記述言語：英語   掲載種別：研究論文（学術雑誌）  

Among the four bioactive cationic homo-poly(amino acids) discovered in nature, two are mirror-image isomers of poly(2,4-diaminobutyric acid) (poly-Dab) whose biosynthesis has long been unexplained. Their structural analogy plausibly suggested that they could share a common biosynthetic pathway utilizing ε-poly(l-lysine) synthetase-like enzymology but with an unprecedented process for enantiomeric inversion of polymer building blocks. To investigate this possibility, we comparatively explored the biosynthesis of poly-l-Dab and its mirror-image isomer poly-d-Dab in Streptomyces celluloflavus USE31 and Streptoalloteichus hindustanus NBRC15115, respectively, through genome mining, genetic inactivation, and heterologous expression combined with biochemical assays. While they shared the same biosynthetic pathway, the poly-d-Dab biosynthetic gene cluster additionally harbored the racemase gene. The critical finding that poly-d-Dab synthetase, in contrast to the synthetase generating the l-isomer, selectively activated d-Dab through adenylation conclusively demonstrated that free diffusible d-Dab preactivationally generated by the racemase is directly activated to be incorporated into the polymer. Our study thus represents the first demonstration of the stereoselective biosynthesis of a nonribosomal peptide governed by adenylation activity for a d-amino acid other than alanine. In silico sequence comparison between poly-Dab synthetases allowed us to identify amino acid residues potentially responsible for the discrimination of Dab enantiomers. Our results will provide significant insight not only for the future discovery of novel bioactive cationic poly(amino acids) but also for the creation of designer nonribosomal peptides with d-configuration.

DOI： 10.1021/acschembio.0c00321

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記述言語：日本語   出版者・発行元：(公社)日本生物工学会  

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DOI： 10.1007/s00726-018-2671-y

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Frontiers Media S.A.  

The Lactobacillus sakei strain LK-145 isolated from Moto, a starter of sake, produces potentially large amounts of three D-amino acids, D-Ala, D-Glu, and D-Asp, in a medium containing amylase-digested rice as a carbon source. The comparison of metabolic pathways deduced from the complete genome sequence of strain LK-145 to the type culture strain of Lactobacillus sakei strain LT-13 showed that the L- and D-amino acid metabolic pathways are similar between the two strains. However, a marked difference was observed in the putative cysteine/methionine metabolic pathways of strain LK-145 and LT-13. The cystathionine β-lyase homolog gene malY was annotated only in the genome of strain LT-13. Cystathionine β-lyase is an important enzyme in the cysteine/methionine metabolic pathway that catalyzes the conversion of L-cystathionine into L-homocysteine. In addition to malY, most genome-sequenced strains of L. sakei including LT-13 lacked the homologous genes encoding other putative enzymes in this pathway. Accordingly, the cysteine/methionine metabolic pathway likely does not function well in almost all strains of L. sakei. We succeeded in cloning and expressing the malY gene from strain LT-13 (Ls-malY) in the cells of Escherichia coli BL21 (DE3) and characterized the enzymological properties of Ls-MalY. Spectral analysis of purified Ls-MalY showed that Ls-MalY contained a pyridoxal 5'-phosphate (PLP) as a cofactor, and this observation agreed well with the prediction based on its primary structure. Ls-MalY showed amino acid racemase activity and cystathionine β-lyase activity. Ls-MalY showed amino acid racemase activities in various amino acids, such as Ala, Arg, Asn, Glu, Gln, His, Leu, Lys, Met, Ser, Thr, Trp, and Val. Mutational analysis revealed that the ε-amino group of Lys233 in the primary structure of Ls-MalY likely bound to PLP, and Lys233 was an essential residue for Ls-MalY to catalyze both the amino acid racemase and β-lyase reactions. In addition, Tyr123 was a catalytic residue in the amino acid racemase reaction but strongly affected β-lyase activity. These results showed that Ls-MalY is a novel bifunctional amino acid racemase with multiple substrate specificity
both the amino acid racemase and β-lyase reactions of Ls-MalY were catalyzed at the same active site.

DOI： 10.3389/fmicb.2018.00403

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Tokyo  

We successfully expressed the l-aspartate oxidase homolog gene (accession no: OCC_06611) of Thermococcus litoralis DSM 5473 in the soluble fraction of Escherichia coli BL21 (DE3) using a pET21b vector with 6X His tag at its C-terminus. The gene product (Tl-LASPO) showed l-aspartate oxidase activity in the presence of FAD in vitro, and this report is the first that details an l-aspartate oxidase derived from a Thermococcus species. The homologs of Tl-LASPO existed mainly in archaea, especially in the genus of Thermococcus, Pyrococcus, Sulfolobus, and Halobacteria. The quaternary structure of Tl-LASPO was homotrimeric with a subunit molecular mass of 52 kDa. The enzyme activity of Tl-LASPO increased with temperature up to 70 °C. Tl-LASPO was active from pH 6.0 to 9.0, and its highest activity was at pH 8.0. Tl-LASPO was stable at 80 °C for 1 h. The highest kcat/Km value was observed in assays at 70 °C. Tl-LASPO was highly specific for l-aspartic acid. Tl-LASPO utilized fumaric acid, 2,6-dichlorophenolindophenol, and ferricyanide in addition to FAD as a cofactor under anaerobic conditions. The absorption spectrum of holo-Tl-LASPO exhibited maxima at 380 and 450 nm. The FAD dissociation constant, Kd, of the FAD-Tl-LASPO complex was determined to be 5.9 × 10−9 M.

DOI： 10.1007/s00792-017-0977-4

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

The present study reports the complete genome sequence of Leuconostoc mesenteroides strain LT-38, which is a non-spore-forming Gram-positive lactic acid bacterium. The genome is composed of a 2,022,184-bp circular chromosome and contains 2,005 putative protein-coding genes.

DOI： 10.1128/genomeA.00670-17

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

This announcement reports the complete genome sequence of strain LK-145 of Lactobacillus sakei isolated from a Japanese sake cellar as a potent strain for the production of large amounts of D-amino acids. Three putative genes encoding an amino acid racemase were identified.

DOI： 10.1128/genomeA.00656-17

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

Here, we report the complete genome sequence of strain LK-151 of Leuconostoc mesenteroides, which was isolated from a Japanese sake cellar and has the potential to produce large amounts of D-amino acids, namely, D-Ala and D-Glu. The genome contains 4 genes related to D-amino acid production.

DOI： 10.1128/genomeA.00661-17

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

Maleylacetate reductase plays a crucial role in catabolism of resorcinol by catalyzing the NAD(P)H-dependent reduction of maleylacetate, at a carbon-carbon double bond, to 3-oxoadipate. The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP-10005, GraC, has been elucidated by the X-ray diffraction method at 1.5 angstrom resolution. GraC is a homodimer, and each subunit consists of two domains: an N-terminal NADH-binding domain adopting an alpha/beta structure and a C-terminal functional domain adopting an alpha-helical structure. Such structural features show similarity to those of the two existing families of enzymes in dehydroquinate synthase-like superfamily. However, GraC is distinct in dimer formation and activity expression mechanism from the families of enzymes. Two subunits in GraC have different structures from each other in the present crystal. One subunit has several ligands mimicking NADH and the substrate in the cleft and adopts a closed domain arrangement. In contrast, the other subunit does not contain any ligand causing structural changes and adopts an open domain arrangement. The structure of GraC reveals those of maleylacetate reductase both in the coenzyme, substrate-binding state and in the ligand-free state. The comparison of both subunit structures reveals a conformational change of the Tyr326 loop for interaction with His243 on ligand binding. Structures of related enzymes suggest that His243 is likely a catalytic residue of GraC. Mutational analyses of His243 and Tyr326 support the catalytic roles proposed from structural information. The crystal structure of GraC characterizes the maleylacetate reductase family as a third family in the dehydroquinate synthase-like superfamily. (C) 2016 Wiley Periodicals, Inc.

DOI： 10.1002/prot.25046

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

The intrinsic D-amino acid profile of mouse macrophages extracted from the peritoneal cavity was analyzed using high performance liquid chromatography. Six D-amino acids (D-Asp, D-Ser, D-Ala, D-Leu, D-Gln and D-Lys) were detected in cell lysates of mouse macrophages. The content and the D/D + L ratio differed depending on the type of D-amino acid and were approximately 3.5-22 nmol/g cells, and approximately 1-20%, respectively. The D-amino acid composition of RAW 264.7 cells, which is a model macrophage cell line, was similar to that of the mouse macrophage. These results suggest that macrophages and RAW 264.7 cells with macrophage-like functions have a similar D-amino acid profile. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jpba.2015.04.028

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：INT UNION CRYSTALLOGRAPHY  

Aspartate racemase catalyzes the interconversion between L-aspartate and D-aspartate and belongs to the PLP-independent racemases. The enzyme from the lactic acid bacterium Lactobacillus sakei NBRC 15893, isolated from kimoto, is considered to be involved in D-aspartate synthesis during the brewing process of Japanese sake at low temperatures. The enzyme was crystallized at 293K by the sitting-drop vapour-diffusion method using 25%(v/v) PEG MME 550, 5%(v/v) 2-propanol. The crystal belonged to space group P3(1)21, with unit-cell parameters a = b = 104.68, c = 97.29 angstrom, and diffracted to 2.6 angstrom resolution. Structure determination is under way.

