Yoshimichi Takai, Rumana Yesmin Hasi, Naoko Matsumoto, Chiho Fujita, Hanif Ali, Junji Hayashi, Ryushi Kawakami, Mutsumi Aihara, Toshiki Ishikawa, Hiroyuki Imai, Mayuko Wakida, Kazuya Ando and Tamotsu Tanaka : Degradation of glycosylinositol phosphoceramide during plant tissue homogenization, The Journal of Biochemistry, Vol.175, No.1, 115-124, 2024.
(要約)
A convenient method for the determination of plant sphingolipids (glycosylinositol phosphoceramide, GIPC; glucosylceramide, GluCer; phytoceramide 1-phosphate, PC1P and phytoceramide, PCer) was developed. This method includes the extraction of lipids using 1-butanol, alkali hydrolysis with methylamine and separation by TLC. The amounts of sphingolipids in the sample were determined based on the relative intensities of standard sphingolipids visualized by primulin/UV on TLC. Using this method, we found that almost all GIPCs were degraded in response to tissue homogenization in cruciferous plants (cabbage, broccoli and Arabidopsis thaliana). The decrease in GIPCs was compensated for by increases in PC1P and PCer, indicating that GIPC was degraded by hydrolysis at the D and C positions of GIPC, respectively. In carrot roots and leaves, most of GIPC degradation was compensated for by an increase in PCer. In rice roots, the decrease in GIPCs was not fully explained by the increases in PC1P and PCer, indicating that enzymes other than phospholipase C and D activities operated. As the visualization of lipids on TLC is useful for detecting the appearance or disappearance of lipids, this method will be available for the characterization of metabolism of sphingolipids in plants.
Ryushi Kawakami, Takami Naoki, Junji Hayashi, Kazunari Yoneda, Ohmori Taketo, Toshihisa Ohshima and Haruhiko Sakuraba : First crystal structure of an NADP+-dependent L-arginine dehydrogenase belonging to the μ-crystallin family, International Journal of Biological Macromolecules, Vol.249, 2023.
Hanif Ali, Miyu Kobayashi, Katsuya Morito, Rumana Yesmin, Mutsumi Aihara, Junji Hayashi, Ryushi Kawakami, Koichiro Tsuchiya, Kazunori Sango and Tamotsu Tanaka : Peroxisomes attenuate cytotoxicity of very long-chain fatty acids, Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, Vol.1868, No.2, 159259, 2023.
(要約)
One of the major functions of peroxisomes in mammals is oxidation of very long-chain fatty acids (VLCFAs). Genetic defects in peroxisomal β-oxidation result in the accumulation of VLCFAs and lead to a variety of health problems, such as demyelination of nervous tissues. However, the mechanisms by which VLCFAs cause tissue degeneration have not been fully elucidated. Recently, we found that the addition of small amounts of isopropanol can enhance the solubility of saturated VLCFAs in an aqueous medium. In this study, we characterized the biological effect of extracellular VLCFAs in peroxisome-deficient Chinese hamster ovary (CHO) cells, neural crest-derived pheochromocytoma cells (PC12), and immortalized adult Fischer rat Schwann cells (IFRS1) using this solubilizing technique. C20:0 FA was the most toxic of the C16-C26 FAs tested in all cells. The basis of the toxicity of C20:0 FA was apoptosis and was observed at 5 μM and 30 μM in peroxisome-deficient and wild-type CHO cells, respectively. The sensitivity of wild-type CHO cells to cytotoxic C20:0 FA was enhanced in the presence of a peroxisomal β-oxidation inhibitor. Further, a positive correlation was evident between cell toxicity and the extent of intracellular accumulation of toxic FA. These results suggest that peroxisomes are pivotal in the detoxification of apoptotic VLCFAs by preventing their accumulation.
Glycosylinositol phosphoceramide (GIPC) is a major sphingolipid in the plasma membranes of plants. Previously, we found an enzyme activity that produces phytoceramide 1-phosphate (PC1P) by hydrolysis of the D position of GIPC in cabbage and named this activity as GIPC-phospholipase D (PLD). Here, we purified GIPC-PLD by sequential chromatography from radish roots. Peptide mass fingerprinting analysis revealed that the potential candidate for GIPC-PLD protein was nonspecific phospholipase C3 (NPC3), which has not been characterized as a PLD. The recombinant NPC3 protein obtained by heterologous expression system in Escherichia coli produced PC1P from GIPC and showed essentially the same enzymatic properties as those we characterized as GIPC-PLD in cabbage, radish and Arabidopsis thaliana. From these results, we conclude that NPC3 is one of the enzymes that degrade GIPC.
Ryushi Kawakami, Tatsuya Ohshida, Junji Hayashi, Kazunari Yoneda, Toshio Furumoto, Toshihisa Ohshima and Haruhiko Sakuraba : Crystal structure of a novel type of ornithine δ-aminotransferase from the hyperthermophilic archaeon Pyrococcus horikoshii, International Journal of Biological Macromolecules, Vol.208, 731-740, 2022.
