功した．本酵素の補酵素について立体特異性を明らかにした．


A nicotinamide mononucleotide adenylyltransferase with unique adenylyl group donor specificity from a hyperthermophilic archaeon, Pyrococcus horikoshii OT3

超好熱菌由来のニコチンアミドモノヌクレオチドアデニリルトランスフェラーゼが高温環境下では，ATPだけでなくADPやAMPもアデニリルドナーとして利用可能なことを示した．この新規な反応性の応用面での有用性を示した．


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．


Cloning, Expression, and Characterization of the First Archaeal ATP-Dependent Glucokinase from Aerobic Hyperthermophilic Archaeon, Aeropyrum pernix

超好熱古細菌由来のATP依存性グルコキナーゼについて，初めて遺伝子クローニング，発現系の構築および産物の性質決定を行った．進化的に最古と言われる超好熱菌の酵素を解析し，グルコキナーゼの進化において重要な知見を与えた．


Stable Ammonia-specific NAD Synthetase from Bacillus stearothermophilus: Purification, Characterization, Gene Cloning, and Applications

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.

好熱菌由来のアンモニア依存性NADキナーゼの精製を行い，性質の決定，遺伝子クローニングに成功した．本酵素が，アンモニアの定量に有用であることを明らかにした．

Bioscience, Biotechnology, and Biochemistry


Crystal structure of the ADP-dependent glucokinase from Pyrococcus horikoshii at 2.0-Å resolution: A large conformational change in ADP-dependent glucokinase

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.

超好熱古細菌の解糖系に存在するADP依存性グルコキナーゼのX線内結晶構造解析に成功した．本酵素が，立体構造上リボキナーゼに類似していることや，基質の結合に伴い構造が大きく変化することを明らかにした．


Crystallization and preliminary X-ray diffraction analysis of glutamate dehydrogenase from an aerobic hyperthermophilic archaeon, Aeropyrum pernix K1

超好熱古細菌の2大キングダムの1つであるクレンアーキオータ由来のグルタミン酸脱水素酵素の結晶化に初めて成功した．X線結晶構造の初期解析を行った．

Acta Crystallographica. Section D, Biological Crystallography


Crystallization and preliminary X-ray analysis of substrate complexes of leucine dehydrogenase from Thermoactinomyces intermedius

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.


Stabilization of Flavobacterium meningosepticum Glycerol Kinase by Introduction of a Hydrogen Bond

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.

高脂血症診断用試薬として多用されるグリセロールキナーゼの立体構造情報に基づき，水素結合の導入可能な部位を推定し，変異酵素を作製した．その結果，酵素活性に影響を及ばさずに安定性を向上させることに成功した．


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．


Fishing for bioactive substances from scorpionfish and some sea urchins

Venom proteins from the dorsal spine of two scorpionfish, Hypodytes rubripinnis and Synanceia verrucosa were assayed for mitogenicity and cytotoxicity. The two venoms had both mitogenic and cytotoxic activity on murine splenocytes and murine P388 leukemic cells. In H. rubripinnis, the second gel chromatographic fraction showed cytotoxic activity on P388 leukemic cells. On native PAGE, the glycoprotein isolated by concavalin A sepharose chromatography appeared to have a molecular mass of 110 kDa. In addition, two D-galactose-binding lectins (SUL-I and SUL-II) and a heparin-binding lectin (TGL-I) were purified from the globiferous pedicellariae of the toxopneustid sea urchins, Toxopneustes pileolus and Tripneustes gratilla, respectively. SUL-I (Nakagawa et al., 1999a) had mitogenic activity and cytotoxic activity but SUL-II and TGL-I did not. SUL-I did not show sequence homology to SUL-II. A hemolytic lectin with a molecular mass of 29 kDa was isolated from the coelomic fluid of T. gratilla. The hemolytic activity of the lectin was dependent on Ca2+ concentration and inhibited by lactose. The present results suggest that some species of scorpionfish and sea urchins may be novel sources for biologically active substances such as anti-tumor compounds or new lectins.

スコルピオンフィシュの仲間であるハオコゼおよびオニダルマオコゼの背鰭毒棘のタンパク画分から生理活性物質の分離を試みた．また，有毒ウニとして知られているラッパウニおよびシラヒゲウニの毒棘より，生理活性物質としてレクチンを探索した．ハオコゼおよびオニダルマオコゼからは細胞障害性のタンパク毒を部分精製した．一方，ウニ類からは中型のレクチンを精製した．


Temperature Dependence of Kinetic Parameters for Hyperthermophilic Glutamate Dehydrogenase from Aeropyrum pernix K1

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.