DOI： 10.1107/S2053230X15010572

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER JAPAN KK  

We successfully cloned a novel branched-chain amino acid aminotransferase (Ts-BcAT; EC 2.6.1.42) gene from the Thermococcus sp. CKU-1 genome and expressed it in the soluble fraction of Escherichia coli Rosetta (DE3) cells. Ts-BcAT is a homodimer with an apparent molecular mass of approximately 92 kDa. The primary structure of Ts-BcAT showed high homology with the fold-type I, subgroup I aminotransferases, but showed little homology with BcATs known to date, i.e., those of Escherichia coli and Salmonella typhimurium, which belong to the fold-type IV, subgroup III aminotransferases. The maximum enzyme activity of Ts-BcAT was detected at 95 A degrees C, and Ts-BcAT did not lose any enzyme activity, even after incubation at 90 A degrees C for 5 h. Ts-BcAT was active in the pH range from 4.0 to 11.0, the optimum pH was 9.5, and the enzyme was stable between pH 6 and 7. The exceptionally low pK (a) of the nitrogen atom in the Lys258 epsilon-amino group in the internal aldimine bond of Ts-BcAT was determined to be 5.52 +/- A 0.05. Ts-BcAT used 21 natural and unnatural amino acids as a substrate in the overall transamination reaction. l-Leucine and other aliphatic amino acids are efficient substrates, while polar amino acids except glutamate were weak substrates. Phylogenetic analysis revealed that Ts-BcAT is a novel fold-type I, subgroup I branched-chain aminotransferase.

DOI： 10.1007/s00792-014-0642-0

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

We performed sensory evaluations on 141 bottles of sake and analyzed the relationship between the d-amino acid concentrations, and the taste of the sake using principal component analysis, which yielded seven principal components (PC1-7) that explained 100 % of the total variance in the data. PC1, which explains 33.6 % of the total variance, correlates most positively with strong taste and most negatively with balanced tastes. PC2, which explains 54.4 % of the total variance, correlates most positively with a sweet taste and most negatively with bitter and sour tastes. Sakes brewed with "Kimoto yeast starter" and "Yamahaimoto" had high scores for PC1 and PC2, and had strong taste in comparison with sakes brewed with "Sokujo-moto". When present at concentrations below 50 μM, d-Ala did not affect the PC1 score, but all the sakes showed a high PC1 score, when the d-Ala was above 100 μM. Similar observations were found for the d-Asp and d-Glu concentrations with regard to PC1, and the threshold concentrations of d-Asp and d-Glu that affected the taste were 33.8 and 33.3 μM, respectively. Certain bacteria present in sake, especially lactic acid bacteria, produce d-Ala, d-Asp and d-Glu during storage, and these d-amino acids increased the PC1 score and produced a strong taste (Nojun). When d- and l-Ala were added to the sakes, the value for the umami taste in the sensory evaluation increased, with the effect of d-Ala being much stronger than that of l-Ala. The addition of 50-5,000 μM dl-Ala did not effect on the aroma of the sakes at all. © 2012 Springer-Verlag.

DOI： 10.1007/s00726-012-1359-y

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

We measured all of the D- and L-amino acids in 141 bottles of sakes using HPLC. We used two precolumn derivatization methods of amino acid enantiomer detection with o-phthalaldehyde and N-acetyl-L-cysteine, as well as (+)-1-(9-fluorenyl)ethyl chloroformate/1-aminoadamantane and one postcolumn derivatization method with o-phthalaldehyde and N-acetyl-L-cysteine. We found that the sakes contained the D-amino acids forms of Ala, Asn, Asp, Arg, Glu, Gin, His, Ile, Leu, Lys, Ser, Tyr, Val, Phe, and Pro. We were not able to detect D-Met, D-Thr D-Trp in any of the sakes analyzed. The most abundant D-Ala, D-Asp, and D-Glu ranged from 66.9 to 524.3 mu M corresponding to relative 34.4, 12.0, and 14.6% D-enantiomer. The basic parameters that generally determine the taste of sake such as the sake meter value (SMV; "Nihonshudo"), acidity ("Sando"), amino acid value ("Aminosando"), alcohol content by volume, and rice species of raw material show no significant relationship to the D-amino acid content of sake. The brewing water ("Shikomimizu") and brewing process had effects on the D-amino acid content of the sakes: the D-amino acid contents of the sakes brewed with deep-sea water "Kaiyoushinosousui", "Kimoto yeast starter", "Yamahaimoto", and the long aging process "Choukijukusei" are high compared with those of other sakes analyzed. Additionally, the D-amino acid content of sakes that were brewed with the adenine auxotroph of sake yeast ("Sekishoku seishu kobo", Saccharomyces cerevisiae) without pasteurization ("Hiire") increased after storage at 25 degrees C for three months. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jchromb.2011.04.006

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

A number of D-amino acids occur in nature, and there is growing interest in their function and metabolism, as well as in their production and use. Here we use the well-established L-amino-acid-producing bacterium Corynebacterium glutamicum to study whether D-amino acid synthesis is possible and whether mechanisms for the export of these amino acids exist. In contrast to Escherichia coli, C. glutamicum tolerates D-amino acids added extracellularly. Expression of argR (encoding the broad-substrate-specific racemase of Pseudomonas taetrolens) with its signal sequence deleted results in cytosolic localization of ArgR in C. glutamicum. The isolated enzyme has the highest activity with lysine (100%) but also exhibits activity with serine (2%). Upon overexpression of argR in an L-arginine, L-ornithine, or L-lysine producer, equimolar mixtures of the D-and L-enantiomers accumulated extracellularly. Unexpectedly, argR overexpression in an L-serine producer resulted in extracellular accumulation of a surplus of D-serine (81 mM D-serine and 37 mM L-serine) at intracellular concentrations of 125 mM D-serine plus 125 mM L-serine. This points to a nonlimiting ArgR activity for intracellular serine racemization and to the existence of a specific export carrier for D-serine. Export of D-lysine relies fully on the presence of lysE, encoding the exporter for L-lysine, which is apparently promiscuous with respect to the chirality of lysine. These data show that D-amino acids can also be produced with C. glutamicum and that in special cases, due to specific carriers, even a preferential extracellular accumulation of this enantiomer is possible.

DOI： 10.1128/JB.01295-10

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

The crystal structure of a L-threonine dehydrogenase (L-ThrDH; EC 1.1.1.103) from the psychrophilic bacterium Flavobacterium frigidimaris KUC-1, which shows no sequence similarity to conventional L-ThrDHs, was determined in the presence of NAD and a substrate analog, glycerol. The asymmetric unit consisted of two subunits related by a two-fold rotation axis. Each monomer consisted of a Rossmann-fold domain and a carboxyl-terminal catalytic domain. The overall fold of F. frigidimaris L-ThrDH showed significant similarity to that of UDP-galactose 4-epimerase (GalE); however, structural comparison of the enzyme with E. coli and human GalEs showed clear topological differences in three loops (loop 1, loop 2 and the NAD-binding loop) around the substrate and NAD binding sites. In F. frigidimaris L-ThrDH, loops 1 and 2 insert toward the active site cavity, creating a barrier preventing the binding of UDP-glucose. Alternatively, loop 1 contributes to a unique substrate binding pocket in the F. frigidimaris enzyme. The NAD binding loop, which tightly holds the adenine ribose moiety of NAD in the Escherichia coli and human GalEs, is absent in F. frigidimaris L-ThrDH. Consequently, the cofactor binds to F. frigidimaris L-ThrDH in a reversible manner, unlike its binding to GalE. The substrate binding model suggests that the reaction proceeds through abstraction of the beta-hydroxyl hydrogen of L-threonine via either a proton shuttle mechanism driven by Tyr143 and facilitated by Ser118 or direct proton transfer driven by Tyr143. The present structure provides a clear bench mark for distinguishing GalE-like L-ThrDHs from GalEs.

DOI： 10.1111/j.1742-4658.2010.07916.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BIOMED CENTRAL LTD  

Background: Corynebacterium glutamicum is able to grow with lactate as sole or combined carbon and energy source. Quinone-dependent L-lactate dehydrogenase LldD is known to be essential for utilization of L-lactate by C. glutamicum. D-lactate also serves as sole carbon source for C. glutamicum ATCC 13032.
Results: Here, the gene cg1027 was shown to encode the quinone-dependent D-lactate dehydrogenase (Dld) by enzymatic analysis of the protein purified from recombinant E. coli. The absorption spectrum of purified Dld indicated the presence of FAD as bound cofactor. Inactivation of dld resulted in the loss of the ability to grow with D-lactate, which could be restored by plasmid-borne expression of dld. Heterologous expression of dld from C. glutamicum ATCC 13032 in C. efficiens enabled this species to grow with D-lactate as sole carbon source. Homologs of dld of C. glutamicum ATCC 13032 are not encoded in the sequenced genomes of other corynebacteria and mycobacteria. However, the dld locus of C. glutamicum ATCC 13032 shares 2367 bp of 2372 bp identical nucleotides with the dld locus of Propionibacterium freudenreichii subsp. shermanii, a bacterium used in Swiss-type cheese making. Both loci are flanked by insertion sequences of the same family suggesting a possible event of horizontal gene transfer.
Conclusions: Cg1067 encodes quinone-dependent D-lactate dehydrogenase Dld of Corynebacterium glutamicum. Dld is essential for growth with D-lactate as sole carbon source. The genomic region of dld likely has been acquired by horizontal gene transfer.

