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Characterization and improvement of substrate-binding affinity of d-aspartate oxidase of the thermophilic fungus Thermomyces dupontii

  • Shouji TakahashiEmail author
  • Kohei Osugi
  • Yuya Shimekake
  • Akira Shinbo
  • Katsumasa Abe
  • Yoshio Kera
Biotechnologically relevant enzymes and proteins
  • 107 Downloads

Abstract

d-Aspartate oxidase (DDO) is a valuable enzyme that can be utilized in the determination of acidic d-amino acids and the optical resolution of a racemic mixture of acidic amino acids, which require its higher stability, higher catalytic activity, and higher substrate-binding affinity. In the present study, we identified DDO gene (TdDDO) of a thermophilic fungus, Thermomyces dupontii, and characterized the recombinant enzyme expressed in Escherichia coli. In addition, we generated a variant that has a higher substrate-binding affinity. The recombinant TdDDO expressed in E. coli exhibited oxidase activity toward acidic d-amino acids and a neutral d-amino acid, d-Gln, with the highest activity toward d-Glu. The Km and kcat values for d-Glu were 2.16 mM and 217 s−1, respectively. The enzyme had an optimum pH and temperature 8.0 and 60 °C, respectively, and was stable between pH 5.0 and 10.0, with a T50 of ca. 51 °C, which was much higher than that in DDOs from other origins. Enzyme stability decreased following a decrease in protein concentration, and externally added FAD could not repress the destabilization. The mutation of Phe248, potentially located in the active site of TdDDO, to Tyr residue, conserved in DDOs and d-amino acid oxidases, markedly increased substrate-binding affinity. The results showed the great potential of TdDDO and the variant for practical applications.

Keywords

d-Aspartate oxidase Thermomyces dupontii Thermophilic fungus Thermostable E. coli expression Site-directed mutagenesis 

Notes

Acknowledgments

This study was supported by the Grant-in-Aid for Scientific Research (C) (23580106) to S. Takahashi from the Japan Society for the Promotion of Science.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Research involving human participants

This article does not contain any studies with human participants or animals performed by any of the authors.