超好熱古細菌由来のグルタミン酸脱水素酵素のカイネティックパラメータが温度依存的に変化することを見出した．これにより，超好熱菌が生育する高温環境における酵素反応のパラメータ解析が重要であることを提唱した．

Bioscience, Biotechnology, and Biochemistry


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．


Transcriptional Regulation of Phosphoenolpyruvate Synthase by Maltose in the Hyperthermophilic Archaeon, Pyrococcus furiosus

The phosphoenolpyruvate synthase (EC 2.7.9.2) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the Pi-dependent formation of pyruvate, ATP and water from phosphoenolpyruvate and AMP [Sakuraba, H., Arch. Biochem. Biophys., 364, 125-128 (1999)]. In this study, the P. furiosus phosphoenolpyruvate synthase was purified to homogeneity and the N-terminal amino acid sequence was determined to be AYRFIKWFEELS. The sequence coincided completely with the N-terminal amino acid sequence of the translation product of the mlrA gene that was found to be upregulated at the transcriptional level by the alpha-linked glucose disaccharide maltose [Robinson, K.A. and Schreier, H.J., Gene, 151, 173-176 (1994)]. The mlrA gene was cloned and expressed in Escherichia coli. The recombinant cells produced a hyperthermostable phosphoenolpyruvate synthase. This indicates that the mlrA gene encodes the enzyme. When P. furiosus was grown on the medium supplemented with maltose, the specific activity of the enzyme markedly increased (about 10-fold) compared with that produced by the cells grown on the medium without maltose. Northern blot analysis revealed enhanced transcription of the mlrA gene in the presence of maltose. These results indicate that transcriptional regulation of phosphoenolpyruvate synthase by maltose is present in P. furiosus.

超好熱古細菌 Pyrococcus furiosus に見出したAMPからATPを合成する新規キナーゼ遺伝子の転写誘導が，培地へのマルトースの添加により著しく上昇することを明らかにした．この結果から，超好熱古細菌のエネルギー代謝において本酵素がATP生産に機能することを提唱した．

Journal of Bioscience and Bioengineering


Increasing the thermostability of Flavobacterium meningosepticum glycerol kinase by changing Ser329 to Asp in the subunit interface region

高脂血症診断用試薬として多用されるグリセロールキナーゼに対し1アミノ酸残基置換を行うだけでその安定性を高めた成果を報告している．導入部位は酵素の活性部位から離れたところにあり，酵素活性に影響を及ばさず安定性の向上に成功した．他の酵素にもこの手法の応用が期待できる．


A novel hyperthermophilic archaeal glyoxylate reductase from Thermococcus litoralis --- Characterization, gene cloning, nucleotide sequence and expression in Escherichia coli

A novel NADH-dependent glyoxylate reductase has been found in a hyperthermophilic archaeon Thermococcus litoralis DSM 5473. This is the first evidence for glyoxylate metabolism and its corresponding enzyme in hyperthermophilic archaea. NADH-dependent glyoxylate reductase was purified approximately 560-fold from a crude extract of the hyperthermophile by five successive column chromatographies and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 76 kDa, and the enzyme consisted of a homodimer with a subunit molecular mass of approximately 37 kDa. The optimum pH and temperature for enzyme activity were approximately 6.5 and 90 degrees C, respectively. The enzyme was extremely thermostable; the activity was stable up to 90 degrees C. The glyoxylate reductase catalyzed the reduction of glyoxylate and hydroxypyruvate, and the relative activity for hydroxypyruvate was approximately one-quarter that of glyoxylate in the presence of NADH as an electron donor. NADPH exhibited rather low activity as an electron donor compared with NADH. The Km values for glyoxylate, hydroxypyruvate, and NADH were determined to be 0.73, 1.3 and 0.067 mM, respectively. The gene encoding the enzyme was cloned and expressed in Escherichia coli. The nucleotide sequence of the glyoxylate reductase gene was determined and found to encode a peptide of 331 amino acids with a calculated relative molecular mass of 36,807. The amino-acid sequence of the T. litoralis enzyme showed high similarity with those of probable dehydrogenases in Pyrococcus horikoshii and P. abyssi. The purification of the enzyme from recombinant E. coli was much simpler compared with that from T. litoralis; only two steps of heat treatment and dye-affinity chromatography were needed.

