Skip to main content
SpringerLink
Log in

Stabilization of quaternary structure of water-soluble quinoprotein glucose dehydrogenase

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Water-soluble quinoprotein glucose dehydrogease (PQQGDH-B) is a dimeric enzyme whose application for glucose sensing is the focus of much attention. We attempted to increase the thermal stability of PQQGDH-B by introducing a disulfide bond at the dimer interface. The Ser residue at position 415 was selected for substitution with Cys, as structural information revealed that its side chains face each other at the dimer interface of PQQGDH-B. PQQGDH-B with Ser415Cys showed 30-fold greater thermal stability at 55°C than did the wild-type enzyme without any decrease in catalytic activity. After incubation at 70°C for 10 min, Ser415Cys retained 90% of the GDH activity of the wild-type enzyme. Disulfide bond formation between the mutant subunits was confirmed by analyses with sodium dodecylsulfate-polyacrylamide gel electrophoresis in the presence and absence of reductants. Our results indicate that the introduction of one Cys residue in each monomer of PQQGDH-B resulted in formation of a disulfide bond at the dimer interface and thus achieved a large increase in the thermal stability of the enzyme.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. D’Costa, E.J., Higgins, I.J., and Turner, A.P.F. (1986) Quinoprotein glucose dehydrogenase and its application in an amperometric glucose sensor. Biosensors, 2, 71–87.

    Article  PubMed  CAS  Google Scholar 

  2. Yokoyama, K., Sode, K., Tamiya, E., and Karube, I. (1989) Integrated biosensor for glucose and galactose. Anal. Chim. Acta. 218, 137–142.

    Article  CAS  Google Scholar 

  3. Smolander, M., Livio, H. -L., and Rasanen, L. (1992) Mediated amperometric determination of xylose and glucose with an immobilized aldose dehydrogenase electrode. Biosensors Bioelectronics 7, 637–643.

    Article  PubMed  CAS  Google Scholar 

  4. Sode, K., Nakasono, S., Tanaka, M., and Matsunaga, T. (1993) Subzero temperature operating biosensor utilizing an organic solvent and quinoprotein glucose dehydrogenase. Biotechnol. Bioeng., 42, 251–254.

    Article  CAS  Google Scholar 

  5. Ye, L., Hammerle, M., Olsthoorn, A.J.J., Schuhmann, W., Schmidt, H.-L., Duine, J.A., and Heller, A. (1993) High current density “wired” quinoprotein glucose dehydrogenase electrode. Anal. Chem. 65, 238–241.

    Article  CAS  Google Scholar 

  6. Katz, E., Schlereth, D.D., and Schmidt, H.-L. (1994) Reconstitution of the quinoprotein glucose dehydrogenase from its apoenzyme on a gold electrode surface modified with a monolayer of pyrroloquinoline quinone. Electroanal. Chem. 368, 165–171.

    Article  CAS  Google Scholar 

  7. Kost, G.J., Vu, H.-T., Lee, J.H., Bourgeois, P., Kiechle, F.L., Martin, C., et al. (1998) Multi-center study of oxygen-insensitive handheld glucose point-of-care testing in critical care/hospital/ambulatory patients in the United States and Canada. Crit. Care. Med. 26, 581–590.

    Article  PubMed  CAS  Google Scholar 

  8. Laurinavicius, V., Kurtinaitiene, B., Liauksminas, V., Jankauskaite, A., Simkus, R., Meskys, R., et al. (2000) Reagentless biosensor based on PQQ-dependent glucose dehydrogenase and partially hydrolyzed polyarbutin. Talanta 52, 485–493.

    Article  CAS  Google Scholar 

  9. Razumiene, J., Meskys, R., Gureviciene, V., Laurinavicius, V., Reshetova, M.D. and Ryabov, A.D. (2000) 4-Ferrocenyllphenol as an electron transfer mediator on PQQ-dependent alcohol and glucose dehydrogenase-catalyzed reactions. Electrochem. Commun. 2, 307–311.