DOI： 10.1186/1471-2180-10-321

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

We found that Corynebacterium glutamicum ATCC 13032::argF extracellularly produced a large amount of D-ornithine when cultivated in a CGXII medium containing 1 mm L-arginine. This is the first report that C. glutamicum ATCC 13032 or its mutant produces a D-amino acid extracellularly. C. glutamicum ATCC 13032::argF produced 13 mm D-ornithine in 45 h of cultivation.

DOI： 10.1271/bbb.100523

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

We succeeded in constructing the Glu219Ala/Asp225Ala (i.e., E219A/D225A) serine racemase (SerR) by site-directed mutagenesis, and the effects of Mg2+ on the catalytic efficiency and the structure were compared between the E219A/D225A-SerR and the wild-type protein. This is the first example of a serine racemase whose amino acid residues in the Mg2+-binding site were replaced with other amino acids by site-directed mutagenesis. Neither the serine racemase nor the dehydratase activities of the E219A/D225A-SerR were affected by the addition of Mg2+, and Glu219 and Asp225 of the SerR are the essential amino acid residues for Mg2+ to affect both kinds of enzyme activities. Therefore, Glu219 and Asp225 mediate the effects of Mg2+ on the activity and are important for the SerR to form the Mg2+- binding site. Judging from the difference of the K-eq values between the E219A/D225A-SerR and the SerR. Mg2+ might affect the equilibrium states in the racemase reaction. The fluorescence quenching analysis of the E219A/D225A-SerR showed that Me2+ hound to Glu219 and Asp225 of the SerR probably causes a conformational change in the ternary structure of the SerR.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-V C H VERLAG GMBH  

We found that a single intramolecular disulfide bond between the cysteines C47 and C73 exists in the primary structure of arginine racemase (ArgR) from Pseudomonas taetrolens NBRC 3460, and this is the first example of a pyridoxal phosphate (PLP)-dependent amino acid racemase that contains a disulfide bond. The amino acid racemase activity was still detected, when the disulfide bond of ArgR was disrupted by site-directed mutagenesis or reduced with dithiothreitol (DTT). The thermal and pH profiles and the quaternary structure of ArgR did not change when the disulfide bond of ArgR was disrupted by site-directed mutagenesis. The substrate specificity and the overall structure did not change when the disulfide bond of ArgR was reduced with DTT after the protein was matured. However, these properties changed when the disulfide bond of ArgR was disrupted by site-directed mutagenesis before protein maturation. The total activity of ArgR decreased when the disulfide bond of ArgR was disrupted by site-directed mutagenesis before the protein was matured or when ArgR was expressed in the cytoplasm. Based on these results, we can conclude that the disulfide bond of ArgR is essential for ArgR to fold and mature as an amino acid racemase with broad substrate specificity.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

The pyridoxal-5'-phosphate (PLP)-dependent amino acid racemases occur in almost every bacterium but may differ considerably with respect to substrate specificity. We here isolated the cloned broad substrate specificity racemase ArgR of Pseudomonas taetrolens from Escherichia coli by classical procedures. The racemase was biochemically characterized and amongst other aspects it was confirmed that it is mostly active with lysine, arginine and ornithine, but merely weakly active with alanine, whereas the alanine racemase of the same organism studied in comparison acts on alanine only. Unexpectedly, sequencing the amino-terminal end of ArgR revealed processing of the protein, with a signal peptide cleaved off. Subsequent localization studies demonstrated that in both P. taetrolens and E. coli ArgR activity was almost exclusively present in the periplasm, a feature so far unknown for any amino acid racemase. An ArgR-derivative carrying a carboxy-terminal His-tag was made and this was demonstrated to localize even in an E. coli mutant devoid of the twin-arginine translocation (Tat) pathway in the periplasm. These data indicate that ArgR is synthesized as a prepeptide and translocated in a Tat-independent manner. We therefore propose that ArgR translocation depends on the Sec system and a post-translocational insertion of PLP occurs. As further experiments showed, ArgR is necessary for the catabolism of d-arginine and d-lysine by P. taetrolens.

DOI： 10.1007/s00253-009-1942-7

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

Germinated, unpolished rice was found to contain a substantial amount of D-serine, with the ratio of the D-enantiomer to the L-enantiomer being higher for serine than for other amino acids. The relative amount of D-serine (D/(D + L)%) reached approximately 10% six days after germination. A putative serine racemase gene (serr, clone No. 001-110-B03) was found in chromosome 4 of the genomic DNA of Oryza sativa L ssp. Japonica cv. Nipponbare. This was expressed as serr in Escherichia coli and its gene product (SerR) was purified to apparent homogeneity. SerR is a homodimer with a subunit molecular mass of 34.5 kDa, and is highly specific for serine. In addition to a serine racemase reaction, SerR catalyzes D-and L-serine dehydratase reactions, for which the specific activities were determined to be 2.73 and 1.42 nkatal/mg, respectively. The optimum temperature and pH were respectively determined for the racemase reaction (35 degrees C and pH 9.0) and for the dehydratase reaction (35 degrees C and pH 9.5). SerR was inhibited by PLP-enzyme inhibitors. ATP decreased the serine racemase activity of SerR but increased the serine dehydratase activity. Kinetic analysis showed that Mg2+ increases the catalytic efficiency of the serine racemase activity of SerR and decreases that of the serine dehydratase activity. Fluorescence-quenching analysis of the tryptophan residues in SerR indicated that the structure of SerR is distorted by the addition of Mg2+, and this structural change probably regulates the two enzymatic activities. (C) 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.phytochem.2009.01.003

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BLACKWELL PUBLISHING  

Flavobacterium frigidimaris KUC-1 is a novel psychrotolerant bacterium isolated from Antarctic seawater. Malate dehydrogenase (MDH) is an essential metabolic enzyme in the citric acid cycle and has been cloned, overexpressed and purified from F. frigidimaris KUC-1. In contrast to the already known dimeric form of MDH from the psychrophile Aquaspirillium arcticum, F. frigidimaris MDH exists as a tetramer. It was crystallized at 288 K by the hanging-drop vapour-diffusion method using ammonium sulfate as the precipitating agent. The crystal diffracted to a maximum resolution of 1.80 angstrom. It contains one tetrameric molecule in the asymmetric unit.

DOI： 10.1107/S1744309107051524

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An NAD(+)-dependent alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, Flavobacterium frigidimaris KUC-1 was purified to homogeneity with an overall yield of about 20% and characterized enzymologically. The enzyme has an apparent molecular weight of 160k and consists of four identical subunits with a molecular weight of 40k. The pI value of the enzyme and its optimum pH for the oxidation reaction were determined to be 6.7 and 7.0, respectively. The enzyme contains 2 gram-atoms Zn per subunit. The enzyme exclusively requires NAD(+) as a coenzyme and shows the pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NAD(+). F. frigidimaris KUC-1 alcohol dehydrogenase shows as high thermal stability as the enzymes from thermophilic microorganisms. The enzyme is active at 0 to over 85 degrees C and the most active at 70 degrees C. The half-life time and k (cat) value at 60 degrees C were calculated to be 50 min and 27,400 min(-1), respectively. The enzyme also shows high catalytic efficiency at low temperatures (0-20 degrees C) (k (cat)/K (m) at 10 degrees C; 12,600 mM(-1 )min(-1)) similar to other cold-active enzymes from psychrophiles. The alcohol dehydrogenase gene is composed of 1,035 bp and codes 344 amino acid residues with an estimated molecular weight of 36,823. The sequence identities were found with the amino acid sequences of alcohol dehydrogenases from Moraxella sp. TAE123 (67%), Pseudomonas aeruginosa (65%) and Geobacillus stearothermophilus LLD-R (56%). This is the first example of a cold-active and thermostable alcohol dehydrogenase.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

The suborder Corynebacterianeae comprises bacteria like Mycobacterium tuberculosis and Corynebacterium glutamicum, and these bacteria contain in addition to the linear fatty acids, unique alpha-branched beta-hydroxy fatty acids, called mycolic acids. Whereas acetyl-coenzyme A (CoA) carboxylase activity is required to provide malonyl-CoA for fatty acid synthesis, a new type of carboxylase is apparently additionally present in these bacteria. It activates the alpha-carbon of a linear fatty acid by carboxylation, thus enabling its decarboxylative condensation with a second fatty acid to afford mycolic acid synthesis. We now show that the acetyl-CoA carboxylase of C. glutamicum consists of the biotinylated alpha-subunit AccBC, the beta-subunit AccD1, and the small peptide AccE of 8.9 kDa, forming an active complex of approximately 812,000 Da. The carboxylase involved in mycolic acid synthesis is made up of the two highly similar beta-subunits AccD2 and AccD3 and of AccBC and AccE, the latter two identical to the subunits of the acetyl-CoA carboxylase complex. Since AccD2 and AccD3 orthologues are present in all Corynebacterianeae, these polypeptides are vital for mycolic acid synthesis forming the unique hydrophobic outer layer of these bacteria, and we speculate that the two beta-subunits present serve to lend specificity to this unique large multienzyme complex.

