Advertisement

Extremophiles

, Volume 22, Issue 6, pp 975–981 | Cite as

Enzymological characteristics of a novel archaeal dye-linked d-lactate dehydrogenase showing loose binding of FAD

  • Takenori Satomura
  • Junji Hayashi
  • Tatsuya Ohshida
  • Haruhiko Sakuraba
  • Toshihisa Ohshima
  • Shin-ichiro Suye
Original Paper

Abstract

A gene-encoding a dye-linked d-lactate dehydrogenase (Dye-DLDH) homolog was identified in the genome of the hyperthermophilic archaeon Thermoproteus tenax. The gene was expressed in Escherichia coli and the product was purified to homogeneity. The recombinant protein exhibited highly thermostable Dye-DLDH activity. To date, four types of Dye-DLDH have been identified in hyperthermophilic archaea (in Aeropyrum pernix, Sulfolobus tokodaii, Archaeoglobus fulgidus, and Candidatus Caldiarchaeum subterraneum). The amino acid sequence of T. tenax Dye-DLDH showed the highest similarity (45%) to A. pernix Dye-DLDH, but neither contained a known FAD-binding motif. Nonetheless, both homologs required FAD for enzymatic activity, suggesting that FAD binds loosely to the enzyme and is easily released unlike in other Dye-DLDHs. Our findings indicate that Dye-DLDHs from T. tenax and A. pernix are a novel type of Dye-DLDH characterized by loose binding of FAD.

Keywords

d-Lactate Dye-linked dehydrogenase FAD Hyperthermophilic archaea Thermostable enzyme 

Abbreviations

Bis–tris

bis(2-Hydroxyethyl)iminotris(hydroxymethyl)methane

DCIP

2,6-DichloroindophenolHEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

INT

p-Iodonitrotetrazolium violetMTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

Notes

Acknowledgements

We thank Ms. Manami Oi, Mr. Kazuya Umebayashi, and Ms. Sayuri Yoshihara for the technical assistance. We would like to thank Editage (www.editage.jp) for English language editing.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