    Article  CAS  Google Scholar 

  10. Schmidt, B. (1997) A new class of enzymes for application in diagnostics. Clinica. Chim. Acta. 26, 33.

    Article  Google Scholar 

  11. Mullen, W.H., Churchhouse, J. and Vadgama, P. (1985) Enzyme electrode for glucose based on the quinoprotein glucose dehydrogenase. Analyst 110, 925.

    Article  PubMed  CAS  Google Scholar 

  12. Takahashi, Y., Igarashi, S., Nakazawa, Y., Tsugawa, W., and Sode, K. (2000) Construction and characterization of glucose enzyme sensor employing engineered water soluble PQQ glucose dehydrogenase with improved thermal stability. Electrochemistry 68, 907–911.

    CAS  Google Scholar 

  13. Sode, K. and Sano, H. (1994) Glu742 substitution to Lys enhanced the EDTA tolerance of Escherichia coli PQQ glucose dehydrogenase. Biotechnol. Lett., 16, 455–460.

    Article  CAS  Google Scholar 

  14. Sode, K., Yoshida, H., Matsumura, K., Kikuchi, T., Watanabe, M., Yasutake, N., et al. (1995) Elucidation of the region responsible for EDTA tolerance in PQQ glucose dehydrogenases by constructing Escherichia coli and Acinetobacter calcoaceticus chimeric enzymes. Biochem. Biophys. Res. Commun. 211, 268–273.

    Article  PubMed  CAS  Google Scholar 

  15. Sode, K., Watanabe, K., Ito, S., Matsumura, K. and Kikuchi, T., (1995) Thermostable chimeric PQQ glucose dehydrogenase. FEBS Lett. 364, 325–327.

    Article  PubMed  CAS  Google Scholar 

  16. Sode, K. and Yoshida, H. (1997) Construction and characterization of a chimeric Escherichia coli PQQ glucose dehydrogenase (PQQGDH) with increased EDTA tolerance. Denki Kagaku. 65, 444–451.

    CAS  Google Scholar 

  17. Yoshida, H. and Sode, K. (1997) Thr424 to Asn substitution alters bivalent metal specificity of pyrroloquinoline quinone glucose dehydrogenase. J. Biochem. Mol. Biol. Biophys. 1, 89–93.

    CAS  Google Scholar 

  18. Sode, K. and Kojima, K. (1997) Improved substrate specificity and dynamic range for glucosse measurement of Escherichia coli PQQ glucose dehydrogenase by site directed mutagenesis. Biotechnol. Lett. 19, 1073–1077.

    Article  CAS  Google Scholar 

  19. Yoshida, H., Kojima, K., Witarto, A.B., and Sode, K. (1999) Engineering a chimeric pyrroloquinoline quinone glucose dehydrogenase: improvement of EDTA tolerance, thermal stability and substrate specificity. Protein Eng. 12, 63–70.

    Article  PubMed  CAS  Google Scholar 

  20. Witarto, A.B., Ohuchi, S., Narita, M. and Sode, K. (1999) Secondary structure study of pyrroloquinoline quinone glucose dehydrogenase. J. Biochem. Mol. Biol. Biophys. 2, 209–213.

    CAS  Google Scholar 

  21. Witarto, A.B., Ohtera, T. and Sode, K. (1999) Site-directed mutagenesis study on the thermal stability of a chimeric PQQ glucose dehydrogenase and its structural interpretation. Appl. Biochem. Biotechnol. 77–79, 159–168.

    Article  PubMed  Google Scholar 

  22. Yoshida, H., Iguchi, T., Sode, K. (2000) Construction of mulit-chimeric pyrroloquinoline quinone glucose dehydrogenase with improved enzymatic properties and application in glucose monitoring Biotechnol. Lett. 22, 1505–1510.