DOI： 10.1128/JB.00254-07

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1128/JB.01675-06

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.M607270200

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Reversible 2,6-dihydroxybenzoate decarboxylase from Rhizobium sp. strain MTP-10005 belongs to a nonoxidative decarboxylase family. We have determined the structures of the following three forms of the enzyme: the native form, the complex with the true substrate (2,6-dihydroxybenzoate), and the complex with 2,3-dihydroxybenzaldehyde at 1.7-, 1.9-, and 1.7-angstrom resolution, respectively. The enzyme exists as a tetramer, and the subunit consists of one (alpha beta)(8) triose-phosphate isomerase-barrel domain with three functional linkers and one C-terminal tail. The native enzyme possesses one Zn2+ ion liganded by Glu(8), His(10), His(164), Asp(287), and a water molecule at the active site center, although the enzyme has been reported to require no cofactor for its catalysis. The substrate carboxylate takes the place of the water molecule and is coordinated to the Zn2+ ion. The 2-hydroxy group of the substrate is hydrogen-bonded to Asp(287), which forms a triad together with His(218) and Glu(221) and is assumed to be the catalytic base. On the basis of the geometrical consideration, substrate specificity is uncovered, and the catalytic mechanism is proposed for the novel Zn2+-dependent decarboxylation.

DOI： 10.1074/jbc.M607270200

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

We constructed the high-expression system of the alr gene from Corynebacterium glutamicum ATCC 13032 in Escherichia coli BL21 (DE3) to characterize the enzymological and structural properties of the gene product, Alr. The Alr was expressed in the soluble fractions of the cell extract of the E. coli clone and showed alanine racemase activity. The purified Alr was a dimer with a molecular mass of 78 kDa. The Alr required pyridoxal 5'-phosphate (PLP) as a coenzyme and contained 2 mol of PLP per mol of the enzyme. The holoenzyme showed maximum absorption at 420 nm, while the reduced form of the enzyme showed it at 3 10 nm. The Alr was specific for alanine, and the optimum pH was observed at about nine. The Alr was relatively thermostable, and its half-life time at 60 degrees C was estimated to be 26 min. The K-m and V-max values were determined as follows: L-alanine to D-alanine, K-m (L-alanine) 5.01 mM and V-max 306 U/Mg; D-alanine to L-alanine, K-m (D-alanine) 5.24 mM and V-max 345 U/mg. The K-eq value was calculated to be 1.07 and showed good agreement with the theoretical value for the racemization reaction. The high substrate specificity of the Alr from C glutamicum ATCC 13032 is expected to be a biocatalyst for D-alanine production from the L-counter part. (c) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jbiotec.2006.04.002

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER  

New series historic deacetylase inhibitors comprising a hydroxamic acid or 2-aminobenzamide group as a zinc-chelating function were synthesized and evaluated for antiproliferative activities against a panel of human cancer cells. The 2-aminobenzamide series inhibitors generally had the potency in cell growth inhibitions comparable to that of MS-275. Among them, the compound having a (3,4-difluorobenzyl)(2-hydroxyethyl) amino group at one end and a 2-aminobenzamide group at the other of molecule showed the most promising profile as an anticancer drug candidate, since it had a comparatively low toxicity as did MS-275 against a normal fibroblast cell CCD-1059SK. Additionally, the derivative exhibited a high recovery in human plasma stability test. (c) 2006 Elsevier SAS. All rights reserved.

DOI： 10.1016/j.ejmech.2006.02.002

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

We demonstrated several kinds Of D-amino acids in plant seedlings, and moreover alanine racemase (E.C.5.1.1.1) in alfalfa (Medicago sativa L.) seedlings. This is the first evidence for the presence of amino acid racemase in plant. The enzyme was effectively induced by the addition Of L- or D-alanine, and we highly purified the enzyme to show enzymological properties. The enzyme exclusively catalyzed racemization Of L- and D-alanine. The K-m and V-max values of enzyme for L-alanine were 29.6 x 10(-3) M and 1.02 mol/s/kg, and those for D-alanine are 12.0 x 10(-3) M and 0.44 mol/s/kg, respectively. The K-eq value was estimated to be about I and indicated that the enzyme catalyzes a typical racemization of both enantiomers of alanine. The enzyme was inactivated by hydroxylamine, phenylhydrazine and some other pyridoxal 5'-phosphate enzyme inhibitors. Accordingly, the enzyme required pyridoxal 5'-phosphate as a coenzyme, and enzymologically resembled bacterial alanine racemases studied so far. (c) 2006 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.phytochem.2006.02.017

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

A cDNA encoding a homolog of mammalian serine racemase, a unique enzyme in eukaryotes, was isolated from Arabidopsis thaliana and expressed in Escherichia coli cells. The gene product, of which the amino acid residues for binding pyridoxal 5'-phosphate (PLP) are conserved in this as well as mammalian serine racemases, catalyzes not only serine racemization but also dehydration of serine to pyruvate. The enzyme is a homodimer and requires PLP and divalent cations, Ca2+, Mg2+, Mn2+, Fe2+, or Ni2+, at alkaline pH for both activities. The racemization process is highly specific toward L-serine, whereas L-alanine, L-arginine, and L-glutamine were poor substrates. The V-max/K-m values for racemase activity of L- and D-serine are 2.0 and 1.4 nmol/mg/min/mM, respectively, and those values for L- and D-serine on dehydratase activity are 13 and 5.3 nmol/mg/min/mM, i.e. consistent with the theory of racemization reaction and the specificity of dehydration toward L-serine. Hybridization analysis showed that the serine racemase gene was expressed in various organs of A. thaliana. (c) 2006 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.phytochem.2006.01.003

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記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.80.3_152

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER IRELAND LTD  

Tunicamycin, an inhibitor of the glycosylation of newly biosynthesized proteins, induces endoplasmic reticulum (ER) stress and subsequent apoptosis, and caspase family proteases are activated during the process of ER stress-mediated apoptosis. In the present study, we showed that thapsigargin (Th), an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA), also induced ER stress-mediated apoptosis, and nerve growth factor (NGF) prevented the apoptosis in PC 12 cells. We also found that LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), reduced the survival of cells treated with NGF for 24 It in the presence of Th. We discovered that the activities of caspase-3, -9 and -12 were increased time-dependently after the treatment with Th, and NGF suppressed the Th-triggered activation of caspase-3, -9 and -12. LY294002 diminished the effect of NGF on the inactivation of all these caspases. These results indicate that the NGF-induced PI3-K signaling pathway prevents Th-triggered ER stress-specific apoptosis via inhibition of caspase-mediated apoptotic signal. (C) 2005 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2005.07.030

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER IRELAND LTD  

Tunicamycin, an inhibitor of the glycosylation of newly biosynthesized proteins, induces endoplasmic reticulum (ER) stress and subsequent apoptosis, and caspase family proteases are activated during the process of ER stress-mediated apoptosis. In the present study, we showed that thapsigargin (Th), an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA), also induced ER stress-mediated apoptosis, and nerve growth factor (NGF) prevented the apoptosis in PC 12 cells. We also found that LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), reduced the survival of cells treated with NGF for 24 It in the presence of Th. We discovered that the activities of caspase-3, -9 and -12 were increased time-dependently after the treatment with Th, and NGF suppressed the Th-triggered activation of caspase-3, -9 and -12. LY294002 diminished the effect of NGF on the inactivation of all these caspases. These results indicate that the NGF-induced PI3-K signaling pathway prevents Th-triggered ER stress-specific apoptosis via inhibition of caspase-mediated apoptotic signal. (C) 2005 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2005.07.030

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

We purified the psychrophilic and thermolabile malate dehydrogenase to homogeneity from a novel psychrotolerant, Flavobacterium frigidimaris KUC-1, isolated from Antarctic seawater. The enzyme was a homotetramer with a molecular weight of about 123k and that of the subunit was about 32 k. The enzyme required NAD(P)(+) as a coenzyme and catalyzed the oxidation Of L-malate and the reduction of oxalacetate specifically. The reaction proceeded through an ordered bi-bi mechanism. The enzyme was highly susceptible to heat treatment, and the half-life time at 40 degrees C was estimated to be 3.0 min. The k(cat)/K-m (mu m(-1).s(-1)) values for L-malate and NAD(+) at 30 degrees C were 289 and 2,790, respectively. The enzyme showed pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of the coenzyme. The enzyme contained 311 amino acid residues and much lower numbers of proline and arginine residues than other malate dehydrogenases.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER GMBH, URBAN & FISCHER VERLAG  

We described the polyphasic characterization of the psychrotolerant isolated from Antarctic seawater. The strain was closely related to Flarobacterium hydatis, F. pectinororum, and F. saccharophilum on the basis of the 16S rDNA sequence analysis. However, DNA-DNA hybridization experiments showed that the DNA-similarities between strain KUC-1(T) and the reference strains of Flavobacterium were less than 30%. Therefore, we can definite a new species of Flavobacterium phylogenetically, and strain KUC-1(T) can be considered to be a new species of Flavobacterium. i.e. F. frigidimaris (KUC-1(T): JCM 12218(T) and DSM 15937(T): mol% G + C or DNA or the type strain is 34.5 mol%), Useful phenotypical features for discrimination of F. frigidimaris from other Flavobacterium species, such as a resistance to NaCl, optimum growth temperature. and cellular fatty acid composition, were also determined. (c) 2005 Elsevier GmbH. All rights reserved.