References

  1. Ariyoshi M, Katane M, Hamase K, Miyoshi Y, Nakane M, Hoshino A, Okawa Y, Mita Y, Kaimoto S, Uchihashi M, Fukai K, Ono K, Tateishi S, Hato D, Yamanaka R, Honda S, Fushimura Y, Iwai-Kanai E, Ishihara N, Mita M, Homma H, Matoba S (2017) d-Glutamate is metabolized in the heart mitochondria. Sci Rep 7:43911.  https://doi.org/10.1038/srep43911 Google Scholar
  2. Arroyo M, Menendez M, Garcia JL, Campillo N, Hormigo D, de la Mata I, Castillon MP, Acebal C (2007) The role of cofactor binding in tryptophan accessibility and conformational stability of His-tagged d-amino acid oxidase from Trigonopsis variabilis. Biochim Biophys Acta 1774(5):556–565.  https://doi.org/10.1016/j.bbapap.2007.03.009 Google Scholar
  3. Balu DT, Coyle JT (2015) The NMDA receptor ‘glycine modulatory site’ in schizophrenia: d-serine, glycine, and beyond. Curr Opin Pharmacol 20:109–115.  https://doi.org/10.1016/j.coph.2014.12.004 Google Scholar
  4. Bruckner H, Westhauser T (2003) Chromatographic determination of l- and d-amino acids in plants. Amino Acids 24(1–2):43–55.  https://doi.org/10.1007/s00726-002-0322-8 Google Scholar
  5. Cava F, Lam H, de Pedro MA, Waldor MK (2011) Emerging knowledge of regulatory roles of d-amino acids in bacteria. Cell Mol Life Sci 68(5):817–831.  https://doi.org/10.1007/s00018-010-0571-8 Google Scholar
  6. de Oliveira TB, Gomes E, Rodrigues A (2015) Thermophilic fungi in the new age of fungal taxonomy. Extremophiles 19(1):31–37.  https://doi.org/10.1007/s00792-014-0707-0 Google Scholar
  7. Dib I, Slavica A, Riethorst W, Nidetzky B (2006) Thermal inactivation of d-amino acid oxidase from Trigonopsis variabilis occurs via three parallel paths of irreversible denaturation. Biotechnol Bioeng 94(4):645–654.  https://doi.org/10.1002/bit.20854 Google Scholar
  8. Dunlop DS, Neidle A, McHale D, Dunlop DM, Lajtha A (1986) The presence of free d-aspartic acid in rodents and man. Biochem Biophys Res Commun 141(1):27–32.  https://doi.org/10.1016/s0006-291x(86)80329-1 Google Scholar
  9. Errico F, Napolitano F, Nistico R, Usiello A (2012) New insights on the role of free d-aspartate in the mammalian brain. Amino Acids 43(5):1861–1871.  https://doi.org/10.1007/s00726-012-1356-1 Google Scholar
  10. Errico F, Napolitano F, Squillace M, Vitucci D, Blasi G, de Bartolomeis A, Bertolino A, D'Aniello A, Usiello A (2013) Decreased levels of d-aspartate and NMDA in the prefrontal cortex and striatum of patients with schizophrenia. J Psychiatr Res 47(10):1432–1437.  https://doi.org/10.1016/j.jpsychires.2013.06.013 Google Scholar
  11. Errico F, Mothet JP, Usiello A (2015) d-Aspartate: an endogenous NMDA receptor agonist enriched in the developing brain with potential involvement in schizophrenia. J Pharm Biomed Anal 116:7–17.  https://doi.org/10.1016/j.jpba.2015.03.024 Google Scholar
  12. Fukunaga S, Yuno S, Takahashi M, Taguchi S, Kera Y, Odani S, Yamada RH (1998) Purification and properties of d-glutamate oxidase from Candida boidinii 2201. J Ferment Bioeng 85(6):579–583.  https://doi.org/10.1016/S0922-338x(98)80008-1 Google Scholar
  13. Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual, 4th edn. Cold spring Harbor laboratory Press, Cold Spring HarborGoogle Scholar
  14. Hashimoto A, Nishikawa T, Hayashi T, Fujii N, Harada K, Oka T, Takahashi K (1992) The presence of free d-serine in rat brain. FEBS Lett 296(1):33–36.  https://doi.org/10.1016/0014-5793(92)80397-y Google Scholar
  15. Katane M, Homma H (2011) d-Aspartate - an important bioactive substance in mammals: a review from an analytical and biological point of view. J Chromatogr B Anal Technol Biomed Life Sci 879(29):3108–3121.  https://doi.org/10.1016/j.jchromb.2011.03.062 Google Scholar
  16. Katane M, Saitoh Y, Seida Y, Sekine M, Furuchi T, Homma H (2010) Comparative characterization of three d-aspartate oxidases and one d-amino acid oxidase from Caenorhabditis elegans. Chem Biodivers 7(6):1424–1434.  https://doi.org/10.1002/cbdv.200900294 Google Scholar
  17. Katane M, Saitoh Y, Maeda K, Hanai T, Sekine M, Furuchi T, Homma H (2011) Role of the active site residues arginine-216 and arginine-237 in the substrate specificity of mammalian d-aspartate oxidase. Amino Acids 40(2):467–476.  https://doi.org/10.1007/s00726-010-0658-4 Google Scholar
  18. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26(2):283–291.  https://doi.org/10.1107/s0021889892009944 Google Scholar
  19. Maalej I, Belhaj I, Masmoudi NF, Belghith H (2009) Highly thermostable xylanase of the thermophilic fungus Talaromyces thermophilus: purification and characterization. Appl Biochem Biotechnol 158(1):200–212.  https://doi.org/10.1007/s12010-008-8317-x Google Scholar
  20. Maheshwari R, Bharadwaj G, Bhat MK (2000) Thermophilic fungi: their physiology and enzymes. Microbiol Mol Biol Rev 64(3):461–488.  https://doi.org/10.1128/mmbr.64.3.461-488.2000 Google Scholar
  21. Mallek-Fakhfakh H, Belghith H (2016) Physicochemical properties of thermotolerant extracellular β-glucosidase from Talaromyces thermophilus and enzymatic synthesis of cello-oligosaccharides. Carbohydr Res 419:41–50.  https://doi.org/10.1016/j.carres.2015.10.014 Google Scholar