(要約)
Ornithine δ-aminotransferase (Orn-AT) activity was detected for the enzyme annotated as a γ-aminobutyrate aminotransferase encoded by PH1423 gene from Pyrococcus horikoshii OT-3. Crystal structures of this novel archaeal ω-aminotransferase were determined for the enzyme in complex with pyridoxal 5'-phosphate (PLP), in complex with PLP and l-ornithine (l-Orn), and in complex with N-(5'-phosphopyridoxyl)-l-glutamate (PLP-l-Glu). Although the sequence identity was relatively low (28%), the main-chain coordinates of P. horikoshii Orn-AT monomer showed notable similarity to those of human Orn-AT. However, the residues recognizing the α-amino group of l-Orn differ between the two enzymes. In human Orn-AT, Tyr55 and Tyr85 recognize the α-amino group, whereas the side chains of Thr92* and Asp93*, which arise from a loop in the neighboring subunit, form hydrogen bonds with the α-amino group of the substrate in P. horikoshii enzyme. Site-directed mutagenesis suggested that Asp93* plays critical roles in maintaining high affinity for the substrate. This study provides new insight into the substrate binding of a novel type of Orn-AT. Moreover, the structure of the enzyme with the reaction-intermediate analogue PLP-l-Glu bound provides the first structural evidence for the "Glu switch" mechanism in the dual substrate specificity of Orn-AT.
Ali Hanif, Morito Katsuya, Rumana Hasi Yesmin, Mutsumi Aihara, Junji Hayashi, Ryushi Kawakami, Kaori Kanemaru, Koichiro Tsuchiya, Sango Kazunori and Tamotsu Tanaka : Characterization of uptake and metabolism of very long-chain fatty acids in peroxisome-deficient CHO cells, Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, Vol.1867, No.2, 159088, 2022.
(要約)
Fatty acids (FAs) longer than C20 are classified as very long-chain fatty acids (VLCFAs). Although biosynthesis and degradation of VLCFAs are important for the development and integrity of the myelin sheath, knowledge on the incorporation of extracellular VLCFAs into the cells is limited due to the experimental difficulty of solubilizing them. In this study, we found that a small amount of isopropanol solubilized VLCFAs in aqueous medium by facilitating the formation of the VLCFA/albumin complex. Using this solubilizing technique, we examined the role of the peroxisome in the uptake and metabolism of VLCFAs in Chinese hamster ovary (CHO) cells. When wild-type CHO cells were incubated with saturated VLCFAs (S-VLCFAs), such as C23:0 FA, C24:0 FA, and C26:0 FA, extensive uptake was observed. Most of the incorporated S-VLCFAs were oxidatively degraded without acylation into cellular lipids. In contrast, in peroxisome-deficient CHO cells uptake of S-VLCFAs was marginal and oxidative metabolism was not observed. Extensive uptake and acylation of monounsaturated (MU)-VLCFAs, such as C24:1 FA and C22:1 FA, were observed in both types of CHO cells. However, oxidative metabolism was evident only in wild-type cells. Similar manners of uptake and metabolism of S-VLCFAs and MU-VLCFAs were observed in IFRS1, a Schwan cell-derived cell line. These results indicate that peroxisome-deficient cells limit intracellular S-VLCFAs at a low level by halting uptake, and as a result, peroxisome-deficient cells almost completely lose the clearance ability of S-VLCFAs accumulated outside of the cells.
Ryushi Kawakami, Chinatsu Kinoshita, Tomoki Kawase, Mikio Sato, Junji Hayashi, Haruhiko Sakuraba and Toshihisa Ohshima : Characterization of novel moderate-substrate specificity amino acid racemase from the hyperthermophilic archaeon Thermococcus litoralis., Bioscience, Biotechnology, and Biochemistry, Vol.85, No.7, 1650-1657, 2021.
(要約)
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.
Junji Hayashi, Yoshiaki Ichiki, Akiko Kanda, Kazuyoshi Takagi and Mamoru Wakayama : Identification, characterization, and cloning of a novel aminoacylase, L-pipecolic acid acylase from Pseudomonas species, The Journal of General and Applied Microbiology, 2021.
(要約)
L-Pipecolic acid is utilized as a vital component of specific chemical compounds, such as immunosuppressive drugs, anticancer reagents, and anesthetic reagents. We isolated and characterized a novel L-aminoacylase, N-acetyl-L-pipecolic acid-specific aminoacylase (LpipACY), from Pseudomonas sp. AK2. The subunit molecular mass of LpipACY was 45 kDa and was assumed to be a homooctamer in solution. The enzyme exhibited high substrate specificity toward N-acetyl-L-pipecolic acid and a high activity for N-acetyl-L-pipecolic acid and N-acetyl-L-proline. This enzyme was stable at a high temperature (60°C for 10 min) and under an alkaline pH (6.0-11.5). The N-terminal and internal amino acid sequences of the purified enzyme were STTANTLILRNG and IMASGGV, respectively. These sequences are highly consistent with those of uncharacterized proteins from Pseudomonas species, such as amidohydrolase and peptidase. We also cloned and overexpressed the gene coding LpipACY in Escherichia coli. Moreover, the recombinant LpipACY exhibited properties similar to native enzyme. Our results suggest that LpipACY is a potential enzyme for the enzymatic synthesis of L-pipecolic acid. This study provides the first description of the enzymatic characterization of L-pipecolic acid specific amino acid acylase.
