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Thermal Stabilization of Xylose Isomerase from Thermoanaerobacterium thermosulfurigenes

Bio/Technology volume 11, pages 1157–1161 (1993)Cite this article

Abstract

The thermostability of D-xylose isomerase from Thermoanaerobacterium thermosulfurigenes was enhanced by site-directed substitutions of aromatic amino acids in the active site. This enhancement may be explained as the consequence of the reduction of the area of water-accessible hydrophobic surface. The kinetics of thermoinactivation of the enzyme in aqueous solution was also investigated, and we report that in addition to the well known divalent cations, the monovalent cation, K+, also protects the enzyme against thermoinactivation. The kinetic data suggest that the formation of incorrect conformations of the enzyme (“scrambled structure”) is the dominant factor governing the process of thermoinactivation at elevated temperature (80–90°C).

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References

  1. Chen, W.-P. 1980. Glucose isomerase (a review). Process Biochem. 15: 36–41.

    CAS  Google Scholar 

  2. Lee, C., Lee, C. and Zeikus, J.G. 1991. Purification and characterization of thermostable glucose isomerase from Clostridium thermosulfurogenes and Thermuanaerobacter strain B6A. Biochem. J. 274: 565–571.

    Article  Google Scholar 

  3. Henrick, K., Collyer, C.A. and Blow, D.M. 1989. Structures of D-xylose isomerase from Anhrobacter strain B3728 containing the inhibitors xylitol and D-sorbitol at 2.5 Å and 2.3 Å resolution, respectively. J. Mol. Biol. 208: 129–157.

    Article  CAS  Google Scholar 

  4. Collyer, C.A., Henrick, K. and Blow, D.M. 1990. Mechanism for aldose-ketose interconversion by D-xylose isomerase involving ring opening followed by a 1,2-hydride shift. J. Mol. Biol. 212: 211–235.

    Article  CAS  Google Scholar 

  5. Carrell, H.L., Glusker, J.P., Burger, V., Manfre, F., Tritsch, D. and Biellmann, J.F. 1989. X-ray analysis of D-xylose isomerase at 1.9Å: native enzyme in complex with substrate and with a mechanism-designed inactivator. Proc. Natl. Acad. Sci. USA 86: 4440–4444.

    Article  CAS  Google Scholar 

  6. Whitlow, M., Howard, A.J., Finzel, B.C., Poulos, T.L., Winborne, E. and Gilliland, G.L. 1991. A metal-mediated hydride shift mechanism for xylose isomerase based on the 1.6 Å Streptomyces rubiginosus with xylitol and D-xylose. Proteins 9: 153–173.

    Article  CAS  Google Scholar 

  7. Farber, G.K., Glasfeld, A., Tiraby, G., Ringe, D. and Petsko, G.A. 1989. Crystallographic studies of the mechanism of xylose isomerase. Biochemistry 28: 7289–7297.

    Article  CAS  Google Scholar 

  8. Jenkins, J., Janin, J., Rey, F., Chiadmi, M., van Tilbeurgh, H., Lasters, I., De Mareyer, M., Van Belle, D., Wodak, S.J., Lauwreys, M., Stanssens, P., Mrabet, N.T., Snauwaert, J., Matthyssens, G. and Lamberi, A.-M. 1992. Protein engineering of xylose (glucose) isomerase from Aainoplanes missouriensis. 1. crystallography and site-directed mutagenesis of metal binding sites. Biochemistry 31: 5449–5458.

    Article  CAS  Google Scholar 

  9. Lee, C., Bagdasarian, M., Meng, M. and Zeikus, J.G. 1990. Catalytic mechanism of xylose (glucose) isomerase from Clostridium thermosulfurogenes. J. Biol. Chem. 265: 19082–19090.

    CAS  PubMed  Google Scholar 

  10. Matsumura, M., Signor, G. and Matthews, B.W. 1989. Substantial increase of protein stability by multiple disulfide bonds. Nature 342: 291–293.

    Article  CAS  Google Scholar 

  11. Matthews, B.W., Nicholson, H. and Becktel, W.J. 1987. enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc. Natl. Acad. Sci. USA 84: 6663–6667.

    Article  CAS  Google Scholar 

  12. Nicholson, H., Becktel, W.J. and Matthews, B.W. 1988. Enhanced protein stability from designed mutations that interact with α-helix dipoles. Nature 336: 651–656.