超好熱菌にはじめてグリオキシル酸レダクターゼを見出し，これを精製し，遺伝子クローニング，アミノ酸配列の決定を行った．この結果より，本菌がグリオキシル酸代謝経路を持つ事を提唱した．また，本酵素がグリオキシル酸およびヒドロキシピルビン酸の定量用酵素として有用であることを示した．


Glutamate dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1: enzymatic characterization, identification of the encoding gene and phylogenetic implications.

NADP-dependent glutamate dehydrogenase (L-glutamate: NADP oxidoreductase, deaminating, EC 1.4.1.4) from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820) was purified to homogeneity for characterization. The enzyme retained its full activity on heating at 95 degrees C for 30 min, and the maximum activity in L-glutamate deamination was obtained around 100 degrees C. The enzyme showed a strict specificity for L-glutamate and NADP on oxidative deamination and for 2-oxoglutarate and NADPH on reductive amination. The Km values for NADP, L-glutamate, NADPH, 2-oxoglutarate, and ammonia were 0.039, 3.3, 0.022, 1.7, and 83 mM, respectively. On the basis of the N-terminal amino acid sequence, the encoding gene was identified in the A. pernix K1 genome, cloned, and expressed in Escherichia coli. Analysis of the nucleotide sequence revealed an open reading frame of 1257 bp starting with a minor TTG codon and encoding a protein of 418 amino acids with a molecular weight of 46170. Phylogenetic analysis revealed that the glutamate dehydrogenase from A. pernix K1 clustered with those from aerobic Sulfolobus solfataricus, Sulfolobus shibatae, and anaerobic Pyrobaculum islandicum in Crenarchaeota, and it separated from another cluster of the enzyme from Thermococcales in Euryarchaeota. The branching pattern of the enzymes from A. pernix K1, S. solfataricus, S. shibatae, and Pb. islandicum in the phylogenetic tree coincided with that of 16S rDNAs obtained from the same organisms.

超好熱古細菌の2大キングダムの1つであるクレンアーキオータ由来のグルタミン酸脱水素酵素を初めて精製し，性質を決定した．本酵素のアミノ酸配列と他の超好熱菌由来のものを比較し，超好熱古細菌におけるグルタミン酸脱水素酵素の進化が水平伝播によらないことを提唱した．また，TCAサイクルが存在するグループの酵素は，存在しないグループの酵素と系統樹上で離れた位置に存在することを明らかにした．


Biochemical Characterization, Cloning, and Sequencing of ADP-Dependent (AMP-Forming) Glucokinase from Two Hyperthermophilic Archaea, Pyrococcus furiosus and Thermococcus litoralis.

The ADP-dependent (AMP-forming) glucokinases from the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus litoralis catalyze the phosphorylation of glucose using ADP as the essential phosphoryl group donor. Both enzymes were purified to homogeneity and characterized with regard to each other. The enzymes had similar enzymological properties as to substrate specificity, coenzyme specificity, optimum pH, and thermostability. However, a difference was observed in the subunit composition; while the T. litoralis enzyme is a monomer with a molecular mass of 52 kDa, the P. furiosus enzyme has a molecular mass of about 100 kDa and consists of two subunits with identical molecular masses of 47 kDa. The genes encoding these enzymes were cloned and sequenced. The gene for the P. furiosus enzyme contains an open reading frame for 455 amino acids with a molecular weight of 51,265, and that for the T. litoralis enzyme contains an open reading frame for 467 amino acids with a molecular weight of 53,621. About 59% similarity in amino acid sequence was observed between these two enzymes, whereas they did not show similarity with any ATP-dependent kinases that have been reported so far. In addition, two phosphate binding domains, and adenosine and glucose binding motifs commonly conserved in the eukaryotic hexokinase family were not observed.