Tasneem Chemama, Junji Hayashi, Mamoru Wakayama and Narumol Thongwai : Characteristics of D-lactate Dehydrogenase from the High Potential D-lactic Acid Producer Leuconostoc pseudomesenteroides TC49 Isolated from Thailand, Chiang Mai Journal of Science, Vol.48, No.1, 42-55, 2021.
Chiharu Sano, Takafumi Itoh, Putthapong Phumsombat, Junji Hayashi, Mamoru Wakayama and Takao Hibi : Mutagenesis and structure-based analysis of the role of Tryptophan525 of γ-glutamyltranspeptidase from Pseudomonas nitroreducens., Biochemical and Biophysical Research Communications, 2021.
(要約)
γ-Glutamyltranspeptidase (GGT) is a ubiquitous enzyme that catalyzes the hydrolysis of the γ-glutamyl linkage of γ-glutamyl compounds and the transfer of their γ-glutamyl moiety to acceptor substrates. Pseudomonas nitroreducens GGT (PnGGT) is used for the industrial synthesis of theanine, thus it is important to determine the structural basis of hydrolysis and transfer reactions and identify the acceptor site of PnGGT to improve the efficient of theanine synthesis. Our previous structural studies of PnGGT have revealed that crucial interactions between three amino acid residues, Trp385, Phe417, and Trp525, distinguish PnGGT from other GGTs. Here we report the role of Trp525 in PnGGT based on site-directed mutagenesis and structural analyses. Seven mutant variants of Trp525 were produced (W525F, W525V, W525A, W525G, W525S, W525D, and W525K), with substitution of Trp525 by nonaromatic residues resulting in dramatically reduced hydrolysis activity. All Trp525 mutants exhibited significantly increased transfer activity toward hydroxylamine with hardly any effect on acceptor substrate preference. The crystal structure of PnGGT in complex with the glutamine antagonist, 6-diazo-5-oxo-l-norleucine, revealed that Trp525 is a key residue limiting the movement of water molecules within the PnGGT active site.
Takafumi Itoh, Niphawan Panti, Junji Hayashi, Yosuke Toyotake, Daisuke Matsui, Shigekazu Yano, Mamoru Wakayama and Takao Hibi : Crystal structure of the catalytic unit of thermostable GH87 α-1,3-glucanase from Streptomyces thermodiastaticus strain HF3-3., Biochemical and Biophysical Research Communications, Vol.533, No.4, 1170-1176, 2020.
(要約)
α-1,3-Glucan is a homopolymer composed of D-glucose (Glc) and it is an extracellular polysaccharide found in dental plaque due to Streptococcus species. α-1,3-Glucanase from Streptomyces thermodiastaticus strain HF3-3 (Agl-ST) has been identified as a thermostable α-1,3-glucanase, which is classified into glycoside hydrolase family 87 (GH87) and specifically hydrolyzes α-1,3-glucan with an endo-action. The enzyme has a potential to inhibit the production of dental plaque and to be used for biotechnological applications. Here we show the structure of the catalytic unit of Agl-ST determined at 1.16 Å resolution using X-ray crystallography. The catalytic unit is composed of two modules, a β-sandwich fold module, and a right-handed β-helix fold module, which resembles other structural characterized GH87 enzymes from Bacillus circulans str. KA-304 and Paenibacillus glycanilyticus str. FH11, with moderate sequence identities between each other (approximately 27% between the catalytic units). However, Agl-ST is smaller in size and more thermally stable than the others. A disulfide bond that anchors the C-terminal coil of the β-helix fold, which is expected to contribute to thermal stability only exists in the catalytic unit of Agl-ST.
Rumana Yesmin Hasi, Dai Majima, Katsuya Morito, Hanif Ali, Kentaro Kogure, Meera Nanjundan, Junji Hayashi, Ryushi Kawakami, Kaori Kanemaru and Tamotsu Tanaka : Isolation of glycosylinositol phosphoceramide and phytoceramide 1-phosphate in plants and their chemical stabilities., Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, Vol.1152, 122213, 2020.
