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Akira Hachimori - One of the best experts on this subject based on the ideXlab platform.
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Cloning and expression of the inorganic pyrophosphatase gene from Thermophilic Bacterium PS-3
IUBMB Life, 1996Co-Authors: Satoshi Maruyama, T Ichiba, Masanaga Maeshima, Masaki Nishimura, Motoko Aoki, Junichi Sekiguchi, Akira HachimoriAbstract:SUMMARY. The Thermophilic Bacterium PS-3 ppa gene encoding inorganic pyrophosphatase (PPiase) has been cloned and sequenced. The deduced amino acid sequence was exactly the same as that determined on Edman degradation (Ichiba, T., Takenaka, 0., Samejima, T. and Hachimori, A., J. Siochem. 108: 572-578, 1990) except that amino acid-70 is threonine instead of proline and two more amino acids, Asn-Lys, are present at the carboxyl terminus. The structural gene containing BamH I and Hind III restriction cleaving sites at the 5' and 3' ends, respectively, was amplified by the PCR method. Escherichia coli 0/4109 was transformed with a synthetic DNA, and we found that Thermophilic ppa was expressed in E. coli, as judged on enzyme assaying, SDS-PAGE and immune assaying~
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The role of histidine-118 of inorganic pyrophosphatase from Thermophilic Bacterium PS-3.
Biochemical Journal, 1991Co-Authors: Naoto Hirano, T Ichiba, Akira HachimoriAbstract:Treatment of the inorganic pyrophosphatase from Thermophilic Bacterium PS-3 with diethyl pyrocarbonate resulted in the almost complete loss of its activity, which followed pseudo-first-order kinetics. The presence of Mg2+ prevented the inactivation. Enzyme inactivated with diethyl pyrocarbonate was re-activated by hydroxylamine. The inactivation parallelled the amount of modified histidine residue, and a plot of the activity remaining against the amount of modified histidine residue suggested that the modification of one of two histidine residues totally inactivated the enzyme. The site involved was found to be located in a single lysyl endopeptidase-digest peptide derived from the ethoxy[14C]carbonylated enzyme. Amino acid analysis and sequence analysis of the peptide revealed that it comprised residues 96-119 of the inorganic pyrophosphatase from Thermophilic Bacterium PS-3. These results, when compared with those reported for the Escherichia coli and yeast enzymes, imply that His-118 of the inorganic pyrophosphatase from Thermophilic Bacterium PS-3 is located near the Mg(2+)-binding site and thus affects the binding of Mg2+.
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Primary structure of the inorganic pyrophosphatase from Thermophilic Bacterium PS-3.
The Journal of Biochemistry, 1990Co-Authors: T Ichiba, Osamu Takenaka, Tatsuya Samejima, Akira HachimoriAbstract:The complete amino acid sequence of the inorganic pyrophosphatase from Thermophilic Bacterium PS-3 was determined by automated Edman analysis of the intact protein and of peptides derived from digests obtained with lysylendopeptidase, Staphylococcus aureus strain V8 protease, and arginylendopeptidase. The monomer peptide chain comprises 164 amino acid residues and has a calculated molecular weight of 18,792. The sequence is identical at about 46% of the amino acid positions with that of the Escherichia coli enzymes.
Helena Santos - One of the best experts on this subject based on the ideXlab platform.
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a highly thermostable trehalase from the Thermophilic Bacterium rhodothermus marinus
Extremophiles, 2007Co-Authors: Carla D Jorge, Maria Manuel Sampaio, Jakob K Kristjanson, Gudmundur Oli Hreggvidsson, Helena SantosAbstract:Trehalases play a central role in the metabolism of trehalose and can be found in a wide variety of organisms. A periplasmic trehalase (α,α-trehalose glucohydrolase, EC 3.2.1.28) from the Thermophilic Bacterium Rhodothermus marinus was purified and the respective encoding gene was identified, cloned and overexpressed in Escherichia coli. The recombinant trehalase is a monomeric protein with a molecular mass of 59 kDa. Maximum activity was observed at 88°C and pH 6.5. The recombinant trehalase exhibited a K m of 0.16 mM and a V max of 81 μmol of trehalose (min)−1 (mg of protein)−1 at the optimal temperature for growth of R. marinus (65°C) and pH 6.5. The enzyme was highly specific for trehalose and was inhibited by glucose with a K i of 7 mM. This is the most thermostable trehalase ever characterized. Moreover, this is the first report on the identification and characterization of a trehalase from a Thermophilic Bacterium.
M Iu Grabovich - One of the best experts on this subject based on the ideXlab platform.