超好熱古細菌の解糖系に存在する2種のADP依存性グルコキナーゼの遺伝子クローニングに成功し，そのアミノ酸配列を初めて決定した．その結果，既知のグルコキナーゼとは全く相同性を示さない超好熱菌に特徴的な新規酵素であることが判明した．また，本酵素が糖尿病の診断薬酵素として有用であることを示した．これらは現在，旭化成より販売されている．


The RadA protein from a hyperthermophilic archaeon Pyrobaculum islandicum is a DNA-dependent ATPase that exhibits two disparate catalytic modes, with a transition temperature at 75℃

The radA gene is an archaeal homolog of bacterial recA and eukaryotic RAD51 genes, which are critical components in homologous recombination and recombinational DNA repair. We cloned the radA gene from a hyperthermophilic archaeon, Pyrobaculum islandicum, overproduced the radA gene product in Escherichia coli and purified it to homogeneity. The purified P. islandicum RadA protein maintained its secondary structure and activities in vitro at high temperatures, up to 87 degrees C. It also showed high stability of 18.3 kcal.mol-1 (76.5 kJ.mol-1) at 25 degrees C and neutral pH. P. islandicum RadA exhibited activities typical of the family of RecA-like proteins, such as the ability to bind ssDNA, to hydrolyze ATP in a DNA-dependent manner and to catalyze DNA strand exchange. At 75 degrees C, all DNAs tested stimulated ATPase activity of the RadA. The protein exhibited a break in the Arrhenius plot of ATP hydrolysis at 75 degrees C. The cooperativity of ATP hydrolysis and ssDNA-binding ability of the protein above 75 degrees C were higher than at lower temperatures, and the activation energy of ATP hydrolysis was lower above this break point temperature. These results suggest that the ssDNA-dependent ATPase activity of P. islandicum RadA displays a temperature-dependent capacity to exist in two different catalytic modes, with 75 degrees C being the critical threshold temperature.


An AMP-Dependent(ATP-Forming) Kinase in the Hyperthermophilic Archaeon Pyrococcus furiosus: Characterization and Novel Physiological Role.

超好熱古細菌 Pyrococcus furiosus にAMPからATPを合成する新規キナーゼが存在することを初めて見出し，これを単離精製し，その酵素化学的諸性質を明らかにした．また，これまで不明であった超好熱古細菌のエネルギー代謝における本酵素の新しい役割を提唱した．


The NAD-dependent glutamate dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum: cloning, sequencing, and expression of the enzyme gene

The NAD-dependent glutamate dehydrogenase (GluDH) gene from the hyperthermophilic archaeon Pyrobaculum islandicum was cloned and expressed in Escherichia coli. Analysis of the nucleotide sequence revealed an open reading frame of 1266 bp encoding a protein of 421 amino acids with a molecular weight of 46,905. In the alignment of the amino acid sequence with those of mesophilic Clostridium symbiosum NAD-dependent GluDH and hyperthermophilic NADP-dependent enzymes from Thermococcus profundus and Pyrococcus furiosus, substitutions in the residues involved in dinucleotide binding were observed. On the other hand, the residues involved in glutamate binding were well conserved. This is the first description of the primary structure of NAD-dependent GluDH in hyperthermophilic archaea.

超好熱菌で初めて見出したNAD依存性グルタミン酸脱水素酵素の遺伝子をクローニングし，アミノ酸配列を決定し，基質結合部位，補酵素結合部位を予測した．高度耐熱性に関与すると言われるアミノ酸残基が他の超好熱古細菌の酵素と異なることを明らかにした．また，本酵素の補酵素特異性と一次構造に相関があることを見出した．

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology


A Novel Glycerol Kinase from Flavobacterium meningosepticum: Characterization, Gene Cloning and Primary Structure

Bioscience, Biotechnology, and Biochemistry


Enzymological Characteristics of the Hyperthermostable NAD-Dependent Glutamate Dehydrogenase from the Archaeon Pyrobaculum islandicum and Effects of Denaturants and Organic Solvents


Construction of a new leucine dehydrogenase with preferred specificity for NADP+ by site-directed mutagenesis of the strictly NAD+-specific enzyme

On the basis of sequence comparison between NAD+-dependent leucine dehydrogenase (LeuDH) from Thermoactinomyces intermedius and NADP+-dependent dehydrogenases, a set of amino acid residues that are supposed to determine the coenzyme specificity of LeuDH were assigned. Systematic replacement of these amino acids by others was done with the aim to switch its natural coenzyme specificity to a new one preferring NADP+. Single D203A, double D203A-I204R and triple D203A-I204R-D210R mutation enzymes were constructed. The wild-type LeuDH is inactive with NADP+. However, D203A single mutant exhibited dual specificity for NAD+ and NADP+ with essentially identical k(cat)/Km values for both coenzymes, but the values were three orders of magnitude lower than that of the wild-type enzyme. Introduction of positive charge at 204 together with the removal of the negative charge at 203 in the double mutant D203A-I204R provided the enzyme with significantly high affinity for NADP+. The best k(cat)/Km value for NADP+ was shown for the triple mutant D203A-I204R-D210R: more than 2% of the k(cat)/Km value of the wild-type enzyme. Thus, we succeeded in constructing a mutant LeuDH with a new coenzyme specificity preferring NADP+ which is highly active (specific activity, 19 micromol/mg/min).