(要約)
Glycosylinositol phosphoceramide (GIPC) is a sphingophospholipid in plants. Recently, we identified that GIPC is hydrolyzed to phytoceramide 1-phosphate (PC1P) by an uncharacterized phospholipase D activity following homogenization of certain plant tissues. We now developed methods for isolation of GIPC and PC1P from plant tissues and characterized their chemical stabilities. Hydrophilic solvents, namely a lower layer of a mixed solvent system consisting of isopropanol/hexane/water (55:20:25, v/v/v) was efficient solvent for extraction and eluent in column chromatography. GIPC was isolated by Sephadex column chromatography followed by TLC. A conventional method, such as the Bligh and Dyer method, was applicable for PC1P extraction. Specifically, PC1P was isolated by TLC following mild alkali treatment of lipid extracts of plants. The yields of GIPC and PC1P in our methods were both around 50-70%. We found that PC1P is tolerant against heat (up to 125 °C), strong acid (up to 10 M HCl), and mild alkali (0.1 M KOH). In contrast, significant degradation of GIPC occurred at 100 °C and 1.0 M HCl treatment, suggesting the instability of the inositol glycan moiety in these conditions. These data will be useful for further biochemical and nutritional studies on these sphingolipids.
Takafumi Itoh, Rattanaporn Intuy, Wasana Suyotha, Junji Hayashi, Shigekazu Yano, Koki Makabe, Mamoru Wakayama and Takao Hibi : Structural insights into substrate recognition and catalysis by glycoside hydrolase family 87 α-1,3-glucanase from Paenibacillus glycanilyticus FH11., The FEBS Journal, 2019.
(要約)
The α-1,3-glucanase from Paenibacillus glycanilyticus FH11 (Agl-FH1), a member of the glycoside hydrolase family 87 (GH87), hydrolyzes α-1,3-glucan with an endo-action. GH87 enzymes are known to degrade dental plaque produced by oral pathogenic Streptococcus species. In this study, the kinetic analyses revealed that this enzyme hydrolyzed α-1,3-tetraglucan into glucose and α-1,3-triglucan with β-configuration at the reducing end by an inverting mechanism. The crystal structures of the catalytic domain (CatAgl-FH1) complexed with or without oligosaccharides at 1.4-2.5 or 1.6 Å resolutions, respectively, are also presented. The initial crystal structure of CatAgl-FH1 was determined by native single-wavelength anomalous diffraction. The catalytic domain was composed of two modules, a β-sandwich fold module, and a right-handed β-helix fold module. The structure of the β-sandwich was similar to those of the carbohydrate-binding module family 35 members. The glycerol or nigerose enzyme complex structures demonstrated that this β-sandwich fold module is a novel carbohydrate-binding module with the capabilities to bind saccharides and to promote the degradation of polysaccharides. The structures of the inactive mutant in complexes with oligosaccharide showed that at least eight subsites for glucose binding were located in the active cleft of the β-helix fold and the architecture of the active cleft was suitable for the recognition and hydrolysis of α-1,3-glucan by the inverting mechanism. The structural similarity to GH28 and GH49 enzymes and the results of site-directed mutagenesis indicated that three Asp residues, Asp1045, Asp1068, and Asp1069, are the most likely candidates for the catalytic residues of Agl-FH1. DATABASE: Structural data are available in RCSB Protein Data Bank under the accession numbers 6K0M (CatAgl-FH1), 6K0N (WT/nigerose), 6K0P (D1045A/nigerose), 6K0Q (D1068A/nigerose), 6K0S (D1069A/ nigerose), 6K0U (D1068A/oligo), and 6K0V (D1069A/oligo). ENZYMES: Agl-FH1, α-1,3-glucanase (EC3.2.1.59) from Paenibacillus glycanilyticus FH11.
Tatsuya Ohshida, Junji Hayashi, Kazunari Yoneda, Toshihisa Ohshima and Haruhiko Sakuraba : Unique active site formation in a novel galactose 1-phosphate uridylyltransferase from the hyperthermophilic archaeon Pyrobaculum aerophilum., Proteins, 2019.
(要約)
A gene encoding galactose 1-phosphate uridylyltransferase (GalT) was identified in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The expressed enzyme was highly thermostable and retained about 90% of its activity after incubation for 10 minutes at temperatures up to 90°C. Two different crystal structures of P. aerophilum GalT were determined: the substrate-free enzyme at 2.33 Å and the UDP-bound H140F mutant enzyme at 1.78 Å. The main-chain coordinates of the P. aerophilum GalT monomer were similar to those in the structures of the E. coli and human GalTs, as was the dimeric arrangement. However, there was a striking topological difference between P. aerophilum GalT and the other two enzymes. In the E. coli and human enzymes, the N-terminal chain extends from one subunit into the other and forms part of the substrate-binding pocket in the neighboring subunit. By contrast, the N-terminal chain in P. aerophilum GalT extends to the substrate-binding site in the same subunit. Amino acid sequence alignment showed that a shorter surface loop in the N-terminal region contributes to the unique topology of P. aerophilum GalT. Structural comparison of the substrate-free enzyme with UDP-bound H140F suggests that binding of the glucose moiety of the substrate, but not the UDP moiety, gives rise to a large structural change around the active site. This may in turn provide an appropriate environment for the enzyme reaction.