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Metabolism of the Thermophilic Bacterium Oceanithermus profundus
Microbiology, 2008Co-Authors: D V Fedosov, D A Podkopaeva, M L Miroshnichenko, E. A. Bonch-osmolovskaya, Alexander V. Lebedinsky, M Iu GrabovichAbstract:The metabolism of the novel facultatively anaerobic Thermophilic Bacterium Oceanithermus profundus was studied during growth on maltose, acetate, pyruvate, and hydrogen. The utilization of carbohydrates was shown to proceed via the glycolytic pathway. Under microaerobic growth conditions, the metabolism of O. profundus grown on maltose depended on the substrate concentration. At an initial maltose concentration of 1.4 mM, O. profundus carried out oxygen respiration, and in the presence of 3.5 mM maltose, facilitated fermentation occurred, with the formation of acetate and ethanol and limited involvement of oxygen. The use of pyruvate and acetate occurred via the TCA cycle. In cells grown on acetate, the activity of glyoxylate pathway enzymes was revealed. Depending on the energy-yielding process providing for growth (oxygen respiration or nitrate reduction), cells contained cytochromes a and c or b, respectively. The results obtained demonstrate the plasticity of the metabolism of O. profundus, which thus appears to be well-adjusted to the rapidly changing conditions in deep-sea hydrothermal vents.
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Metabolism of the Thermophilic Bacterium Oceanithermus profundus
Mikrobiologiia, 2008Co-Authors: D V Fedosov, D A Podkopaeva, M L Miroshnichenko, E A Bonch-osmolovskaia, A V Lebedinskiĭ, M Iu GrabovichAbstract:The metabolism of the novel facultatively anaerobic Thermophilic Bacterium Oceanithermus profundus was studied during growth on maltose, acetate, pyruvate, and hydrogen. The utilization of carbohydrates was shown to proceed via the glycolytic pathway. Under microaerobic growth conditions, the metabolism of O. profundus grown on maltose depended on the substrate concentration. At an initial maltose concentration of 1.4 mM, O. profundus carried out oxygen respiration, and in the presence of 3.5 mM maltose, facilitated fermentation occurred, with the formation of acetate and ethanol and limited involvement of oxygen. The use of pyruvate and acetate occurs via the TCA cycle. In cells grown on acetate, the activity of glyoxylate pathway enzymes was revealed. Depending on the energy-yielding process providing for growth (oxygen respiration or nitrate reduction), cells contained cytochromes a and c or b, respectively. The results obtained demonstrate the plasticity of the metabolism of O. profundus, which thus appears to be well-adjusted to the rapidly changing conditions in deep-sea hydrothermal vents.
Carla D Jorge - One of the best experts on this subject based on the ideXlab platform.
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a highly thermostable trehalase from the Thermophilic Bacterium rhodothermus marinus
Extremophiles, 2007Co-Authors: Carla D Jorge, Maria Manuel Sampaio, Jakob K Kristjanson, Gudmundur Oli Hreggvidsson, Helena SantosAbstract:Trehalases play a central role in the metabolism of trehalose and can be found in a wide variety of organisms. A periplasmic trehalase (α,α-trehalose glucohydrolase, EC 3.2.1.28) from the Thermophilic Bacterium Rhodothermus marinus was purified and the respective encoding gene was identified, cloned and overexpressed in Escherichia coli. The recombinant trehalase is a monomeric protein with a molecular mass of 59 kDa. Maximum activity was observed at 88°C and pH 6.5. The recombinant trehalase exhibited a K m of 0.16 mM and a V max of 81 μmol of trehalose (min)−1 (mg of protein)−1 at the optimal temperature for growth of R. marinus (65°C) and pH 6.5. The enzyme was highly specific for trehalose and was inhibited by glucose with a K i of 7 mM. This is the most thermostable trehalase ever characterized. Moreover, this is the first report on the identification and characterization of a trehalase from a Thermophilic Bacterium.
Karel Sigler - One of the best experts on this subject based on the ideXlab platform.
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N-Acylated Bacteriohopanehexol-Mannosamides from the Thermophilic Bacterium Alicyclobacillus acidoterrestris
Lipids, 2011Co-Authors: Tomáš Řezanka, Lucie Siristova, Karel Melzoch, Karel SiglerAbstract:Identification of molecular species of various N -acylated bacteriohopanehexol-mannosamides from the Thermophilic Bacterium Alicyclobacillus acidoterrestris by semipreparative HPLC and by RP-HPLC with ESI is described. We used triple-quadrupole type mass spectrometer, ^1H and ^13C NMR for analyzing this complex lipid. CD spectra of two compounds (model compound—7-deoxy- d - glycero - d - allo -heptitol obtained by stereospecific synthesis, and an isolated derivative of hopane) were also measured and the absolute configuration of both compounds was determined. On the basis of all the above methods, we identified the full structure of a new class of bacteriohopanes, represented by various N -acylated bacteriohopanehexol-mannosamides.
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Direct ESI-MS analysis of O-acyl glycosylated cardiolipins from the Thermophilic Bacterium Alicyclobacillus acidoterrestris.
Chemistry and Physics of Lipids, 2009Co-Authors: Tomáš Řezanka, Lucie Siristova, Karel Melzoch, Karel SiglerAbstract:Abstract We used direct ESI-MS analysis to identify derivatives of cardiolipin molecular species (i.e. O-acyl glycosylated cardiolipins) from the Thermophilic Bacterium Alicyclobacillus acidoterrestris. We used triple-quadrupole type mass spectrometer for analysis of this complex lipid and enzymatic hydrolysis and 1H and 13C NMR for the identification of these cardiolipin derivatives. These techniques enabled us to identify and quantify the specific molecular species profiles of derivatives of cardiolipin directly from lipid extracts of the Bacterium including the identification of the sugar moiety as α- d -mannose and all five acyls including their positional isomers.