4-α-Glucanotransferase from the hyperthermophilic archaeon Thermococcus litoralis. --- Enzyme purification and characterization, and gene cloning, sequencing and expression in Escherichia coli

4-Alpha-Glucanotransferase was purified from cells of Thermococcus litoralis, a hyperthermophilic archaeon. The molecular mass of the enzyme was estimated to be approximately 87 kDa by gel filtration. The optimal temperature for its activity was 90 degrees C. The enzyme catalyzed the transglycosylation of maltooligosaccharides, yielding maltooligosaccharides of various lengths and glucose. When maltoheptaose was used as the substrate, glucoamylase-resistant and glucoamylase-sensitive saccharides were produced. On incubation of amylose with the T. litoralis enzyme, glucoamylase-resistant but alpha-amylase-sensitive molecules were produced, but the amount of reducing sugar showed only slight increases. These results indicate that the T. litoralis enzyme catalyzes not only intermolecular transglycosylation to produce linear alpha-1,4-glucan, but also intramolecular transglycosylation to produce cyclic alpha-1,4-glucan (cycloamylose), similarly to potato 4-alpha-glucanotransferase (called disproportionating enzyme). The gene encoding the T. litoralis 4-alpha-glucanotransferase was cloned, sequenced and expressed in Escherichia coli. The nucleotide sequence of the gene encoded a 659-amino acid protein with a calculated molecular mass of 77,883 Da. The amino acid sequence of the T. litoralis enzyme showed high similarity with those of alpha-amylases of Pyrococcus furiosus, a hyperthermophilic archaeon, and Dictyoglomus thermophilum, an extremely thermophilic bacterium, but little similarity with those of other known 4-alpha-glucanotransferases.


Preparation of Optically Active α-Hydroxy Esters: Stereoselective Reduction of α-Keto Esters Using Thermophilic Actinomycetes.

Journal of Fermentation and Bioengineering


Stereoselective reduction of α- and β-keto esters with aerobic thermophiles, Bacillus strains.


Biomechanical effects of shock waves on Escherichia coli and λphaseDNA.


Determination of Phosphate Using Immobilized Enzyme Reactor -- Chemiluminescence Detector by FIA


Gene cloning, purification, and characterization of thermostable and halophilic leucine dehydrogenase from a halophilic thermophile, Bacillus licheniformis TSN9.


食品甘味料としての異性化糖の教材化

食品甘味料としての異性化糖の教材化


Construction and Characterization of Chimeric Enzyme Consisting of an Amino-Terminal Domain of Phenylalanine Dehydrogenase and a Carboxy-Terminal Domain of Leucine Dehydrogenase


The purification, characterization, cloning and sequencing of the gene for a halostable and thermostable leucine dehydrogenase from Thermoactinomyces intermedius

Leucine dehydrogenase has been purified to homogeneity from a moderate thermophilic actinomycete, Thermoactinomyces intermedius IFO 14230. The enzyme can be stored without loss of its activity at a low temperature (e.g., 4 degrees C) for over two years. The enzyme was more thermostable at higher concentrations of salts such as NaCl and KCl. It retained about 90% of activity on incubation at 70 degrees C for at least 40 min in the presence of 3 M NaCl. The Michaelis constants for NAD, L-leucine, NADH, 2-oxoisocaproate and ammonia were determined to be 0.36, 2.0, 0.042, 0.63 and 118 mM, respectively, from initial-velocity analyses. The enzyme showed pro-S stereospecificity for hydrogen transfer of NADH in the reductive amination. The enzyme gene was cloned into Escherichia coli and its complete DNA sequence was determined. The leucine dehydrogenase gene (leudh) consists of a 1098-bp open reading frame and encodes 366 amino acid residues corresponding to a subunit (M(r) 40586) of the octameric enzyme. The amino acid sequence of the enzyme showed 80.7% similarity with that of the Bacillus stearothermophilus enzyme. The enzyme was overproduced in E. coli JM 109 having a recombinant plasmid, pULDH2, which was constructed from pUC18 and the leudh gene. The enzyme was purified from the cell extract to homogeneity in one day, with 78% recovery.