Rumana Yesmin Hasi, Makoto Miyagi, Takashi Kida, Tatsuya Fukuta, Kentaro Kogure, Junji Hayashi, Ryushi Kawakami, Kaori Kanemaru and Tamotsu Tanaka : Quantitative Analysis of Glycosylinositol Phosphoceramide and Phytoceramide 1-Phosphate in Vegetables, Journal of Nutritional Science and Vitaminology, Vol.65, No.Supplement, S175-S179, 2019.
(要約)
Previously, we found an unidentified sphingolipid in cabbage, and determined it as phytoceramide 1-phosphate (PC1P). PC1P is found to be produced from glycosylinositol phosphoceramide (GIPC) by the action of phospholipase D (PLD) activity. Although GIPC is abundant sphingolipid, especially in cruciferous vegetables, amount of daily intake, digestibility and nutritional activity of GIPC are not well understood. Here, we investigated amounts of GIPC and PC1P in vegetables. GIPC was found in all vegetables examined (13 kinds) at levels 3-20 mg/100 g (wet weight). On the other hand, PC1P was present in limited vegetables which show higher GIPC-PLD activity, such as inner cabbage leaves (5.2 mg/100 g). Because PC1P is formed during homogenization by activated GIPC-PLD, level of PC1P in boiled cabbage leaves was very low. Although digestibility of GIPC is unknown at present, a portion of dietary GIPC is considered to be converted to PC1P during mastication by plant-derived GIPC-PLD activity in some vegetables.
Kodchakorn Phetsri, Makoto Furukawa, Risa Yamashiro, Yuka Kawamura, Junji Hayashi, Ryuta Tobe, Yosuke Toyotake and Mamoru Wakayama : Comparative Biochemical Characterization of L-Asparaginases from Four Species of Lactic Acid Bacteria, Journal of Biotechnology and Biomedicine, Vol.2, No.3, 112-124, 2019.
Hasi Yesmin Rumana, Makoto Miyagi, Katsuya Morito, Toshiki Ishikawa, Maki Kawai-Yamada, Hiroyuki Imai, Tatsuya Fukuta, Kentaro Kogure, Kaori Kanemaru, Junji Hayashi, Ryushi Kawakami and Tamotsu Tanaka : Glycosylinositol phosphoceramide-specific phospholipase D activity catalyzes transphosphatidylation, The Journal of Biochemistry, Vol.166, No.5, 441-448, 2019.
(要約)
Glycosylinositol phosphoceramide (GIPC) is the most abundant sphingolipid in plants and fungi. Recently, we detected GIPC-specific phospholipase D (GIPC-PLD) activity in plants. Here, we found that GIPC-PLD activity in young cabbage leaves catalyzes transphosphatidylation. The available alcohol for this reaction is a primary alcohol with a chain length below C4. Neither secondary alcohol, tertiary alcohol, choline, serine nor glycerol serves as an acceptor for transphosphatidylation of GIPC-PLD. We also found that cabbage GIPC-PLD prefers GIPC containing two sugars. Neither inositol phosphoceramide, mannosylinositol phosphoceramide nor GIPC with three sugar chains served as substrate. GIPC-PLD will become a useful catalyst for modification of polar head group of sphingophospholipid.
Rattanaporn Intuy, Takafumi Itoh, Wasana Suyotha, Junji Hayashi, Shigekazu Yano, Koki Makabe, Mamoru Wakayama and Takao Hibi : X-ray crystallographic analysis of the catalytic domain of α-1,3-glucanase FH1 from Paenibacillus glycanilyticus overexpressed in Brevibacillus choshinensis., Acta Crystallographica. Section F, Structural Biology Communications, Vol.74, No.Pt 12, 770-773, 2018.
(要約)
2, with unit-cell parameters a = b = 132.6, c = 76.1 Å. The space group and unit-cell parameters suggest that there is one molecule in the asymmetric unit.
Takenori Satomura, Junji Hayashi, Tatsuya Ohshida, Haruhiko Sakuraba, Toshihisa Ohshima and Shin-Ichiro Suye : Enzymological characteristics of a novel archaeal dye-linked D-lactate dehydrogenase showing loose binding of FAD., Extremophiles, Vol.22, No.6, 975-981, 2018.
(要約)
A gene-encoding a dye-linked D-lactate dehydrogenase (Dye-DLDH) homolog was identified in the genome of the hyperthermophilic archaeon Thermoproteus tenax. The gene was expressed in Escherichia coli and the product was purified to homogeneity. The recombinant protein exhibited highly thermostable Dye-DLDH activity. To date, four types of Dye-DLDH have been identified in hyperthermophilic archaea (in Aeropyrum pernix, Sulfolobus tokodaii, Archaeoglobus fulgidus, and Candidatus Caldiarchaeum subterraneum). The amino acid sequence of T. tenax Dye-DLDH showed the highest similarity (45%) to A. pernix Dye-DLDH, but neither contained a known FAD-binding motif. Nonetheless, both homologs required FAD for enzymatic activity, suggesting that FAD binds loosely to the enzyme and is easily released unlike in other Dye-DLDHs. Our findings indicate that Dye-DLDHs from T. tenax and A. pernix are a novel type of Dye-DLDH characterized by loose binding of FAD.
