Advertisement

Amino Acids

, Volume 38, Issue 1, pp 247–255 | Cite as

d-Amino acid dehydrogenase from Helicobacter pylori NCTC 11637

  • Minoru Tanigawa
  • Tomomitsu Shinohara
  • Makoto Saito
  • Katsushi Nishimura
  • Yuichiro Hasegawa
  • Sadao Wakabayashi
  • Morio Ishizuka
  • Yoko Nagata
Original Article

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.

Keywords

d-Amino acid dehydrogenase d-Proline Gene cloning Helicobacter pylori Bacterial respiration 

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

Notes

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.

References

  1. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem 72:248–254. doi: 10.1016/0003-2697(76)90527-3 CrossRefPubMedGoogle Scholar
  2. Dixon M, Kleppe K (1965) d-Amino acid oxidase II. Specificity, competitive inhibitions and reaction sequence. Biochim Biophys Acta 96:368–382Google Scholar
  3. Dunn BE, Cohen H, Blaser MJ (1997) Helicobacter pylori. Clin Microbiol Rev 10:720–741PubMedGoogle Scholar
  4. Fischer L, Gabler M, Horner R, Wagner F (1996) Microbial d-amino acid oxidases (EC 1.4.3.3). Ann NY Acad Sci 799:683–688CrossRefPubMedGoogle Scholar
  5. Franklin FCH, Venables WA (1976) Biochemical, genetic, and regulatory studies of alanine catabolism in scherichia coli K12. Mol Gen Genet 149:229–237. doi: 10.1007/BF00332894 CrossRefPubMedGoogle Scholar
  6. Frisell WR, Hellerman L (1956) The sulfhydryl character of d-amino acid oxidase. J Biol Chem 225:53–62Google Scholar
  7. Frisell WR, Lowe HJ, Hellerman L (1956) Flavoenzyme catalysis. Substrate-competitive inhibition of d-amino acid oxidase. J Biol Chem 223:75–83PubMedGoogle Scholar
  8. Fukui K, Watanabe F, Shibata T, Miyake Y (1987) Molecular cloning and sequence analysis of cDNAs encoding porcine kidney d-amino acid oxidase. Biochemistry 26:3612–3618. doi: 10.1021/bi00386a054 CrossRefPubMedGoogle Scholar
  9. Kelly DJ (1998) The physiology and metabolism of the human gastric pathogen Helicobacter pylori. Adv Microb Physiol 44:137–189. doi: 10.1016/S0065-2911(08)60131-9 CrossRefGoogle Scholar
  10. Krishnan N, Becker DB (2006) Oxygen reactivity of PutA from Helicobacter species and proline-linked oxidative stress. J Bacteriol 188:1227–1235. doi: 10.1128/JB.188.4.1227-1235.2006 CrossRefPubMedGoogle Scholar
  11. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the bacteriophage T4. Nature 227:680–685. doi: 10.1038/227680a0 CrossRefPubMedGoogle Scholar
  12. Łobocka M, Henning J, Wild J, Kłopotowski T (1994) Organization and expression of the Escherichia coli K-12 dad operon encoding the smaller subunit of d-amino acid dehydrogenase and the catabolic alanine racemase. J Bacteriol 176:1500–1510PubMedGoogle Scholar
  13. Marshall VP, Sokatch JR (1968) Oxidation of d-amino acids by a particulate enzyme from Pseudomonas aeruginosa. J Bacteriol 95:1419–1425PubMedGoogle Scholar
  14. Menzel R, Roth J (1981) Enzymatic properties of the purified putA protein from Salmonella typhimurium. J Biol Chem 256:9762–9766PubMedGoogle Scholar
  15. Nagata K, Nagata Y, Sato T, Fujino A, Nakajima K, Tamura T (2003) l-Serine, d- and l-proline and alanine as respiratory substrates of Helicobacter pylori: correlation between in vitro and in vivo amino acid levels. Microbiol 149:2023–2030. doi: 10.1099/mic.0.26203-0 CrossRefGoogle Scholar
  16. Nagata Y, Sato T, Enomoto N, Isii Y, Sasaki K, Yamada T (2007) High concentrations of d-amino acids in human gastric juice. Amino Acids 32:137–140. doi: 10.1007/s00726-006-0262-9 CrossRefPubMedGoogle Scholar
  17. Olsiewski PJ, Kaczorowski CJ, Walsh CT (1980) Purification and properties of d-amino acid dehydrogenase, an inducible membrane-bound iron-sulfur flavoenzyme from Escherichia coli. J Biol Chem 255:4487–4494PubMedGoogle Scholar
  18. Raunio RP, Straus LP, Jenkins WT (1973) d-Alanine oxidase from Escherichia coli: Participation in the oxidation of l-alanine. J Bacteriol 115:567–573PubMedGoogle Scholar
  19. Ronchi S, Minchiotti L, Galliano M, Curti B, Swenson RP, Williams CH Jr, Massey V (1982) The primary structure of d-amino acid oxidase from pig kidney. II. Isolation and sequence of overlap peptides and the complete sequence. J Biol Chem 257:8824–8834PubMedGoogle Scholar
  20. Saito M, Nishimura K, Hasegawa Y, Shinohara T, Wakabayashi S, Kurihara T, Ishizuka M, Nagata Y (2007) Alanine racemase from Helicobacter pylori NCTC 11637: purification, characterization and gene cloning. Life Sci 80:788–794. doi: 10.1016/j.lfs.2006.11.005 CrossRefPubMedGoogle Scholar
  21. Satomura T, Kawakami R, Sakuraba H, Ohshima T (2002) Dye-linked d-proline dehydrogenase from hyperthermophilic archaeon Pyrobaculum islandicum is a novel FAD-dependent amino acid dehydrogenase. J Biol Chem 277:12861–12867. doi: 10.1074/jbc.M112272200 CrossRefPubMedGoogle Scholar
  22. Scarpulla RC, Soffer RL (1978) Membrane-bound proline dehydrogenase from Escherichia coli. J Biol Chem 253:5997–6001PubMedGoogle Scholar
  23. Tsuda M, Karita M, Morshed MG, Okita K, Nakazawa T (1994) A urease-negative mutant of Helicobacter pylori constructed by allelic exchange mutagenesis lacks the ability to colonize the nude mouse stomach. Infect Immun 62:3586–3589PubMedGoogle Scholar
  24. Tsukada K (1966) d-Amino acid dehydrogenases of Pseudomonas fluorescence. J Biol Chem 241:4522–4528PubMedGoogle Scholar
  25. Wild J, Walczac W, Krajewska-Grynkiewicz K, Klopotowski T (1974) d-Amino acid dehydrogenase: the enzyme of the first step of d-histidine and d-methionine racemisation in Salmonella typhimurium. Mol Gen Genet 128:131–146. doi: 10.1007/BF02654486 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Minoru Tanigawa
    • 1
  • Tomomitsu Shinohara
    • 1
  • Makoto Saito
    • 1
  • Katsushi Nishimura
    • 1
    • 2
  • Yuichiro Hasegawa
    • 1
  • Sadao Wakabayashi
    • 3
  • Morio Ishizuka
    • 4
  • Yoko Nagata
    • 1
  1. 1.Department of Materials and Applied Chemistry, College of Science and TechnologyNihon UniversityTokyoJapan
  2. 2.Department of Applied Chemistry, Junior CollegeNihon UniversityChibaJapan
  3. 3.Department of Life Sciences, Graduate School of Life ScienceUniversity of HyogoHyogoJapan
  4. 4.Department of Applied Chemistry, Faculty of Science and EngineeringChuo UniversityTokyoJapan

Personalised recommendations