  22. Martineau M, Baux G, Mothet JP (2006) d-Serine signalling in the brain: friend and foe. Trends Neurosci 29(8):481–491.  https://doi.org/10.1016/j.tins.2006.06.008 Google Scholar
  23. Miyoshi Y, Koga R, Oyama T, Han H, Ueno K, Masuyama K, Itoh Y, Hamase K (2012) HPLC analysis of naturally occurring free d-amino acids in mammals. J Pharm Biomed Anal 69:42–49.  https://doi.org/10.1016/j.jpba.2012.01.041 Google Scholar
  24. Mothet JP, Parent AT, Wolosker H, Brady RO, Linden DJ, Ferris CD, Rogawski MA, Snyder SH (2000) d-Serine is an endogenous ligand for the glycine site of the N-methyl-d-aspartate receptor. Proc Natl Acad Sci U S A 97(9):4926–4931.  https://doi.org/10.1073/pnas.97.9.4926 Google Scholar
  25. Negri A, Massey V, Williams CH Jr (1987) d-Aspartate oxidase from beef kidney. Purification and properties. J Biol Chem 262(21):10026–10034Google Scholar
  26. Nuzzo T, Sacchi S, Errico F, Keller S, Palumbo O, Florio E, Punzo D, Napolitano F, Copetti M, Carella M, Chiariotti L, Bertolino A, Pollegioni L, Usiello A (2017) Decreased free d-aspartate levels are linked to enhanced d-aspartate oxidase activity in the dorsolateral prefrontal cortex of schizophrenia patients. NPJ Schizophr 3:16.  https://doi.org/10.1038/s41537-017-0015-7 Google Scholar
  27. Pollegioni L, Fukui K, Massey V (1994) Studies on the kinetic mechanism of pig kidney d-amino acid oxidase by site-directed mutagenesis of tyrosine 224 and tyrosine 228. J Biol Chem 269(50):31666–31673Google Scholar
  28. Pollegioni L, Iametti S, Fessas D, Caldinelli L, Piubelli L, Barbiroli A, Pilone MS, Bonomi F (2003) Contribution of the dimeric state to the thermal stability of the flavoprotein d-amino acid oxidase. Protein Sci 12(5):1018–1029.  https://doi.org/10.1110/ps.0234603 Google Scholar
  29. Rosini E, Caldinelli L, Piubelli L (2017) Assays of d-amino acid oxidase activity. Front Mol Biosci 4:102.  https://doi.org/10.3389/fmolb.2017.00102 Google Scholar
  30. Rossmann MG, Moras D, Olsen KW (1974) Chemical and biological evolution of a nucleotide-binding protein. Nature 250(5463):194–199.  https://doi.org/10.1038/250194a0 Google Scholar
  31. Sasabe J, Aiso S (2016) Abnormal d-serine metabolism in amyotrophic lateral sclerosis. In: Yoshimura T, Nishikawa T, Homma H (eds) d-Amino acids: physiology, metabolism, and application. Springer, Tokyo, pp 137–149.  https://doi.org/10.1007/978-4-431-56077-7_9 Google Scholar
  32. Setoyama C, Miura R (1997) Structural and functional characterization of the human brain d-aspartate oxidase. J Biochem 121(4):798–803.  https://doi.org/10.1093/oxfordjournals.jbchem.a021655 Google Scholar
  33. Still JL, Buell MV, Knox WE, Green DE (1949) Studies on the cyclophorase system: VII. d-aspartic oxidase. J Biol Chem 179(2):831–837Google Scholar
  34. Stolk AC (1965) Thermophilic species of Talaromyces benjamin and Thermoascus miehe. Antonie Van Leeuwenhoek 31:262–276.  https://doi.org/10.1007/BF02045906 Google Scholar
  35. Stolk AC, Samson RA (1972) The genus Talaromyces: studies on Talaromyces and related genera II. Stud Mycol 2:1–65Google Scholar
  36. Takahashi S, Takahashi T, Kera Y, Matsunaga R, Shibuya H, Yamada RH (2004) Cloning and expression in Escherichia coli of the d-aspartate oxidase gene from the yeast Cryptococcus humicola and characterization of the recombinant enzyme. J Biochem 135(4):533–540.  https://doi.org/10.1093/jb/mvh068 Google Scholar
  37. Takahashi S, Furukawara M, Omae K, Tadokoro N, Saito Y, Abe K, Kera Y (2014) A highly stable d-amino acid oxidase of the thermophilic bacterium Rubrobacter xylanophilus. Appl Environ Microbiol 80(23):7219–7229.  https://doi.org/10.1128/AEM.02193-14 Google Scholar
  38. Takahashi S, Shimada K, Nozawa S, Goto M, Abe K, Kera Y (2016) Possible role of a histidine residue in the substrate specificity of yeast d-aspartate oxidase. J Biochem 159(3):371–378.  https://doi.org/10.1093/jb/mvv108 Google Scholar
  39. Tedeschi G, Negri A, Ceciliani F, Ronchi S, Vetere A, D’Aniello G, D’Aniello A (1994) Properties of the flavoenzyme d-aspartate oxidase from Octopus vulgaris. Biochim Biophys Acta 1207(2):217–222.  https://doi.org/10.1016/0167-4838(94)00071-9 Google Scholar
  40. Yamada R, Ujiie H, Kera Y, Nakase T, Kitagawa K, Imasaka T, Arimoto K, Takahashi M, Matsumura Y (1996) Purification and properties of d-aspartate oxidase from Cryptococcus humicolus UJ1. Biochim Biophys Acta 1294(2):153–158.  https://doi.org/10.1016/0167-4838(96)00012-X Google Scholar
  41. Yu JH, Hamari Z, Han KH, Seo JA, Reyes-Dominguez Y, Scazzocchio C (2004) Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genet Biol 41(11):973–981.  https://doi.org/10.1016/j.fgb.2004.08.001 Google Scholar
  42. Zhang X, Li X, Xia L (2015) Heterologous expression of an alkali and thermotolerant lipase from Talaromyces thermophilus in Trichoderma reesei. Appl Biochem Biotechnol 176(6):1722–1735.  https://doi.org/10.1007/s12010-015-1673-4 Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of BioengineeringNagaoka University of TechnologyNagaokaJapan

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