Tatsuya Ohshida, Kohei Koba, Junji Hayashi, Kazunari Yoneda, Taketo Ohmori, Toshihisa Ohshima and Haruhiko Sakuraba : A novel bifunctional aspartate kinase-homoserine dehydrogenase from the hyperthermophilic bacterium, Thermotoga maritima., Bioscience, Biotechnology, and Biochemistry, Vol.82, No.12, 2084-2093, 2018.
(要約)
= 500 μM). In contrast to A. thaliana AK-HseDH, Hse oxidation of the T. maritima enzyme was almost impervious to inhibition by L-threonine. Amino acid sequence comparison indicates that the distinctive sequence of the regulatory domain in T. maritima AK-HseDH is likely responsible for the unique sensitivity to L-threonine. Abbreviations: AK: aspartate kinase; HseDH: homoserine dehydrogenase; AK-HseDH: bifunctional aspartate kinase-homoserine dehydrogenase; AsaDH: aspartate-β-semialdehyde dehydrogenase; ACT: aspartate kinases (A), chorismate mutases (C), and prephenate dehydrogenases (TyrA, T).
Keitaro Take, Hidehisa Fujiki, Wasana Suyotha, Junji Hayashi, Kazuyoshi Takagi, Shigekazu Yano and Mamoru Wakayama : Enzymatic and molecular characterization of an acidic and thermostable chitinase 1 from Streptomyces thermodiastaticus HF 3-3., The Journal of General and Applied Microbiology, Vol.64, No.4, 190-197, 2018.
(要約)
values of Chi1 for the substrate colloidal chitin were 1.23 ± 0.7 mg/mL and 6.33 ± 1.0 U/mg, respectively. Thin-layer chromatography analysis of the enzymatic reaction end products mainly detected diacetylchitobiose. We also cloned the Chi1 gene and purified the recombinant protein; the properties of the recombinant enzyme were nearly identical to those of the native enzyme. Therefore, Chi1 purified from S. thermodiastaticus HF 3-3 is unique, as it is highly stable under broad range of pH values, temperatures, and chemical exposures. Combined, these properties make this enzyme attractive for use in the industrial bioconversion of chitin.
Takenori Satomura, Junji Hayashi, Hiroaki Sakamoto, Takuro Nunoura, Yoshihiro Takaki, Ken Takai, Hideto Takami, Toshihisa Ohshima, Haruhiko Sakuraba and Shin-Ichiro Suye : d-Lactate electrochemical biosensor prepared by immobilization of thermostable dye-linked d-lactate dehydrogenase from Candidatus Caldiarchaeum subterraneum., Journal of Bioscience and Bioengineering, Vol.126, No.4, 425-430, 2018.
(要約)
A stable d-lactate electrochemical sensing system was developed using a dye-linked d-lactate dehydrogenase (Dye-DLDH) from an uncultivated thermophilic archaeon, Candidatus Caldiarchaeum subterraneum. To develop the system, the putative gene encoding the Dye-DLDH from Ca. Caldiarchaeum subterraneum was overexpressed in Escherichia coli, and the expressed product was purified. The recombinant enzyme was a highly thermostable Dye-DLDH that retained full activity after incubation for 10 min at 70°C. The electrode for detection of d-lactate was prepared by immobilizing the thermostable Dye-DLDH and multi-walled carbon nanotube (MWCNT) within Nafion membrane. The electrocatalytic response of the electrode was clearly observed upon exposure to d-lactate. The electrode response to d-lactate was linear within the concentration range of 0.03-2.5 mM, and it showed little reduction in responsiveness after 50 days. This is the first report describing a d-lactate sensing system using a thermostable Dye-DLDH.
HS1 (SmAEH) was homogeneous in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analyses, and was present as a tetramer in gel-filtration experiments. The activity of the SmAEH enzyme was then determined by monitoring the synthesis of the antihypertensive agent dipeptide isoleucyl-tryptophan (Ile-Trp) from isoleucyl methyl ester (Ile-OMe) and tryptophan (Trp). In these experiments, SmAEH had wide substrate specificity for acyl donors, such as Gly-OMe, β-Ala-OMe, Pro-OMe and Trp-OMe and Ile-OMe, and maximal activity were observed under conditions of pH 9.0 and 30 °C. SmAEH also showed the greatest stability at pH 9.0, whereas its activity was reduced by 40% after 10-min incubation at approximately 50 °C. In subsequent activity assays in the presence of various metal ions, Ag
Junji Hayashi, Tomonari Seto, Hironaga Akita, Masahiro Watanabe, Tamotsu Hoshino, Kazunari Yoneda, Toshihisa Ohshima and Haruhiko Sakuraba : Structure-Based Engineering of an Artificially Generated NADP+-Dependent d-Amino Acid Dehydrogenase, Applied and Environmental Microbiology, Vol.83, No.11, 2017.