Purification and Characterizaion of Extremely Thermo-Stable Glutamate Dehydrogenase from a Hyperthermophilic Archaeon, Thermococcus Litoralis


ポリアクリルアミド電気泳動法を利用する酵素の特異性を調べる実験の教材化

ポリアクリルアミド電気泳動法を利用する酵素の特異性を調べる実験の教材化


色素アフィニティクロマトグラフィーの生化学実習のための教材化

色素アフィニティクロマトグラフィーの生化学実習のための教材化


Site-Directed Mutagenesis of a Hexapeptide Segment Involved in Substrate Recognition of Phenylalanine Dehydrogenase from Thermoactinomyces intermedius


Purification and Properties Extremely Thermostable Glutamate Dehydrogenase from Two Hyperthermophilic Archaebacteria, Pyrococcus woesei and Pyrococcus furiosus

Bioscience, Biotechnology, and Biochemistry


Dye-linked L-malate dehydrogenase from thermophilic Bacillus species DSM 465 Purification and characterization

The distribution of dye-linked L-malate dehydrogenase (L-malate: acceptor oxidoreductase, EC 1.1.99.16) was investigated in many thermophilic bacteria. The enzyme occurred widely in thermophilic spore-forming bacteria like bacilli and thermoactinomycetes. The enzyme was purified to homogeneity from a thermophile, Bacillus sp. DSM 465, with a 2.7% overall recovery by DEAE-Toyopearl column chromatography, Sephacryl S-400 column chromatography and preparative slab PAGE. The enzyme had a molecular mass of about 660 kDa and consisted of about ten subunits all with a molecular mass of 66 kDa. The enzyme retained its full activity upon heating at 55 degrees C for at least 60 min and with incubation at pH 5.0-10.0, 55 degrees C, for 10 min. The enzyme exclusively catalyzed L-malate dehydrogenation in the presence of an electron acceptor such as 2,6-dichloroindophenol. The Michaelis constants for L-malate and 2,6-dichloroindophenol were determined to be 1.67 mM and 0.050 mM, respectively. FAD was identified as a prosthetic group of the enzyme by HPLC.


Valine dehydrogenase from a non-spore-forming bacterium, Alcaligenes faecalis: purification and characterization

An NAD-dependent valine dehydrogenase (L-valine:NAD oxidoreductase, deaminating, EC 1.4.1.-), was found in a non-spore-forming bacterium, Alcaligenes faecalis, and purified about 80-fold to be characterized. The molecular mass of the enzyme was estimated to be about 72 kDa and the enzyme consists of two identical subunits with a molecular mass of 40 kDa and is thermolabile. It loses its activity fully on incubation at 50 degrees C for 5 min. The enzyme catalyzes the reversible deamination of L-valine, which is the preferred substrate, and other branched-chain and straight-chain L-amino acids in the presence of NAD. The pH optima are about 10.8 and 8.8 for the oxidative deamination and reductive amination, respectively. The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADH was exclusively transferred to the substrate in the reductive amination. The amino-acid composition markedly differs from those of Bacillus sphaericus and Clostridium thermoaceticum leucine dehydrogenases. The enzyme did not react with the antibody of C. thermoaceticum leucine dehydrogenase. Therefore, the enzyme is clearly different from leucine dehydrogenases from spore-forming bacteria, which act on valine, and the first valine dehydrogenase to be characterized in detail.

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology


A Large-Scale Preparative Electrophoretic Method for the Purification of Pyridine Nucleotide-Linked Dehydrogenases

A large-scale preparative polyacrylamide gel electrophoresis (PAGE) method that uses a 1.5- or a 2.0-cm-thick slab gel has been developed for the purification of NAD-dependent dehydrogenases. With the 2.0-cm-thick gel, a maximum volume (up to about 160 ml) of enzyme sample was applied to a gel plate, resulting in the application of a large amount of protein and enzyme. After the electrophoretic run, the enzyme band on the gel was detected by activity staining and recovered from the gel by extraction with a fairly loose-fitting glass-Teflon homogenizer. NAD-dependent alanine dehydrogenase, leucine dehydrogenase, and glycerol dehydrogenase were purified in high yields (more than 80%) by the preparative PAGE method. The method can be carried out using a simple slab gel apparatus, which is modified from the conventional analytical apparatus for the purpose of preparative PAGE under conditions used for routine analytical runs. Thus, the method may be suitable for use in purifying NAD(P)-dependent dehydrogenases and many other enzymes after conventional chromatography such as dye-ligand affinity chromatography or ion-exchange chromatography.