DOI： 10.1016/j.syapm.2005.01.001

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER GMBH, URBAN & FISCHER VERLAG  

We described the polyphasic characterization of the psychrotolerant isolated from Antarctic seawater. The strain was closely related to Flarobacterium hydatis, F. pectinororum, and F. saccharophilum on the basis of the 16S rDNA sequence analysis. However, DNA-DNA hybridization experiments showed that the DNA-similarities between strain KUC-1(T) and the reference strains of Flavobacterium were less than 30%. Therefore, we can definite a new species of Flavobacterium phylogenetically, and strain KUC-1(T) can be considered to be a new species of Flavobacterium. i.e. F. frigidimaris (KUC-1(T): JCM 12218(T) and DSM 15937(T): mol% G + C or DNA or the type strain is 34.5 mol%), Useful phenotypical features for discrimination of F. frigidimaris from other Flavobacterium species, such as a resistance to NaCl, optimum growth temperature. and cellular fatty acid composition, were also determined. (c) 2005 Elsevier GmbH. All rights reserved.

DOI： 10.1016/j.syapm.2005.01.001

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The Corynebacterianeae such as Corynebacterium glutamicum and Mycobacterium tuberculosis possess several unique and structurally diverse lipids, including the genus-specific mycolic acids. Although the function of a number of genes involved in fatty acid and mycolic acid biosynthesis is known, information relevant to the initial steps within these biosynthetic pathways is relatively sparse. Interestingly, the genomes of Corynebacterianeae possess a high number of accD genes, whose gene products resemble the beta-subunit of the acetyl-CoA carboxylase of Escherichia coli, providing the activated intermediate for fatty acid synthesis. We present here our studies on four putative accD genes found in C. glutamicum. Although growth of the accD4 mutant remained unchanged, growth of the accD1 mutant was strongly impaired and partially recovered by the addition of exogenous oleic acid. Overexpression of accD1 and accBC, encoding the carboxylase alpha-subunit, resulted in an 8-fold increase in malonyl-CoA formation from acetyl-CoA in cell lysates, providing evidence that accD1 encodes a carboxyltransferase involved in the biosynthesis of malonyl-CoA. Interestingly, fatty acid profiles remained unchanged in both our accD2 and accD3 mutants, but a complete loss of mycolic acids, either as organic extractable trehalose and glucose mycolates or as cell wall-bound mycolates, was observed. These two carboxyltransferases are also retained in all Corynebacterianeae, including Mycobacterium leprae, constituting two distinct groups of orthologs. Furthermore, carboxyl fixation assays, as well as a study of a Cg-pks deletion mutant, led us to conclude that accD2 and accD3 are key to mycolic acid biosynthesis, thus providing a carboxylated intermediate during condensation of the mero-chain and alpha-branch directed by the pks-encoded polyketide synthase. This study illustrates that the high number of accD paralogs have evolved to represent specific variations on the well known basic theme of providing carboxylated intermediates in lipid biosynthesis.

DOI： 10.1074/jbc.M408648200

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.M408648200

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1128/JB.186.20.6855-6863.2004

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記述言語：英語   出版者・発行元：ELSEVIER SCIENCE BV  

NAD(P)(+)-dependent aldehyde dehydrogenase (EC 1.2.1.5) and aspartase (EC 4.3.1.1) in the cells of an atypical psychrophile from Antarctic seawater, Cytophaga sp. KUC-1, were paradoxically thermostable, although they derived from a psychrophile. Both enzymes showed the highest activity at about 55 degreesC, and also active even under cold conditions. The enzymes contained more lie residues than the enzymes from mesophiles. The Ile/Ile + Val + Len ratio of the Cytophaga thermostable enzymes was much higher than that of the enzymes from mesophiles. As compared with the enzymes from other microorganisms, the Cytophaga thermostable enzymes have the structural differences in the C-terminal region of the enzymes. Therefore, the C-terminal region might be important for the paradoxical thermostability of the enzymes. The psychrophilic microorganism produces not only psychrophilic enzyme, but thermostable enzyme with psychrophilicity. Therefore, the psychrophilic microorganism is one of the candidates for isolation of novel biocatalysts, which have potential for various industrial applications. (C) 2003 Elsevier B.V. All rights reserved.

DOI： 10.1016/S1381-1177(03)00073-0

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

A psychrophilic bacterium, Cytophaga sp. strain KUC-1, that abundantly produces a NAD+-dependent L-threonine dehydrogenase was isolated from Antarctic seawater, and the enzyme was purified. The molecular weight of the enzyme was estimated to be 139,000, and that of the subunit was determined to be 35,000. The enzyme is a homotetramer. Atomic absorption analysis showed that the enzyme contains no metals. In these respects, the Cytophaga enzyme is distinct from other L-threonine dehydrogenases that have thus far been studied. L-Threonine and DL-threo-3-hydroxynorvaline were the substrates, and NAD(+) and some of its analogs served as coenzymes. The enzyme showed maximum activity at pH 9.5 and at 45 degreesC. The kinetic parameters of the enzyme are highly influenced by temperatures. The K-m for L-threonine was lowest at 20 degreesC. Dead-end inhibition studies with pyruvate and adenosine-5'-diphosphoribose showed that the enzyme reaction proceeds via the ordered Bi Bi mechanism in which NAD(+) binds to an enzyme prior to L-threonine and 2-amino-3-oxobutyrate is released from the enzyme prior to NADH. The enzyme gene was cloned into Escherichia coli, and its nucleotides were sequenced. The enzyme gene contains an open reading frame of 939 by encoding a protein of 312 amino acid residues. The amino acid sequence of the enzyme showed a significant similarity to that of UDP-glucose 4-epimerase from Staphylococcus aureus and belongs to the short-chain dehydrogenase-reductase superfamily. In contrast, L-threonine dehydrogenase from E. coli belongs to the medium-chain alcohol dehydrogenase family, and its amino acid sequence is not at all similar to that of the Cytophaga enzyme. L-Threonine dehydrogenase is significantly similar to an epimerase, which was shown for the first time. The amino acid residues playing an important role in the catalysis of the E. coli and human UDP-glucose 4-epimerases are highly conserved in the Cytophaga enzyme, except for the residues participating in the substrate binding.

DOI： 10.1128/JB.185.15.4483-4489.2003

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JAPANESE BIOCHEMICAL SOC  

We found that a psychrophilic bacterium isolated from Antarctic seawater, Cytophaga sp. KUC-1, abundantly produces aspartase [EC4.3.1.1], and the enzyme was purified to homogeneity. The molecular weight of the enzyme was estimated to be 192,000, and that of the subunit was determined to be 51,000: the enzyme is a homotetramer. L-Aspartate was the exclusive substrate. The optimum pH in the absence and presence of magnesium ions was determined to be pH 7.5 and 8.5, respectively. The enzyme was activated cooperatively by the presence of L-aspartate and by magnesium ions at neutral and alkaline pHs. In the deamination reaction, the K-m value for L-aspartate was 1.09 mM at pH 7.0, and the S-1/2 value was 2.13 mM at pH 8.5. The V-max value were 99.2 U/ mg at pH 7.0 and 326 U/mg at pH 8.5. In the deamination reaction, the Km values for fumarate and ammonium were 0.797 and 25.2 mM, respectively, and V-max was 604 U/ mg. The optimum temperature of the enzyme was 55degreesC. The enzyme showed higher pH and thermal stabilities than that from mesophile: the enzyme was stable in the pH range of 4.5-10.5, and about 80% of its activity remained after incubation at 50degreesC for 60 min. The gene encoding the enzyme was cloned into Escherichia coli, and its nucleotides were sequenced. The gene consisted of an open reading frame of 1,410-bp encoding a protein of 469 amino acid residues. The amino acid sequence of the enzyme showed a high degree of identity to those of other aspartases, although these enzymes show different thermostabilities.

DOI： 10.1093/jb/mvg012

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JAPANESE BIOCHEMICAL SOC  

D-Arginase activity was found in the cells of an isolate, Arthrobacter sp. KUJ 8602, grown in the L-arginine medium, and the enzyme was purified and characterized. Its molecular weight was estimated to be about 232,000 by gel filtration, and that of the subunit was approximately 40,000 by SDS-PAGE, suggesting that the enzyme is a homohexamer. The enzyme acted on not only D-arginine but also 4-guanidinobutyrate, 3-guanidinopropionate and even L-arginine. The V-max/K-m values for 4-guanidinobutyrate and D-arginine were determined to be 87 and 0.81 mumol/min/mg/mM, respectively. Accordingly, the enzyme is regarded as a kind of guanidinobutyrase [EC 3.5.3.7]. The pH optima for 4-guanidinobutyrate and D-arginine were 9.0 and 9.5, respectively. The enzyme was inhibited competitively by 5-aminovalerate, and thiol carboxylates such as mercaptoacetate served as strong mixed-type inhibitors. The enzyme contained about 1 g-atom of firmly bound Zn2+ per mol of subunit, and removal of the metal ions by incubation with 1,10-phenanthroline resulted in loss of activity. The inactivated enzyme was reactivated markedly by incubation with either Zn2+ or Co2+, and slightly by incubation with Mn2+. The nucleotide sequence of enzyme contains an open reading frame that encodes a polypeptide of 353 amino acid residues (M,: 37,933). The predicted amino acid sequence contains sequences involved in the binding of metal ions and the guanidino group of the substrate, which show a high homology with corresponding sequences of Mn2+-dependent amidinohydrolases such as agmatinase from Escherichia coli and L-arginase from rat liver, though the homology of their entire sequences is relatively low (24-43%).