(要約)
-dependent d-amino acid dehydrogenase (DAADH) makes single-step production of d-amino acids from oxo-acid analogs and ammonia possible. We recently succeeded in creating a stable DAADH and demonstrated that it is applicable for one-step synthesis of d-amino acids, such as d-leucine and d-isoleucine. As the next step, the creation of an enzyme exhibiting different substrate specificity and higher catalytic efficiency is a key to the further development of d-amino acid production. In this study, we succeeded in creating a novel mutant exhibiting extremely high catalytic activity for phenylpyruvate amination. Structural insight into the mutant will be useful for further improvement of DAADHs.
Junji Hayashi, Yuta Mutaguchi, Yume Minemura, Noriko Nakagawa, Kazunari Yoneda, Taketo Ohmori, Toshihisa Ohshima and Haruhiko Sakuraba : Crystal structure of the novel amino-acid racemase isoleucine 2-epimerase from Lactobacillus buchneri., Acta Crystallographica. Section D, Structural Biology, Vol.73, No.Pt 5, 428-437, 2017.
(要約)
Crystal structures of Lactobacillus buchneri isoleucine 2-epimerase, a novel branched-chain amino-acid racemase, were determined for the enzyme in the apo form, in complex with pyridoxal 5'-phosphate (PLP), in complex with N-(5'-phosphopyridoxyl)-L-isoleucine (PLP-L-Ile) and in complex with N-(5'-phosphopyridoxyl)-D-allo-isoleucine (PLP-D-allo-Ile) at resolutions of 2.77, 1.94, 2.65 and 2.12 Å, respectively. The enzyme assembled as a tetramer, with each subunit being composed of N-terminal, C-terminal and large PLP-binding domains. The active-site cavity in the apo structure was much more solvent-accessible than that in the PLP-bound structure. This indicates that a marked structural change occurs around the active site upon binding of PLP that provides a solvent-inaccessible environment for the enzymatic reaction. The main-chain coordinates of the L. buchneri isoleucine 2-epimerase monomer showed a notable similarity to those of α-amino-ℇ-caprolactam racemase from Achromobactor obae and γ-aminobutyrate aminotransferase from Escherichia coli. However, the amino-acid residues involved in substrate binding in those two enzymes are only partially conserved in L. buchneri isoleucine 2-epimerase, which may account for the differences in substrate recognition by the three enzymes. The structures bound with reaction-intermediate analogues (PLP-L-Ile and PLP-D-allo-Ile) and site-directed mutagenesis suggest that L-isoleucine epimerization proceeds through abstraction of the α-hydrogen of the substrate by Lys280, while Asp222 serves as the catalytic residue adding an α-hydrogen to the quinonoid intermediate to form D-allo-isoleucine.
Tatsuya Ohshida, Junji Hayashi, Takenori Satomura, Ryushi Kawakami, Toshihisa Ohshima and Haruhiko Sakuraba : First characterization of extremely halophilic 2-deoxy-D-ribose-5-phosphate aldolase, Protein Expression and Purification, Vol.126, 62-68, 2016.
(要約)
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.
Junji Hayashi, Shota Inoue, Kwang Kim, Kazunari Yoneda, Yutaka Kawarabayasi, Toshihisa Ohshima and Haruhiko Sakuraba : Crystal Structures of a Hyperthermophilic Archaeal Homoserine Dehydrogenase Suggest a Novel Cofactor Binding Mode for Oxidoreductases., Scientific Reports, Vol.5, 2015.
(要約)
NAD(P)-dependent dehydrogenases differ according to their coenzyme preference: some prefer NAD, others NADP, and still others exhibit dual cofactor specificity. The structure of a newly identified archaeal homoserine dehydrogenase showed this enzyme to have a strong preference for NADP. However, NADP did not act as a cofactor with this enzyme, but as a strong inhibitor of NAD-dependent homoserine oxidation. Structural analysis and site-directed mutagenesis showed that the large number of interactions between the cofactor and the enzyme are responsible for the lack of reactivity of the enzyme towards NADP. This observation suggests this enzyme exhibits a new variation on cofactor binding to a dehydrogenase: very strong NADP binding that acts as an obstacle to NAD(P)-dependent dehydrogenase catalytic activity.
Shin-ichi Sakasegawa, Junji Hayashi, Yoshiaki Ikura, Shigeru Ueda, Shigeyuki Imamura, Toshihiko Kumazawa, Atsuhisa Nishimura, Toshihisa Ohshima and Haruhiko Sakuraba : Colorimetric inorganic pyrophosphate assay using a double cycling enzymatic method., Analytical Biochemistry: Methods in the Biological Sciences, Vol.416, No.1, 61-66, 2011.