Effect of Shock Waves on Microorganisms: Evaluation Methods of the Effects; Shock Waves

Proceedings of 18th International Synposium on Shock Waves, Sendai, Springer- Verlag


Distribution, purification, and characterization of thermostable phenylalanine dehydrogenase from thermophilic actinomycetes.

Phenylalanine dehydrogenase (L-phenylalanine:NAD oxidoreductase, deaminating; EC 1.4.1.-) was found in various thermophilic actinomycetes. We purified the enzyme to homogeneity from Thermoactinomyces intermedius IFO 14230 by heat treatment and by Red Sepharose 4B, DEAE-Toyopearl, Sepharose CL-4B, and Sephadex G-100 chromatographies with a 13% yield. The relative molecular weight of the native enzyme was estimated to be about 270,000 by gel filtration. The enzyme consists of six subunits identical in molecular weight (41,000) and is highly thermostable: it is not inactivated by incubation at pH 7.2 and 70 degrees C for at least 60 min or in the range of pH 5 to 10.8 at 50 degrees C for 10 min. The enzyme preferably acts on L-phenylalanine and its 2-oxo analog, phenylpyruvate, in the presence of NAD and NADH, respectively. Initial velocity and product inhibition studies showed that the oxidative deamination proceeds through a sequential ordered binary-ternary mechanism. The Km values for L-phenylalanine, NAD, phenylpyruvate, NADH, and ammonia were 0.22, 0.078, 0.045, 0.025, and 106 mM, respectively. The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADH was exclusively transferred to the substrate.


Thermostable phenylalanine dehydrogenase of Thermoactinomyces intermedius --- cloning, expression, and sequencing of its gene


Thermostable Alanine Dehydrogenase from Thermophilic Bacillus sphaericus DSM 462

Alanine dehydrogenase (L-alanine: NAD+ oxidoreductase, deaminating) was simply purified to homogeneity from a thermophile, Bacillus sphaericus DSM 462, by ammonium sulfate fractionation, red-Sepharose 4B chromatography and preparative slab gel electrophoresis. The enzyme had a molecular mass of about 230 kDa and consisted of six subunits with an identical molecular mass of 38 kDa. The enzyme was much more thermostable than that from a mesophile, B. sphaericus, and retained its full activity upon heating at 75 degrees C for at least 60 min and with incubation in pH 5.5-9.5 at 75 degrees C for 10 min. The enzyme can be stored without loss of its activity in a frozen state (-20 degrees C, at pH 7.2) for over 5 months. The optimum pH for the L-alanine deamination and pyruvate amination were around 10.5 and 8.2, respectively. The enzyme exclusively catalyzed the oxidative deamination of L-alanine in the presence of NAD+, but showed low amino acceptor specificity; hydroxypyruvate, oxaloacetate, 2-oxobutyrate and 3-fluoropyruvate are also aminated as well as pyruvate in the presence of NADH and ammonia. Initial velocity and product inhibition studies showed that the reductive amination proceeded through a sequential mechanism containing partially random binding. NADH binds first to the enzyme, and then pyruvate and ammonia bind in a random fashion. The products are sequentially released from the enzyme in the order L-alanine then NAD+. A dead-end inhibition by the formation of an abortive ternary complex which consists of the enzyme, NAD+ and pyruvate was included in the reaction. A possible role of the dead-end inhibition is to prevent the enzyme from functioning in the L-alanine synthesis. The Michaelis constants for the substrates were as follows: NADH, 0.10 mM; pyruvate, 0.50 mM; ammonia, 38.0 mM; L-alanine, 10.5 mM and NAD+, 0.26 mM.


Continous Production of 3-Fluoro-L-alanine with Alanine Dehydrogenase


Enzymatic in Situ Determination of Stereospecificity of NAD-dependent Dehydrogenases


Gene cloning and sequence determination of leucine dehydrogenase from Bacillus stearothermophilus, and structural comparison with other NAD(P)+ dependent dehydrogenase.


Selective enzymatic determination of L-phenylalanine and 3-phenylpyruvate with phenylalanine dehydrogenase.


NAD要求性アミノ酸脱水素酵素の酵素化学的研究とその応用

NAD要求性アミノ酸脱水素酵素の酵素化学的研究とその応用


A spectrophotometric method for the determination of γ-glutamyl cyclotransferase with analine dehydrogenase in the presence of anthglutin.