    Article  CAS  Google Scholar 

  13. Sandberg, W.S. and Terwilliger, T.C. 1989. Influence of interior packing and hydrophobicity on the stability of a protein. Science 245: 54–56.

    Article  CAS  Google Scholar 

  14. Wigley, D.B., Clarke, A.R., Dunn, C.R., Barstow, D.A., Atkinson, T., Chia, W.N., Muirhead, H. and Holbrook, J.J. 1987. The engineering of a more thermally stable lactate dehydrogenase by reduction of the area of water-accessible hydrophobic surface. Biochim. Biophysica. Acta 916: 145–148

    Article  CAS  Google Scholar 

  15. Quax, W.J., Mrabet, N.T., Luiten, R.G.M., Schuurhuizen, P.W., Stanssens, P. and Lasters, I. 1991. Enhancing the thermostability of glucose isomerase by protein engineering. Bio/Technology 9: 738–742.

    CAS  PubMed  Google Scholar 

  16. Sanger, F., Nicklen, S. and Coulson, A.R. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463–5467.

    Article  CAS  Google Scholar 

  17. Dawson, R.M.C., Elliott, D.C., Elliott, W.H. and Jones, K.M. 1986. PH, buffer, and physiological media, p. 417–448. In: Data for Biochemical Research, 3rd Ed. Clarendon Press, Oxford.

    Google Scholar 

  18. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 227: 680–685.

    Article  CAS  Google Scholar 

  19. Tomazic, S.J. and Klibanov, A.M. 1988. Mechanism of irreversible thermal inactivation of Bacillus α-amylases. J. Biol. Chem. 263: 3086–3091.

    CAS  PubMed  Google Scholar 

  20. Kasumi, T., Hayashi, K. and Tsumura, N. 1982. Role of cobalt in stabilizing the molecular structure of glucose isomerase from Streptomyces griseofuscus S-41. Agric. Biol. Chem. 46: 31–39.

    CAS  Google Scholar 

  21. Callens, M., Kersters-Hilderson, H., Vangrysperre, W. and De Bruyne, C.K. 1988. D-Xylose isomerase from Streptomyces violaceoruber: structural and catalytic roles of bivalent metal ions. Enzyme Microb. Technol. 10: 695–700.

    Article  CAS  Google Scholar 

  22. Gaikwad, S.M., Rao, M.B. and Deshpande, V.V. 1992. D-Glucose/xylose isomerase from Streptomyces: differential roles of magnesium and cobalt ions. Enzyme Micro. Technol. 14: 317–320.

    Article  CAS  Google Scholar 

  23. Rangarajan, M., Asboth, B. and Hartley, B.S. 1992. Stability of Arthrobacter D-xylose isomerase to denaturants and heat. Biochem. J. 285: 889–898.

    Article  CAS  Google Scholar 

  24. Argos, P., Rossmann, M.G., Grau, U.M., Zuber, H., Frank, G. and Tratschin, J.D. 1979. Thermal stability and protein structure. Biochemistry 18: 5698–5703.

    Article  CAS  Google Scholar 

  25. Lowe, S., Jain, M.K. and Zeikus, J.G. 1933. Biology, ecology and biotech-nological applications of anaerobic extremophiles adapted to environmental stress in temperature, pH, salinity or substrate. Microbiol. Reviews, In press.

    Google Scholar 

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Author notes

  1. Michael Bagdasarian: Corresponding author.

Authors and Affiliations

  1. Department of Microbiology, Michigan State University, East Lansing, MI, 48824

    Menghsiao Meng, Michael Bagdasarian & J. Gregory Zeikus

  2. Department of Biochemistry, Michigan State University, East Lansing, MI, 48824

    J. Gregory Zeikus

  3. Michigan Biotechnology Institute, Lansing, MI, 48909

    J. Gregory Zeikus

  4. Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, Texas, 75235

    Menghsiao Meng

Authors
  1. Menghsiao Meng
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  2. Michael Bagdasarian
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  3. J. Gregory Zeikus
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Meng, M., Bagdasarian, M. & Zeikus, J. Thermal Stabilization of Xylose Isomerase from Thermoanaerobacterium thermosulfurigenes. Nat Biotechnol 11, 1157–1161 (1993). https://doi.org/10.1038/nbt1093-1157

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