    Article  CAS  Google Scholar 

  23. Okuda, J., Yoshida, H., Kojima, K., Himi, M. and Sode, K. (2000) The role of conserved Hia775/781 in membrane-binding PQQ glucose dehydrogenase of Escherichia coli and Acinetobacter calcoaceticus. J. Biochem. Mol. Biol. Biophys. 4, 415–422.

    CAS  Google Scholar 

  24. Sode, K., Ohtera, T., Shirahane, M., Witarto, A.B., Igarashi, S., and Yoshida, H. (2000) Increasing the thermal stability of the water-soluble pyrroloquinoline quinone glucose dehydrogenase by single amino acid replacement. Enz. Microbial. Technol. 26, 491–496.

    Article  CAS  Google Scholar 

  25. Sode, K., Shirahane, M., and Yoshida H. (1999) Construction and characterization of a linked-dimeric pyrrolquinoline quinone glucose dehydrogenase. Biotechnol. Lett. 21, 707–710.

    Article  CAS  Google Scholar 

  26. Igarashi, S., Ohtera T., Yoshida, H., Witarto, A.B., and Sode, K. (1999) Construction and characterization of mutant water-soluble PQQ glucose dehydrogenases with altered Km value-site-directed mutagenesis studies on the putative active site. Biochem. Biophys. Res. Commun. 264, 820–824.

    Article  PubMed  CAS  Google Scholar 

  27. Geiger, O. and Görisch, H. (1989) Reversible thermal inactivation of the quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. Biochem. J. 261, 415–421.

    PubMed  CAS  Google Scholar 

  28. Sauer, R.T., Hehir, K., Stearman, R.S., Weiss, M.A., Jeitler-Nilsson, A., Suchanek E.G, et al. (1986) An engineered intersubunit disulfide enhances the stability and DNA binding on the N-terminal domain of lambda repressor. Biochemistry 7, 5992–5998.

    Article  Google Scholar 

  29. Heikoop, J.C., van den Boogart, P., Mulders J.W.W., and Grootenhuis P.D.J. (1997) Structure-based design and protein engineering of intersubunit disulfide bonds in gonadotropins. Nat. Biotechnol. 15, 658–662.

    Article  PubMed  CAS  Google Scholar 

  30. Bunting, K.A., Cooper, J.B., Tickle, I.J., and Young, D.B. (2002) Engineering of an intersubunit disulfide bridge in the iron-superoxide dismutase of Mycobacterium tuberculosis. Arch. Biochem. Biophys. 397, 69–76.

    Article  PubMed  CAS  Google Scholar 

  31. Oubrie, A., Rozeboom, H.J., Kalk, K.H., Duine, J.A., and Dijkstra, B.W. (1999) The 1.7Å crystal structure of the apo-form of the soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus reveals a novel internal conserved sequence repeat. J. Mol. Biol. 289, 319–333.

    Article  PubMed  CAS  Google Scholar 

  32. Oubrie, A., Rozeboom, H.J., Kalk, K.H., Olsthoorn, A.J.J., Duine, J.A., and Dijkstra B.W. (1999) Structure and mechanism of soluble quinoprotein glucose dehydrogenase. EMBO J. 18 5187–5194.

    Article  PubMed  CAS  Google Scholar 

  33. Oubrie, A., Rozeboom, H.J., and Dijkstra, B.W. (1999) Active-site structure of the soluble quino-protein glucose dehydrogenase complexed with methylhydrazine: A covalent cofactor-inhibitor complex. Proc. Natl. Acad. Sci. USA 96, 11787–11797.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, 184-8588, Tokyo, Japan

    Koji Sode

Authors
  1. Satoshi Igarashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Koji Sode
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Koji Sode.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Igarashi, S., Sode, K. Stabilization of quaternary structure of water-soluble quinoprotein glucose dehydrogenase. Mol Biotechnol 24, 97–103 (2003). https://doi.org/10.1385/MB:24:2:97

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1385/MB:24:2:97

Index Entries

Search

Navigation