[en] The gene encoding the phosphoglycerate kinase (PGK) from the Antarctic Pseudomonas sp. TACII18 has been cloned and found to be inserted between the genes encoding for glyceraldhyde-3-phosphate dehydrogenase and fructose aldolase. The His-tagged and the native recombinant PGK from the psychrophilic Pseudomonas were expressed in Escherichia coli. The wild-type and the native recombinant enzymes displayed identical properties, such as a decreased thermostability and a 2-fold higher catalytic efficiency at 25 degrees C when compared with the mesophilic PGK from yeast. These properties, which reflect typical features of cold-adapted enzymes, were strongly altered in the His-tagged recombinant PGK. The structural model of the psychrophilic PGK indicated that a key determinant of its low stability is the reduced number of salt bridges, surface charges, and aromatic interactions when compared with mesophilic and thermophilic PGK. Differential scanning calorimetry of the psychrophilic PGK revealed unusual variations in its conformational stability for the free and substrate-bound forms. In the free form, a heat-labile and a thermostable domain unfold independently. It is proposed that the heat-labile domain acts as a destabilizing domain, providing the required flexibility around the active site for catalysis at low temperatures.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Bentahir, Mostafa
Feller, Georges ; Université de Liège - ULiège > Département des sciences de la vie > Labo de biochimie
Low, P. S., Bada, J. L., and Somero, G. N. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 430-432
Johnston, I. A., Walesby, N. J., Davison, W., and Goldspink, G. (1975) Nature 254, 74-75
Privalov, P. L., Tiktopulo, E. J., and Tischenko, V. M. (1979) J. Mol. Biol. 127, 203-216
Fields, P. A., and Somero, G. N. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 11476-11481
Zavodszky, P., Kardos, J., Svingor, and Petsko, G. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7406-7411
Hess, D., Kruger, K., Knappik, A., Palm, P., and Hensel, R. (1995) Eur. J. Biochem. 233, 227-237
Jones, C. E., Fleming, T. M., Cowan, D. A., Littlechild, J. A., and Piper, P. W. (1995) Eur. J. Biochem. 233, 800-808
Watson, H. C., and Littlechild, J. A. (1990) Biochem. Soc. Trans. 18, 187-190
Harlos, K., Vas, M., and Blake, C. F. (1992) Proteins 12, 133-144
Davies, G. J., Gamblin, S. J., Littlechild, J. A., Dauter, Z., Wilson, K. S., and Watson, H. C. (1994) Acta Crystallogr. Sect. D 50, 202-209
Blake, C. C., and Evans, P. R. (1974) J. Mol. Biol. 84, 585-601
Banks, R. D., Blake, C. C., Evans, P. R., Haser, R., Rice, D. W., Hardy, G. W., Merrett, M., and Phillips, A. W. (1979) Nature 279, 773-777
Blake, C. C., and Rice, D. W. (1981) Philos. Trans R. Soc. Lond. B Biol. Sci. 293, 93-104
Bryant, T. N., Watson, H. C., and Wendell, P. L. (1974) Nature 247, 14-17
Watson, H. C., Walker, N. P., Shaw, P. J., Bryant, T. N., Wendell, P. L., Fothergill, L. A., Perkins, R. E., Conroy, S. C., Dobson, M. J., Tuite, M. F., Kingsman, A. J., and Kingsman, S. M. (1982) EMBO J. 1, 1635-1640
May, A., Vas, M., Harlos, K., and Blake, C. (1996) Proteins 24, 292-303
Bernstein, B. E., Michels, P. A., and Hol, W. G. (1997) Nature 385, 275-278
Auerbach, G., Huber, R., Grättinger, M., Zaiss, K., Schurig, H., Jaenicke, R., and Jacob, U. (1997) Structure 5, 1475-1483
Thomas, T. M., and Scopes, R. K. (1998) Biochem. J. 330, 1087-1095
Zaiss, K., and Jaenicke, R. (1999) Biochemistry 38, 4633-4639
Studier, F. W., and Moffatt, B. A. (1986) J Mol. Biol. 189, 113-130
Bücher, T. (1955) Methods Emymol. 1, 415-422
Smith, T. F., Waterman, M. S., and Fitch, W. M. (1981) J. Mol. Evol. 18, 38-46
Devereux, J., Haeberli, P., and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395
Pearlman, D., Case, D., Caldwell, J., Ross, W., Cheathman, T., Ferguson, D., Seibel, G., Singh, U., Weiner, P., and Kollman, P. (1995) AMBER 4.1, University of California, San Francisco, CA
Pace, C. N. (1986) Methods Enzymol. 131, 266-280
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410
Walker, P. A., Littlechild, J. A., Hall, L., and Watson, H. C. (1989) Eur. J. Biochem. 183, 49-55
Rao, B. D., Cohn, M., and Scopes, R. K. (1978) J. Biol. Chem. 253, 8056-8060
Szilagyi, A. N., and Vas, M. (1998) Biochemistry 37, 8551-8563
Gerday, C., Aittaleb, M., Arpigny, J. L., Baise, E., Chessa, J. P., Garsoux, G., Petrescu, I., and Feller, G. (1997) Biochim. Biophys. Acta 1342, 119-131
Feller, G., Narinx, E., Arpigny, J. L., Aittaleb, M., Baise, E., Genicot, S., and Gerday, C. (1996) FEMS Microbiol. Rev. 18, 189-202
Nojima, H., Ikai, A., and Noda, H. (1976) Biochim. Biophys. Acta 427, 20-27
Privalov, P. L. (1982) Adv. Protein Chem. 35, 1-104
Leharne, S. A., and Chowdhry, B. Z. (1998) in Biocalorimetry: Applications of Calorimetry in the Biological Sciences (Chowdhry, L. A., ed) pp. 157-182, Wiley, New York
Alefounder, P. R., and Perham, R. N. (1989) Mol. Microbiol. 3, 723-732
Branny, P., de la Torre, F., and Garel, J. R. (1998) Microbiology 144, 905-914
Gebbia, J. A., Backenson, P. B., Coleman, J. L., Anda, P., and Benach, J. L. (1997) Gene (Amst.) 188, 221-228
Feller, G., and Gerday, C. (1997) Cell. Mol. Life Sci. 53, 830-841
Gerday, C., Aittaleb, M., Arpigny, J. L., Baise, E., Chessa, J. P., François, J. M., Petrescu, I., and Feller, G. (1999) in Cold Adapted Organisms: Ecology, Physiology, Enzymology and Molecular Biology (Margessi, R., and Schinner, F., eds) pp. 257-275, Springer, Berlin
Hochachka, P. W., and Somero, G. N. (1984) Biochemical Adaptation, Princeton University Press, Princeton, NJ
Somero, G. N. (1995) Annu. Rev. Physiol. 57, 43-68
Feller, G., d'Amico, D., and Gerday, C. (1999) Biochemistry 38, 4613-4619
Fontes, C. M., Hall, J., Hirst, B. H., Hazlewood, G. P., and Gilbert, H. J. (1995) Appl. Microbiol. Biotechnol. 43, 52-57
Nicholls, A., Bharadwaj, R., and Honig, B. (1993) Biophys. J. 64, A166