DOI： 10.1093/jb/mvg016

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D-アミノ酸は、非天然型と見なされ生体内に存在するアミノ酸はすべてL-アミノ酸であると考えられてきた。しかし近年、ヒトを含む哺乳動物の特定の部位に高濃度の遊離型D-アミノ酸が存在することが報告され、それらの由来や生理機能に関心が寄せられている。本研究では、従来不分明の状態にある食品、とりわけ飲料中の各種D-アミノ酸に焦点を当て、食品中に含まれるD-アミノ酸の量的分布を明らかにするとともに、D-アミノ酸の生成機構や生理機能を解明する。

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食品中セレンの栄養有効性は種々の要因によって変動するため、食品中セレンの化学分析値と栄養有効性の間にはしばしば齟齬が生ずる。魚肉は日本人の主要なセレン供給源であるが、含有されるセレンの栄養有効性は他の食品由来のセレンに比較して低いと信じられている。魚肉中セレンの低栄養有効性の原因として、しばしば共存する水銀の影響が指摘されている。しかし、水銀含有量が低い魚肉製品でも、含有セレンが低栄養有効性を示すと報告されており、魚肉中セレンの一部が栄養有効性の低い化学種である可能性も否定できない。 従来、食品中の微量セレンの化学種の同定には、化学試薬との反応性を検討するなどの間接的な手法が多く用いられてきたため、食品中セレンを化学種ごとに分別定量することは困難であった。近年、高速液体クロマトグラフィー（HPLC）で分離したセレン化合物を誘導結合プラズマ質量分析器（ICPMS）で特異的に検出する手法が開発・普及しており、種々の食品に含有されるセレンの化学種の同定が可能となりつつある。 本研究では、魚肉の中でもセレン含有量がきわめて高いマグロの血合肉について、その化学種の同定をHPLC-ICPMSを用いて試みた。

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アルコールデヒドロゲナーゼ（AlcDH）とアルデヒドデヒドロゲナーゼ（AldDH）は共にアルコールとアルデヒド代謝に中心的位置を占め、前者は各基質の可逆的,後者は不可逆的脱水素反応を触媒するNAD(P)要求性酵素である。馬肝臓のAlcDHはZnを含み、哺乳類、常温性微生物由来の両酵素に関する研究は多いが極限微生物起源のこれらの酵素については不分明なままである。我々は先に南極海水由来の低温菌Cytophagasp.KUC-1株が著量のAlcDH及びAldDHを生産することを見いだした。本研究ではこの両酵素の酵素科学的諸性質を明らかにするとともに高発現系を構築し、その精製酵素を用いる高感度のアルコール定量法を開発した。

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

We found the occurrence of NAD(P)--dependent aldehyde dehydrogenase (EC1.2.1.5) in the cells of a psychrophile from Antarctic seawater, Cytophaga sp. KUC-1, and purified to homogeneity. About 50% of the enzyme activity remained even after heating at 50°C for 65 min and the highest activity was observed in the range of 55-60°C. The enzyme was thermostable and thermophilic, although it was derived from a psychrophile. The circular dichroism at 222 nm of the enzyme showed a peak at 32°C. This temperature was closely similar to the transition temperature in the Arrhenius plots. The stereospecificity for the hydride transfer at C4-site of nicotinamide moiety of NADH was pro-R. The gene encoding the enzyme consisted of an open reading frame of 1506-bp encoding a protein of 501 amino acid residues. The significant sequence identity (61%) was found between the Cytophaga and the Pseudomonas aeruginosa enzymes, although their thermostabilities are completely different. © 2002 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0006-291X(02)02523-8

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酵素は一般的に基質特異性が高いが性質や構造が類似する基質アナログに対しても，しばしば作用する。酵素反応では基質の-COOHと-SOOHは類似する反応性を示す。例えばアスパラギン酸のアナログであるシステインスルフィン酸は，アスパラギン酸アミノトランスフェラーゼやアスパラギン酸デカルボキシラーゼに対して高い反応性を示す。しかしグルタミン酸のアナログであるホモシステインスルフィン酸(HCS)などについては不分明な状態にある。本研究では，グルタミン酸のアナログとしてHCSやホモシステインスルフォン酸(HCA)を，またグルタミンのアナログとしてホモシステインスルフォンアミド(HCN)を取り上げ，これらを科学的に合成し，様々なグルタミン酸及びグルタミン代謝関連酵素に対する反応性を調べた。

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グアニジノ化合物を尿素とアミノ化合物へ加水分解する反応を触媒するアミジン加水分解酵素は、主にMn2+で活性化することが知られており,中でもL-アルギナーゼ（L-arginine amidinohydrolase, EC 3.5.3.1）は自然界に広く存在する。これらの中で種々のL-アルギナーゼやアグマチナーゼ（agmatine ureohydrolase, EC 3.5.3.11）はすでにクローニングされ、そのほとんどがMn2+要求性アミジン加水分解酵素であり、これらの推定アミノ酸配列は各々部分的に高い相同性を示す。このため、これらの酵素は共通の起源から分岐し,異なる基質特異性を持って進化したと考えられている。現在、L-アルギナーゼ分子と反応速度論的性質に関する研究が報告され,さらにラット肝臓L-アルギナーゼの3次元結晶構造が明らかになり,Mn2+が関与したL-アルギニン加水分解の反応機構が予測された。 一方、グアニジノブチラーゼ（4-guanidinobutyrate amidinohydrolase, EC 3.5.3.7）についてはPseudomonas属細菌やコリネ型細菌での存在が知られており,爬虫類や鳥類の肝臓においてもその活性が認められている。これらの酵素もまた,コリネ型細菌のBrevibacterium由来グアニジノブチラーゼが活性にZn2+を必要とすることを除いて、Mn2+要求性酵素である。しかし,グアニジノブチラーゼの金属イオンや一次構造については不分明なままである。 我々が土壌より単離したD-アルギニン資化性菌Arthrobacter sp. KUJ 6802から精製したグアニジノブチラーゼはMn2+を要求しなかった。本研究では本酵素の一次構造を明らかにし、本酵素の触媒過程における金属イオンの役割を解明することを目的としている。

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：LIPPINCOTT WILLIAMS & WILKINS  

To determine the cytotoxic mode of action of a glutathione (GSH)-doxorubicin (DXR) conjugate, which exhibited point cytotoxicity against various multidrug-resistant as well as DXR-sensitive cell lines, the molecular interaction between covalent GSH-DXR conjugates and glutathione-S-transferase (GST), a possible molecular target of the conjugates, was investigated. The following four GSH molecules with stereoisomeric forms were prepared: L-Glu-L-Cys-Gly (LL-GSH), D-Glu-L-Cys-Gly (DL-GSH), L-Glu-D-Cys-Gly (LD-GSH) anal D-Glu-D-Cys-Gly (DD-GSH). The enzymic activity of GST against each GSH stereoisomer was 88, 38, 8 and 4 nmol/ mg/min, respectively, suggesting that the L-form cysteine residue in the molecule was an important substrate of GST. Addition of DXR conjugated with each isomer (10 muM) to a GSH-containing GST assay mixture inhibited the GST activity to 32% for LL-GSH-DXR, 16% for DL-GSH-DXR and 61% for LD-GSH-DXR as compared with the solvent control. Moreover, IC,, values for these conjugates were 30, 20 and 250 nM, respectively. The cytocidal activity of each conjugate corresponded to the substrate specificity of GST activity for the GSH isomer. These conjugates bound to the GST molecule, and the binding ability was 0.746, 0.627 and 0.462 mol/mol of GST for LL-GSH-DXR, DL-GSH-DXR and LD-GSH-DXR, respectively. These findings suggested that GSH-DXR interacted with the substrate-binding site of the GST molecule and inhibition of GST activity exhibited potent cytotoxicity. [(C) 2001 Lippincott Williams & Wilkins.].

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The transglycosylation of p-nitrophenyl-β-D-cellotrioside to cellotetraose catalyzed by endo-1,4-β-glucanase (cellulase, EC 3.2.1.4) from a psychrotrophic yeast, Phodotorula glutinis KUJ 2731, was increased by addition of a miscible organic solvent in the reaction mixture.Among various organic solvents tested, acetone was most effective.The transglycosylation activity increased with an increase in acetone concentrations, while hydrolysis activity was suppressed .The transglycosylation preferably occurred at acidic pH with the optimum pH at 2 in 10mΜ Gly-HCl buffer. The optimum temperature of transglycosylation was found to be 50℃ in the presence of 40℃ acetone.

