Tomomitsu Shinohara

Nihon University, Edo, Tōkyō, Japan

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Publications (3)8.8 Total impact

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    ABSTRACT: Helicobacter pylori is a microaerophilic bacterium associated with gastric inflammation and peptic ulcers. Knowledge of how pathogenic organisms produce energy is important from a therapeutic point of view. We found d-amino acid dehydrogenase-mediated electron transport from d-proline or d-alanine to oxygen via the respiratory chain in H. pylori. Coupling of the electron transport to ATP synthesis was confirmed by using uncoupler reagents. We reconstituted the electron transport chain to demonstrate the electron flow from the d-amino acids to oxygen using the recombinant cytochrome bc1 complex, cytochrome c-553, and the terminal oxidase cytochrome cbb3 complex. Upon addition of the recombinant d-amino acid dehydrogenase and d-proline or d-alanine to the reconstituted electron transport system, reduction of cytochrome cbb3 and oxygen consumption was revealed spectrophotometrically and polarographically, respectively. Among the constituents of H. pylori's electron transport chain, only the cytochrome bc1 complex had been remained unpurified. Therefore, we cloned and sequenced the H. pylori NCTC 11637 cytochrome bc1 gene clusters encoding Rieske Fe-S protein, cytochrome b, and cytochrome c1, with calculated molecular masses of 18 kDa, 47 kDa, and 32 kDa, respectively, and purified the recombinant monomeric protein complex with a molecular mass of 110 kDa by gel filtration. The absorption spectrum of the recombinant cytochrome bc1 complex showed an α peak at 561 nm with a shoulder at 552 nm.
    Full-text · Article · Dec 2009 · Journal of bacteriology
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    ABSTRACT: Helicobacter pylori is a microaerophilic bacterium, associated with gastric inflammation and peptic ulcers. d-Amino acid dehydrogenase is a flavoenzyme that digests free neutral d-amino acids yielding corresponding 2-oxo acids and hydrogen. We sequenced the H. pylori NCTC 11637 d-amino acid dehydrogenase gene, dadA. The primary structure deduced from the gene showed low similarity with other bacterial d-amino acid dehydrogenases. We purified the enzyme to homogeneity from recombinant Escherichia coli cells by cloning dadA. The recombinant protein, DadA, with 44 kDa molecular mass, possessed FAD as cofactor, and showed the highest activity to d-proline. The enzyme mediated electron transport from d-proline to coenzyme Q1, thus distinguishing it from d-amino acid oxidase. The apparent K m and V max values were 40.2 mM and 25.0 μmol min−1 mg−1, respectively, for dehydrogenation of d-proline, and were 8.2 μM and 12.3 μmol min−1 mg−1, respectively, for reduction of Q1. The respective pH and temperature optima were 8.0 and 37°C. Enzyme activity was inhibited markedly by benzoate, and moderately by SH reagents. DadA showed more similarity with mammalian d-amino acid oxidase than other bacterial d-amino acid dehydrogenases in some enzymatic characteristics. Electron transport from d-proline to a c-type cytochrome was suggested spectrophotometrically.
    No preview · Article · Mar 2009 · Amino Acids
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    ABSTRACT: The Helicobacter pylori NCTC 11637 alanine racemase gene, alr1, was cloned based on a putative alanine racemase gene, alr, of H. pylori 26695. The protein, Alr1, was purified to homogeneity from Escherichia coli MB2795 cells harboring the alr1 gene. The protein exclusively catalyzes the conversion of l-alanine to the d-isomer with K(m) and V(max) values of 100 mM and 909 mumol min(-1) mg(-1), respectively. The values are 16-fold higher than those for the reaction in the reverse direction. The molecular weight of Alr1 is 42,000 by SDS-PAGE, and 68,000 by gel-filtration analysis. The optimal pH and temperature are pH 8.3 and 37 degrees C, respectively, in good accordance with the characteristics shown by the alanine racemase purified from H. pylori NCTC 11637 cells. Pyridoxal 5'-phosphate was suggested to be the cofactor. The physiological function of Alr1 is discussed regarding energy production in the microbial cells.
    No preview · Article · Feb 2007 · Life Sciences