Advertisement

Assays of d-Amino Acid Oxidases

  • Gabriella TedeschiEmail author
  • Loredano Pollegioni
  • Armando Negri
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 794)

Abstract

d-Amino acid oxidase and d-aspartate oxidase are two well-known FAD-containing flavooxidases that catalyze the same reaction (the oxidative deamination) on different d-amino acids. d-aspartate oxidase is specific for acidic d-amino acids (i.e., d-aspartate and d-glutamate) and d-amino acid oxidase is active on neutral and polar d-amino acids (a low activity is also detected on basic d-amino acids). The assay of these flavoenzymes is of utmost importance in different fields because d-amino acids are common constituents of bacterial cell walls, are present in foods and because free d-serine and d-aspartic acid were identified in brain and peripheral tissues of mammals. In this chapter, we report on the most used methods employed to assay the activity of d-amino acid oxidase and d-aspartate oxidase. Interestingly, their activity can be followed using different assays, namely d-amino acid or oxygen consumption, α-keto acid or ammonia production, or using artificial dyes as final indicator of the flavin redox reaction.

Key words

Enzymatic assay d-Amino acid oxidase d-Aspartate oxidase Analytical detection 

Notes

Acknowledgments

The works carried out in the laboratory of L. Pollegioni were supported by grants from Fondo di Ateneo per la Ricerca (University of Insubria) and from Fondazione Cariplo. LP thanks the support of Consorzio Interuniversitario per le Biotecnologie (CIB) and of Centro di Ricerca in Biotecnologie per la Salute Umana (University of Insubria). The work carried out in the laboratory of G. Tedeschi and A. Negri was supported by grants from Ministero dell’Università e della Ricerca Scientifica and from the Consiglio Nazionale delle Ricerche (Italy).

References

  1. 1.
    Friedman M (1999) Chemistry, nutrition, and microbiology of D-amino acids. J Agric Food Chem 47, 34573479.PubMedCrossRefGoogle Scholar
  2. 2.
    Gandolfi I, Palla G, Delprato L et al. (1992) D-Amino acids in milk as related to heat treatments and bacterial activity. J Food Sci 57, 377–379.CrossRefGoogle Scholar
  3. 3.
    Hashimoto A, Oka T (1997) Free D-aspartate and D-serine in the mammalian brain and periphery. Prog Neurobiol 52, 325–353.PubMedCrossRefGoogle Scholar
  4. 4.
    Fujii N (2002). D-Amino acids in living higher organisms. Orig Life Evol Biosph 32, 103127. PubMedCrossRefGoogle Scholar
  5. 5.
    Snyder S H, Kim P M (2000). D-Amino acids as putative neurotransmitters: focus on D-serine. Neurochem Res 25, 553560.PubMedCrossRefGoogle Scholar
  6. 6.
    Pollegioni L, Sacchi S (2010) Metabolism of the neuromodulator D-serine. Cell Mol Life Sci 67, 2387–2404.PubMedCrossRefGoogle Scholar
  7. 7.
    Fantinato S, Pollegioni L, Pilone M S (2001) Engineering, expression and purification of a His-tagged chimeric D-amino acid oxidase from Rhodotorula gracilis. Enz Microb Technol 29, 407–412.CrossRefGoogle Scholar
  8. 8.
    Pollegioni L, Piubelli L, Sacchi S et al. (2007) Physiological functions of D-amino acid oxidases: from yeast to humans. Cell Mol Life Sci 64, 1373–1394.PubMedCrossRefGoogle Scholar
  9. 9.
    Pollegioni L, Sacchi S, Caldinelli L et al. (2007) Engineering the properties of D-amino acid oxidases by a rational and a directed evolution approach. Curr Protein Pept Sci 8, 600–618.PubMedCrossRefGoogle Scholar
  10. 10.
    Boselli A, Piubelli L, Molla G et al. (2007) Investigating the role of active site residues of Rhodotorula gracilis D-amino acid oxidase on its substrate specificity. Biochimie 89, 360–368.PubMedCrossRefGoogle Scholar
  11. 11.
    Negri A, Massey V, Williams C H Jr (1987) D-aspartate oxidase from beef kidney. J Biol Chem 262, 10026–10034.PubMedGoogle Scholar
  12. 12.
    Simonic T, Duga S, Negri A et al. (1997) cDNA cloning and expression of the flavoprotein D-aspartate oxidase from bovine kidney cortex. Biochem J 322, 729–735.PubMedGoogle Scholar
  13. 13.
    Fonda M L, Anderson B M (1969) D-amino acid oxidase. IV. Inactivation by maleimides. J Biol Chem 244, 666–674.Google Scholar
  14. 14.
    Nagata Y, Shimojo T, Akino T (1988) Two spectrophotometric assays for D-amino acid oxidase: for the study of distribution patterns. Int J Biochem 20, 1235–1238.PubMedCrossRefGoogle Scholar
  15. 15.
    Negri A, Tedeschi G, Ceciliani F, Ronchi S (1999) Purification of beef kidney D-aspartate oxidase overexpressed in E. coli and characterization of its redox potentials and oxidative activity towards agonists of excitatory amino acid receptors. Biochim Biophys Acta 143, 212–222.CrossRefGoogle Scholar
  16. 16.
    Mori N, Sano M, Tani Y, Yamada H (1980) Purification and properties of sarcosine oxidase from Cylindrocarpon didymum M-1. Agric Biol Chem 44, 1391–1397.CrossRefGoogle Scholar
  17. 17.
    Sacchi S, Bernasconi M, Martineau M et al. (2008) pLG72 modulates intracellular D-serine levels through its interaction with D-amino acid oxidase: effect on schizophrenia susceptibility. J Biol Chem 283, 22244–22256.PubMedCrossRefGoogle Scholar
  18. 18.
    Tedeschi G, Negri A, Bernardini G et al. (1999) D-Aspartate oxidase is present in ovaries, eggs and embryos but not in testis of Xenopus laevis. Comp Biochem Physiol 124B, 489–494.Google Scholar
  19. 19.
    Molla G, Sacchi S, Bernasconi M G et al. (2006) Characterization of human D-amino acid oxidase. FEBS Letters 580, 2358–2364.PubMedCrossRefGoogle Scholar
  20. 20.
    Pollegioni L, Falbo A, Pilone M S (1992) Specificity and kinetics of Rhodotorula gracilis D-amino acid oxidase. Biochim Biophys Acta 1120, 11–16.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Gabriella Tedeschi
    • 1
    Email author
  • Loredano Pollegioni
    • 2
    • 3
  • Armando Negri
    • 1
  1. 1.Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria – sez. BiochimicaUniversità degli Studi di MilanoMilanoItaly
  2. 2.Dipartimento di Biotecnologie e Scienze MolecolariUniversità degli Studi dell’InsubriaVareseItaly
  3. 3.“The Protein Factory”Politecnico di Milano and Università degli Studi dell’InsubriaVareseItaly

Personalised recommendations