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A new one-pot chemo-enzymatic procedure was developed for enantiomerization of racemates based on enzymatic enantiospecific oxidation of a substrate and chemical non-enantiospecific reduction of the product. The principle is shown as follows for the L-proline production.L-Pro ←ゥゥゥゥゥゥゥゥゥゥゥゥゥ・ ・ ・ ゥゥゥゥゥゥゥゥゥゥゥゥゥゥゥゥ・ ↓ ・D-Pro―D-Amino acid oxidase→△1-Pyrroline-2-carboxylic acidL-Proline and L-pipecolate were produced from racemic proline and pipecolate by means of D-amino acid oxidase and sodium borohydride in high yield in this reaction system [J. W. Huh, K. Yokoigama, N. Esaki, K. Soda, Biosci., Biotechnol., Biochem. 56 (1992) 2081].DL-and L-Lactate were DL-enantiomerized in a one-pot reaction systems containing L-lactate oxidase and sodium borohydride in the similar mannar [S. Mukoyama, K. Yamanaka, T. Oikawa, K. Soda, Nippon Nogei Kagaku Kaishi 73 (1999) 62].Pyruvate was also converted to an equimolar amount of D-lactate in the same system. D-α-Hydroxybutyrate can be produced from the DL- and L-isomers, and α-ketobutyrate in the same manner though slowly.This method is applicable to production of other chiral compounds from the corresponding racemates. Ⓒ2001 Elsevier Science B. V. All right reserved.

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Abstract:┏B/┓ We investigated the total conversion of racemic lactate, L-lactate, and pyruvate into D-lactate, which is very useful as a starting material for the synthesis of chiral compounds and much more valuable than the L-enantiomer by means of coupling of L-specific oxidation of the recemate with L-lactate oxidase and non-enantiospecific reduction of pyruvate to DL-lactate with sodiumborohydride.In this one-pot system, L-lactate was enantiospecifically oxidized to an achiral product, pyruvate, which was chemically reduced to DL-lactate leading to a turnover.Consequently, either DL-lactate, L-lactate, or pyruvate was fully converted to the D-enantiomer.We optimized the reaction conditions: DL-lactate was converted to D-lactate in 99% of the theoretical yield and with more than 99% enantiomeric excess.DL-α-Hydroxybutyrate and α- ketobutyrate were converted also to D-α- hydroxybutyrate in the same way, though slowly.Ⓒ2001

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Academic Press Inc.  

X-ray crystallographic studies revealed that various amino acid dehydrogenases fold into two domains in each subunit, a substrate-binding domain and an NAD(P)+-binding domain (Baker, P. J., Turnbull, A. P., Sedelnikova, S. E., Stillman, T. J., and Rice, D. W. (1995) Structure 3, 693-705). To elucidate the function and folding process of these two domains, we have genetically constructed a fragmentary form of thermostable leucine dehydrogenase of Bacillus stearothermophilus consisting of an N-terminal polypeptide fragment corresponding to the substrate-binding domain including an N-terminus, and a C-terminal fragment corresponding to the NAD+-binding domain. The two peptide fragments were expressed in separate host cells and purified. When both fragments were mixed, the leucine dehydrogenase activity with a specific activity of 1.4% of that of the wild-type enzyme appeared. This suggests that both peptide fragments mutually recognize each other, associate and fold correctly to be catalytically active, although the activity is low. However, the fragmentary form of enzyme produced catalyzed the oxidative deamination of L-leucine, L-isoleucine, and L-valine with broad substrate specificity compared to that of the wild-type enzyme. The fragmentary enzyme retained more than 75% of the initial activity after heating at 50°C for 60 min. The fragmentary enzyme was more stable on heating than separate peptide fragments. These results suggest that the two domains of leucine dehydrogenase probably fold independently, and the two peptide fragments interact and associate with each other to form a functional active site. © 2001 Academic Press.

DOI： 10.1006/bbrc.2001.4252

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

We found the occurrence of valine dehydrogenase in the cell extract of a psychrophilic bacterium, Cytophaga sp. KUC-1, isolated from Antarctic seawater and puriffied the enzyme to homogeneity. The molecular mass of the enzyme was determined to be ≈ 154 kDa by gel filtration and that of the subunit was 43 kDa by SDS/PAGE: the enzyme was a homotetramer. The enzyme required NAD+ as a coenzyme, and catalyzed the oxidative deamination of L-valine, L-isoleucine, L-leucine and the reductive amination of α-ketoisovalerate, α-ketovalerate, α-ketoisocaproate, and α-ketocaproate. The reaction proceeds through an isoordered bi-bi mechanism. The enzyme was highly susceptible to heat treatment and the half-life at 45 °C was estimated to be 2.4 min. The kcat/Km (μ-1·s-1) values for L-valine and NAD+ at 20 °C were 27.48 and 421.6, respectively. The enzyme showed pro-S stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of coenzyme. The gene encoding valine dehydrogenase was cloned into Escherichia coli (Novablue), and the primary structure of the enzyme was deduced on the basis of the nucleotide sequence of the gene encoding the enzyme. The enzyme contains 370 amino-acid residues, and is highly homologous with S. coelicolor ValDH (identity, 46.7%) and S. fradiae ValDH (43.1%). Cytophaga sp. KUC-1 ValDH contains much lower numbers of proline and arginine residues than those of other ValDHs. The changes probably lead to an increase in conformational flexibility of the Cytophaga enzyme molecule to enhance the catalytic activity at low temperatures.

DOI： 10.1046/j.1432-1327.2001.02353.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

We studied production of D-glutamate from L-glutamate using a bioreactor consisting of two columns of sequentially connected immobilized glutamate racemase (EC 5.1.1.3, from Bacillus subtilis no 3336) and L-glutamate oxidase (EC 1.4.3.11, from Streptomyces sp. X119-6): L-glutamate was racemized by the glutamate racemase column, and then L-glutamate was oxidized by the L-glutamate oxidase column. Consequently only D-glutamate remained, and was easily separated from the alpha-ketoglutarate formed by anion-exchange chromatography. Both enzymes were highly stabilized by immobilization. The pH and temperature optima of immobilized glutamate racemase (pH 8, 40 degrees C) were similar to those of immobilized L-glutamate oxidase (pH 7, 50 degrees C). Accordingly, we connected the two columns tandemly to do both enzyme reactions under the same conditions. Actually 4.5 mu mol of D-glutamate was produced and isolated from 10 mu mol of L-glutamate, about 90% of the theoretical yield.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CENTER ACADEMIC PUBL JAPAN  

We synthesized a series of stereoisomers of glutathione (GSH) and glutathione disulfide (GSSG) by the solid-phase method. These peptides were used to examine their reactivities with enzymes acting on glutathione. The glutathione reductase of yeast acted only on LL-GSSG. Glutathione S-transferase catalyzed the conjugation of 1-chloro-2,4-dinitrobenzene with LL-GSH and DL-GSH (Km (mM): for LL-GSH, 0.035; and for DL-GSH, 0.62), but the DD- and LD-diastereomers were inert, gamma-Glutamyl transpeptidase catalyzed the transfer of gamma-glutamyl moiety of LL-GSH and DL-GSH to taurine forming gamma-glutamyl taurine and cysteinyl taurine (Km (mM): for LL-GSH, 0.336; and for DL-GSH, 0.628), but the other diastereomers were not the substrates. The occurrence of L-cysteinyl residue in the tripeptides is required for the glutathione analogue to be a substrate of the enzymes.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：IOS PRESS  

Various vitamin B-6 enzymes play important roles in mammalian and microbial metabolism of selenium amino acids. Selenocysteine is synthesized from selenohomocysteine by catalysis of cystathionine beta-synthase and cystathionine gamma-lyase, which both require pyridoxal phosphate. Selenocysteine beta-lyase, a new B-6-enzyme, exclusively catalyzes beta-elimination of selenocysteine, and occurs in mammalian systems and bacteria. Methionine gamma-lyase, cysteine desulfurase, cysteine sulfinate desulfinase, and D-selenocystine alpha, beta-lyase, which are B-6-enzymes, act on cysteine, cysteine sulfinate, D-cystine, and their derivatives, and their selenium counterparts indiscriminately. Their reaction mechanisms are comparatively described.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

A psychrotrophic yeast, Rhodotorula glutinis KUJ 2731, isolated from soil, effectively produced an extracellular endo-beta-glucanase (EC 3.2.1.4). The enzyme was monomeric, and the molecular mass was about 40,000 Da. The N-terminal amino acid sequence was H-Ser-Leu-Pro-Lys-Leu-Gly-Gly-Val-Asp-Leu-Ala-Gly-Leu-Asp-Ile-Gly-Lys-Asp-Lys-Asn-. alpha-Helix content was calculated to be about 32.6%. The isoelectric point was 8.57. The activation energy was 20.9 kJ/mol, which was much smaller than that of mesophilic enzymes. The enzyme was active at temperatures from 0 to 70 degrees C, with a highest initial velocity at 50 degrees C similar to other psychrotrophic enzymes. The enzyme was inhibited by Hg2+. The enzyme catalyzed hydrolysis of carboxymethyl cellulose with an apparent K-m of 1.1% and V-max of 556 mu mol/min/mg. Products from the enzymatic hydrolysis of carboxymethyl cellulose by the enzyme were glucose, cellobiose, and cellotriose. The enzyme also catalyzed the transglycosylation of p-nitrophenyl-beta-cellotrioside to cellotetraose.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