Biochemical Medicine and Metabolic Biology


Purification and characterization of malate dehydrogenase from the phototrophic bacterium, Rhodopseudomonas capsulata

光合成細菌Rhodopseudomonas capsulataのエネルギー代謝と炭酸固定に関与するクエン酸サイクルの酵素リンゴ酸脱水素酵素の精製と性質について明らかにした．リンゴ酸脱水素酵素の新規反応メカニズムを提唱した．

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology


Purification and characterization of thermostable leucine dehydrogenase from Bacillus stearothermophilus


Improvement for L-Leucine Production in a Continuously Operated Enzyme Membrane Reactor


SCREENING OF THERMOSTABLE LEUCINE AND ALANINE DEHYDROGENASES IN THERMOPHILIC BACILLUS STRAINS


Purification and Partial Characterization of the Soluble NADH Dehydrogenase from the Phototrophic Bacterium

Rhodopseudomonas capsulata. Z. Naturforsch


Modification of Leucine Dehydrogenase by Pyridoxal 5'-Phosphate.


Purification and Properties of α;-Hydroxyglutarate Dehydrogenase of Peptococcus aerogenes. Agric.


A Spectrophotometric Method for the Determination of Aminopeptidase Activity with Leucine Dehydrogenase

Analytical Biochemistry: Methods in the Biological Sciences


A Rapid Method for Purification of Leucine Dehydrogenase by Affinity Chromatography.

Bulletin of the Institute for Chemical Research Kyoto University


Spectrophotometric End Point Method for Assaying Serum Aminopeptidase


Small-Angle X-ray Scattering Studies of L-Leucine Dehydrogenase.


Isolation and Partial Characterization of the Membrane-bound NADH Dehydrogenase from Phototrophic Bacterium Rhodopseudomonas capsulata


Purification and Properties of Alanine Dehydrogenase from Bacillus sphaericus.


Properties of Crystalline Leucine Dehydrogenase from Bacillus sphaericus.


Leucine Dehydrogenase of Bacillus sphaericus: Sulfhydryl Groups and Catalytic Sites.


Determination of Branched- chain L-Amino Acids and Their Keto Analogs with Leucine Dehydrogenase.


A BACTERIAL ANTITUMOR ENZYME, LEUCINE DEHYDROGENASE FROM BACILLUS SPHAERICUS

PROCEEDINGS OF THE FIRST INTERNATIONAL CONGRESS OF IAMS


Development of Biosensors Using Hyperthermophilic Enzymes as an Element:Development of Biosensors Using Hyperthermophilic Enzymes as an Element:Functional Analysis of Polyamine Metabolizing Enzymes and Development for EnzymeElectrode Sensor Using D-Proline Dehydrogenases

超好熱菌酵素を素子とするバイオセンサーの開発:ポリアミン関連酵素の機能解析とD-プロリン脱水素酵素機能電極センサーの開発

An amperometric enzyme sensor give us higher sensitivity and specificity for the substratedetermination. In spite of advantages of enzyme sensor, many enzymes so far found have been toolabile to use as biosensor elements in artificial circumstances for a longer period.Hyperthermophiles, which can grow above 90℃, have been known to produce much more stableenzymes under various artificial conditions. In this work, we carried out screening, biochemicalcharacterization and improvement of production for hyperthermostable enzymes which are more usefulas the elements in the biosensors. We focused on the polyamines as one of the substrates ofbiosensors. Polyamines have been known to play many important roles in cell proliferation,differentiation and transformation. The concentration of the polyamines together with their acetylconjugates remarkably increases in the biological fluids and the affected tissues of cancer patients.Therefore, their polyamines are listed as tumor markers. Gas and ion chromatographies have beenso far used for polyamine determination, but have some problems from the aspects of high sensitivityand easy operation. Thus, we here developed biosensors using hyperthermostable enzymes forpolyamine determination. Such enzyme sensors are more useful for the simple and rapiddetermination of polyamines and application for clinical analysis and food analysis In addition, wetried the construction of biosensor using the hyperthermophilic enzyme, D-Proline dehydrogenase.As the results, we found the thermostable agmatinase and spermidine dehydrogenase inhyperthermophiles, Pyrococcus horikoshii and Sulfolobus tokodaii, respectively. We succeeded theconstruction of novel amperometric sensor for D-proline determination using D-Proline dehydrogenasederived from Pyrobaculum islandicum.

Bulletin of Faculty of Engineering, The University of Tokushima