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d-Amino acid dehydrogenase from Helicobacter pylori NCTC 11637

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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 Km and Vmax 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.

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Abbreviations

DAD:

d-Amino acid dehydrogenase

DadA:

DAD purified from recombinant Escherichia coli

DCIP:

2,6-Dichlorophenolindophenol

PMS:

Phenazine methosulfate

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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Acknowledgments

We would like to express our thanks to Dr. K. Nagata (Department of Bacteriology, Hyogo College of Medicine) for providing H. pylori NCTC 11637 strain and useful advice for culture.

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Authors and Affiliations

  1. Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, Tokyo, 101-8308, Japan

    Minoru Tanigawa, Tomomitsu Shinohara, Makoto Saito, Katsushi Nishimura, Yuichiro Hasegawa & Yoko Nagata

  2. Department of Applied Chemistry, Junior College, Nihon University, Chiba, 274-8501, Japan

    Katsushi Nishimura

  3. Department of Life Sciences, Graduate School of Life Science, University of Hyogo, Hyogo, 678-1297, Japan

    Sadao Wakabayashi

  4. Department of Applied Chemistry, Faculty of Science and Engineering, Chuo University, Bunkyo-Ku, Tokyo, 112-0003, Japan

    Morio Ishizuka

Authors
  1. Minoru Tanigawa
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  2. Tomomitsu Shinohara
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  3. Makoto Saito
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  4. Katsushi Nishimura
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  5. Yuichiro Hasegawa
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  6. Sadao Wakabayashi
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  7. Morio Ishizuka
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  8. Yoko Nagata
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Corresponding author

Correspondence to Yoko Nagata.

Additional information

Database

The sequence of H. pylori NCTC 11637 gene dadA reported in this paper has been submitted to DDBJ, GenBank ™ and BBI databanks under accession No. AB295062 (NCBI accession No. BAF48065).

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Tanigawa, M., Shinohara, T., Saito, M. et al. d-Amino acid dehydrogenase from Helicobacter pylori NCTC 11637. Amino Acids 38, 247–255 (2010). https://doi.org/10.1007/s00726-009-0240-0

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  • DOI: https://doi.org/10.1007/s00726-009-0240-0

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