References

  1. Brizio C, Galluccio M, Wait R, Torchetti EM, Bafunno V, Accardi R, Gianazzae E, Indiverib C, Barile M (2006) Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase. Biochem Biophys Res Commun 344:1008–1016.  https://doi.org/10.1016/j.bbrc.2006.04.003 CrossRefPubMedGoogle Scholar
  2. Brockman HL, Wood WA (1975) d-Lactate dehydrogenase of Peptostreptococcus elsdenii. Methods Enzymol 41:309–312.  https://doi.org/10.1016/S0076-6879(75)41070-9 CrossRefPubMedGoogle Scholar
  3. Dym O, Pratt EA, Ho C, Eisenberg D (2000) The crystal structure of d-lactate dehydrogenase, a peripheral membrane respiratory enzyme. Proc Natl Acad Sci USA 97:9413–9418.  https://doi.org/10.1073/pnas.97.17.9413 CrossRefPubMedGoogle Scholar
  4. Frew JE, Hill HA (1987) Electrochemical biosensors. Anal Chem 59:933A–944A.  https://doi.org/10.1021/ac00142a720 CrossRefPubMedGoogle Scholar
  5. Gregolin C, Singer TP (1963) The lactic dehydrogenase of yeast. III. d(−)Lactic cytochrome c reductase, a zinc flavoprotein from aerobic yeast. Biochim Biophys Acta 67:201–218.  https://doi.org/10.1016/0926-6569(63)90229-3 CrossRefPubMedGoogle Scholar
  6. Kohn LD, Kaback HR (1973) Mechanisms of active transport in isolated bacterial membrane vesicles. XV. Purification and properties of the membrane-bound d-lactate dehydrogenase from Escherichia coli. J Biol Chem 248:7012–7017PubMedGoogle Scholar
  7. Kujo C, Ohshima T (1998) Enzymological characteristics of the hyperthermostable NAD-dependent glutamate dehydrogenase from the archaeon Pyrobaculum islandicum and effects of denaturants and organic solvents. Appl Environ Microbiol 64:2152–2157PubMedPubMedCentralGoogle Scholar
  8. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.  https://doi.org/10.1038/227680a0 CrossRefGoogle Scholar
  9. Pagala VR, Park J, Reed DW, Hartzell PL (2002) Cellular localization of d-lactate dehydrogenase and NADH oxidase from Archaeoglobus fulgidus. Archaea 1:95–104.  https://doi.org/10.1155/2002/297264 CrossRefPubMedGoogle Scholar
  10. Reed DW, Hartzell PL (1999) The Archaeoglobus fulgidus d-lactate dehydrogenase is a Zn2+ flavoprotein. J Bacteriol 181:7578–7580Google Scholar
  11. Sakuraba H, Takamatsu Y, Satomura T, Kawakami R, Ohshima T (2001) Purification, characterization, and application of a novel dye-linked l-proline dehydrogenase from a hyperthermophilic archaeon, Thermococcus profundus. Appl Environ Microbiol 67:1470–1475.  https://doi.org/10.1128/AEM.67.4.1470-1475.2001 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 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.  https://doi.org/10.1074/jbc.M112272200 CrossRefPubMedGoogle Scholar
  13. Satomura T, Kawakami R, Sakuraba H, Ohshima T (2008) A novel flavin adenine dinucleotide (FAD) containing d-lactate dehydrogenase from the thermoacidophilic crenarchaeota Sulfolobus tokodaii strain 7: purification, characterization and expression in Escherichia coli. J Biosci Bioeng 106:16–21.  https://doi.org/10.1263/jbb.106.16 CrossRefPubMedGoogle Scholar
  14. Satomura T, Hayashi J, Sakamoto H, Nunoura T, Takaki Y, Takai K, Takami H, Ohshima T, Sakuraba H, Suye S (2018) D-Lactate electrochemical biosensor prepared by immobilization of thermostable dye-linked d-lactate dehydrogenase from Candidatus Caldiarchaeum subterraneum. J Biosci Bioeng.  https://doi.org/10.1016/j.jbiosc.2018.04.002 (in press) CrossRefPubMedGoogle Scholar
  15. Shibahara T, Satomura T, Kawakami R, Ohshima T, Sakuraba H (2011) Crystallization and preliminary X-ray analysis of a dye-linked d-lactate dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix. Acta Crystallogr F Struct Biol Commun 67:1425–1427.  https://doi.org/10.1107/S1744309111036098 CrossRefGoogle Scholar
  16. Short SA, Kaback HR, Kohn LD (1975) Localization of d-lactate dehydrogenase in native and reconstituted Escherichia coli membrane vesicles. J Biol Chem 250:4291–4296PubMedGoogle Scholar
  17. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680.  https://doi.org/10.1093/nar/22.22.4673 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Wierenga RK, Terpstra P, Hol WGJ (1986) Prediction of the occurrence of the ADP binding βαβ-fold in proteins using an amino acid sequence fingerprint. J Mol Biol 187:101–107.  https://doi.org/10.1016/0022-2836(86)90409-2 CrossRefPubMedGoogle Scholar
  19. Willie A, Jorns MS (1995) Discovery of a third coenzyme in sarcosine oxidase. Biochemistry 34:16703–16707.  https://doi.org/10.1021/bi00051a019 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Japan KK, part of Springer Nature 2018

Authors and Affiliations

  • Takenori Satomura
    • 1
    • 2
  • Junji Hayashi
    • 3
  • Tatsuya Ohshida
    • 4
  • Haruhiko Sakuraba
    • 4
  • Toshihisa Ohshima
    • 5
  • Shin-ichiro Suye
    • 1
    • 2
  1. 1.Division of Engineering, Faculty of EngineeringUniversity of FukuiFukuiJapan
  2. 2.Organization for Life Science Advancement ProgramsUniversity of FukuiFukuiJapan
  3. 3.Department of Biotechnology, College of Life SciencesRitsumeikan UniversityKusatsuJapan
  4. 4.Department of Applied Biological Science, Faculty of AgricultureKagawa UniversityKagawaJapan
  5. 5.Department of Biomedical Engineering, Faculty of EngineeringOsaka Institute of TechnologyOsakaJapan

Personalised recommendations