We purified a novel type of D-mannitol dehydrogenase, which contains a c-type cytochrome and an unknown chromophore in the soluble fraction of an acetic acid bacterium, Acetobacter xylinum KU-1, to homogeneity. The enzyme showed the maximum activity at pH 5 and 40 degrees C. It was stable up to 60 degrees C at pH 6, and was inhibited by Hg2+ and p-quinone (K-i = 0.18 mM). The molecular weight of the enzyme was about 140,000, and those of the subunits were 69,000, 51,000, and 20,000; the enzyme is hetero-trimeric and contained 8 g-atoms of Fe per mole. The alpha-helix was estimated to be about 52.9%. The enzyme catalyzed phenazine methosulfate dependent oxidation of D-mannitol with an apparent K-m of 98 mu M (for D-mannitol) and V-max of 213 mu mol/min/mg. The reduced form of the enzyme showed the absorption maxima at 386, 416, 480, 518, 550, and 586 nm, which are attributable to a c-type cytochrome in the enzyme.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER VERLAG  

A cellulose-producing acetic acid bacterium, Acetobacter xylinum KU-1, abundantly produces an extracellular endo-beta-glucanase (EC 3.2.1.4) in the culture broth. The enzyme was purified to homogeneity by DEAE- and CM- Toyopearl 650M ion-exchange chromatography, Butyl-Toyopearl 650M hydrophobic chromatography, and Toyopearl HW-50 gel filtration. The purified enzyme showed the maximum activity at pH 5 and 50 degrees C: it was stable up to 50 degrees C at pH 5, activated by Co2+, and competitively inhibited by Hg2+; the apparent K-i was 7 mu m. The molecular weight of the enzyme was determined to be about 39,000 by sodium dodesyl sulfate/polyacrylamide gel electrophoresis, and about 41,000 by Toyopearl HW-50 gel filtration; the enzyme is monomeric. The enzyme hydrolyzed carboxy-methylcellulose with an apparent K-m of 30 mg/ml and V-max of 1.2 mu M/min. It hydrolyzed cellohexaose to cellobiose, cellotriose and cellotetraose, and also cellopentaose to cellobiose and cellotriose, but did not act on cellobiose, cellotriose, or cellotetraose.

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記述言語：英語   出版者・発行元：TAYLOR & FRANCIS LTD  

We found that Acetobacter xylinum KU-1 produced cellulose from D-arabitol. The maximum cellulose production was obtained when it was grown in a medium containing 2.0% D-arabitol, 1.0% tryptone, and 1.0% yeast extract (pH 5) at 30 degrees C for 96h statically. The productivity was more than 6 times as much as that of D-glucose [productivity (mg/ml-medium): from D-arabitol, 12.4; from D-glucose, 2.0].

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記述言語：英語   出版者・発行元：TAYLOR & FRANCIS LTD  

We found that Acetobacter xylinum KU-1 produced cellulose from D-mannitol. The optimum culture conditions for cellulose production were 1.5% D-mannitol, 0.5% Polypeptone, 2.0% yeast extract, pH 5, 30 degrees C, and 48 h. The amount of cellulose from D-mannitol was more than 3 times as much as that from D-glucose under the same culture conditions [productivity (mg/ml-medium): from D-mannitol, 4.6; from D-glucose, 1.2].

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記述言語：英語   出版者・発行元：TAYLOR & FRANCIS LTD  

We detected carboxymethyl cellulase activity in a crude extract of Acetobacter xylinum KU-1. The enzyme activity was detected when glycerol, D-fructose, D-mannitol, D-glucose, D-arabitol, D-sorbitol, or carboxynmethyl cellulose was used as a carbon source. The optimum pH was found to be 4.0, while the optimum temperature was 50 degrees C. The enzyme activity was inhibited characteristically by the addition of Hg2+.

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記述言語：英語   出版者・発行元：TAYLOR & FRANCIS LTD  

We detected dye-linked D-mannitol dehydrogenase activity in the crude extract of Acetobacter xylinum KU-1. The enzyme activity was specific for D-mannitol, and not pyridine nucleotide (NAD+, NADP+)-dependent. The optimal pH was found to be 5.0, while the optimal temperature was at 50-degrees-C. The enzyme activity was inhibited by p-quinone noncompetitively.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

Grant-in-Aid for Scientific Research 199204-199304

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JAPAN SOC BIOSCI BIOTECHN AGROCHEM  

Rhodopseudomonas acidophila M402 grown under aerobic-dark condition shows two band of dye-linked aldehyde dehydrogenase activity by polyacrylamide gel electrophoresis. The slower-migrating band coincided with the dye-linked aromatic alcohol dehydrogenase that was active on aromatic and aliphatic alcohols and aldehydes. The faster-migrating band was a new dye-linked aldehyde dehydrogenase. The latter enzyme was purified 125-fold by ultracentrifugation and column chromatographies on DEAE-cellulose, Bio-Gel HTP, and Sepharose CL-6B. The enzyme has a relative molecular mass of 70,000 daltons, and a subunit size of 35,000 dalton indicates the dehydrogenase has a dimeric structure. The isoelectric point was pH 4.74. NAD+ and NADP+ do not act as electron acceptors. The enzyme was active specifically on straight chain aldehydes (C3-C10) rather than on benzaldehyde and its substitutes. Aromatic and aliphatic alcohols were inert for this enzyme. The Michaelis constant (mM) were 1.6, 0.6, 0.9, 3.6, 0.5, 0.3, 0.1, 0.9, 0.6, 0.3, and 0.1 for benzaldehyde, m-hydroxybenzaldehyde, m-anisaldehyde, vanillin, propionaldehyde, butyraldehyde, hexaldehyde, heptaldehyde, octaldehyde, nonaldehyde, and decaldehyde, respectively.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATL ACAD SCIENCES  

We used an automated peptide synthesizer to produce a peptide, metalloselenonein, that contains selenocysteine residues substituted for all cysteine residues in Neurospora crassa copper metallothionein. Metalloselenonein binds 3 mol of Cu(I) per mol. This adduct shows a broad absorption band between 230 and 400 nm and a fluorescence band at 395 nm, which can be attributed to copper-selenolate coordination. The circular dichroism spectrum of the copper-metalloselenonein complex shows a positive band around 245 nm attributable to asymmetry in metal coordination.

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記述言語：日本語  

J-GLOBAL

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：GORDON BREACH SCI PUBL LTD  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Oxford University Press  

The gene coding for the Neurospora crassa copper metallothionein (MT) was synthesized and inserted in the lacZ' gene of pUC18 plasmid to give the same translational reading frame as the latter gene. The MT-β-galactosidase fused gene was expressed in Escherichia coli to produce a fused protein in which the amino and carboxy termini of MT are linked to the β-galactosidase through methionine residues. An MT derivative containing an extra homoserine residue at the carboxy terminus was prepared by cyanogen bromide cleavage of the fused protein followed by a reverse-phase HPLC separation. The spectral features of the MT derivative and its copper complex were similar to those of the corresponding native MTs. © 1988 COPYRIGHT 1988 BY THE JOURNAL OF BIOCHEMISTRY.

DOI： 10.1093/oxfordjournals.jbchem.a122584

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一般に酵素は、熱、酸、アルカリ、有機溶媒などでの処理により立体構造が失われると、容易に失活する。一方、高温で生育する好熱性微生物は本来、耐熱性の酵素を生産する。耐熱性酵素は一般に、普通の酵素とは対照的に熱のみならず、酸、アルカリ、有機溶媒などに対しても高い安定性を示す。耐熱性酵素はPCRなどを始めとする応用面での有用性のみならず、X線解析などを用いた酵素の構造-機能相関の開明など、基礎的研究に際しても様々な特長を示す。好熱菌由来の耐熱性ピリドキサルリン酸酵素特に、D-グルタミン酸、D-アラニンなどの代謝や生合成に関与するD-アミノ酸アミノトランスフェラーゼについて特性と触媒機構を中心に解説する。

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微生物から哺乳類まで広範囲な分野にわたる生物科学の知見を総合的に利用するバイオテクノロジーは本質的に省資源・エネルギー、低公害を特色とし、医学、工学、農学、薬学などの分野で花開きつつある。バイオテクノロジーの中心的存在で、その特色を最もよく示しているのは酵素(生体触媒)である。酵素は巨大なタンパク質としての三次元構造に基づいて多種多様な反応を触媒するのが、その触媒作用を更に高能率化し、多様化しているのは、各種の補酵素と酵素活性化剤としての微量元素である。ここでは酵素活性化因子としての微量元素の役割と活性化機構の最近の進歩を述べる。

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記述言語：日本語   出版者・発行元：関西大学先端科学技術推進機構  

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記述言語：英語   掲載種別：速報，短報，研究ノート等（学術雑誌）   出版者・発行元：John Wiley and Sons Inc.  

DOI： 10.1002/prot.25093

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その他リンク： http://search.jamas.or.jp/link/ui/2013125631

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記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.74.5-6_323

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