(要約)
Pyruvate phosphate dikinase (PPDK, EC 2.7.9.1) from the hyperthermophile Thermotoga maritima was biochemically characterized with the aim of establishing a colorimetric assay for inorganic pyrophosphate (PPi). When heterologously expressed in Escherichia coli, T. maritima PPDK (TmPPDK) was far more stable any other PPDK reported so far: it retained >90% of its activity after incubation for 1 h at 80°C, and >80% of its activity after incubation for 20 min at pHs ranging from 6.5 to 10.5 (50°C). In contrast to PPDKs from protozoa and plants, this TmPPDK showed very long-term stability at low temperature: full activity was retained even after storage for at least 2 years at 4°C. TmPPDK was successfully applied to a novel colorimetric PPi assay, which employed (i) a PPi cycling reaction using TmPPDK and nicotinamide mononucleotide adenylyltransferase (EC 2.7.7.1) from Saccharomyces cerevisiae and (ii) a NAD cycling reaction to accumulate reduced nitroblue tetrazolium (diformazan). This enabled detection of 0.2 μM PPi, making this method applicable for preliminary measurement of PPi levels in PCR products in an automatic clinical analyzer.
Hironaga Akita, Junji Hayashi, Haruhiko Sakuraba and Toshihisa Ohshima : Artificial Thermostable D-Amino Acid Dehydrogenase: Creation and Application., Frontiers in Microbiology, Vol.9, Aug. 2018.
(要約)
-DAPDH and D-AADH, and designed mutations based on the information obtained made it possible to markedly enhance enzyme activity and to create D-AADH homologs with desired reactivity profiles. The methods described here may be an effective approach to artificial creation of biotechnologically useful enzymes.
Takefumi Hattori, Katayama Megumi, Tsuzuki Hiromitsu, Okamoto Yumi, Ida Kyosuke, Yoshizumi Mariko, Abe Masanori, Ryushi Kawakami, Junji Hayashi and Masaomi Yamamura : Phenylpropanoid metabolism in basidiomycete Tricholoma matsutake -cDNA cloning of Sadenosyl-L-methionine-dependent cinnamic acid carboxyl methyltransferase-, 2nd International Lignin Symposium (Kyoto), Sep. 2024.
2.
Nakamoto Akira, Umekawa Midori, Junji Hayashi and Wakayama Mamoru : Construction of D-amino acid derivative production system using Saccharomyces cerevisiae, New Zealand Microbiological Society Conference 2017, Nov. 2017.
3.
Ogiyama Daiki, Wakayama Mamoru and Junji Hayashi : Lactovinegar production by ethanol fermentation of whey using Zymomonas mobile, New Zealand Microbiological Society Conference 2017, Nov. 2017.
4.
Ohshima Toshihisa, Junji Hayashi, Akita Hironaga and Sakuraba Haruhiko : Structuralbased engineering of an artificially created NADP-dependent D-amino acid dehydrogenase, The 3rd International Conference of D-Amino Acid Research 2017, Jul. 2017.
5.
Take Keitaro, Fujiki Hidehisa, Suyotha Wasana, Takagi Kazuyoshi, Junji Hayashi and Wakayama Mamoru : Thermostable and acidic chitinase obtained from Streptomyces thermodiastaticus, New Zealand Microbiological Society Conference 2017, 2017.
6.
Ishiyama Shinnosuke, Yamahata Naoki, Junji Hayashi and Wakayama Mamoru : Development of a new type of liquor made from whey-selections of Aspergillus species and raw materials for malt, and examination of brewing conditions, New Zealand Microbiological Society Conference 2017, 2017.
7.
Junji Hayashi, Ohshima Toshihisa and Sakuraba Haruhiko : Unique cofactor binding mode of homoserine dehydrogenase from hyperthermophilic archaeon Pyrococcus horikoshii, Extremophiles 2016, Sep. 2016.
Intuy Rattanaporn, Takafumi Itoh, Wasana Suyotha, Junji Hayashi, Shigekazu Yano, Koki Makabe, Yosuke Toyotake, Mamoru Wakayama and Takao Hibi : Reaction mechanism and crystallization of catalytic α-1,3-glucanase from Paenibacillus glycanilyticus FH11, Japan Society for Bioscience, Biotechnology, and Agrochemistry, Mar. 2020.
Phumsombat Putthapong, Sano Chiharu, Hibi Takao, Itoh Takafumi, 林 順司, Toyotake Yosuke, Wakayama Mamoru : Immobilization and site directed mutagenesis of Trp525 residue of γ-Glutamyltranspeptidase from Pseudomonas nitroreducens (PnGGT) to improve Theanine production, The Society for Biotechnology, Japan, 2019年9月.