Lys13 plays a crucial role in the functional adaptation of the thermophilic triose-phosphate isomerase from Bacillus stearothermophilus to high temperatures
[en] The thermophilic triose-phosphate isomerases (TIMs) of Bacillus stearothermophilus (bTIM) and Thermotoga maritima (tTIM) have been found to possess a His12-Lys13 pair instead of the Asn12-Gly13 pair normally present in mesophilic TIMs. His12 in bTIM was proposed to prevent deamidation at high temperature, while the precise role of Lys13 is unknown. To investigate the role of the His12 and Lys13 pair in the enzyme's thermoadaptation, we reintroduced the "mesophilic residues" Asn and Gly into both thermophilic TIMs. Neither double mutant displayed diminished structural stability, but the bTIM double mutant showed drastically reduced catalytic activity. No similar behavior was observed with the tTIM double mutant, suggesting that the presence of the His12 and Lys13 cannot be systematically correlated to thermoadaptation in TIMs. We determined the crystal structure of the bTIM double mutant complexed with 2-phosphoglycolate to 2.4-A resolution. A molecular dynamics simulation showed that upon substitution of Lys13 to Gly an increase of the flexibility of loop 1 is observed, causing an incorrect orientation of the catalytic Lys10. This suggests that Lys13 in bTIM plays a crucial role in the functional adaptation of this enzyme to high temperature. Analysis of bTIM single mutants supports this assumption.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Alvarez, Marco
Wouters, Johan; Facultés Universitaires Notre-Dame de la Paix
Maes, Dominique; Vrije Universiteit Brussel
Mainfroid, Véronique
Rentier-Delrue, Françoise ; Université de Liège - ULiège > Département des sciences de la vie > Biologie et génétique moléculaire - GIGA-R : Coordination scientifique
Wyns, Lode; Vrije Universiteit Brussel
Depiereux, Eric; Facultés Universitaires Notre-Dame de la Paix
Martial, Joseph ; Université de Liège - ULiège > Département des sciences de la vie > GIGA-R : Biologie et génétique moléculaire
Language :
English
Title :
Lys13 plays a crucial role in the functional adaptation of the thermophilic triose-phosphate isomerase from Bacillus stearothermophilus to high temperatures
Publication date :
1999
Journal title :
Journal of Biological Chemistry
ISSN :
0021-9258
eISSN :
1083-351X
Publisher :
American Society for Biochemistry and Molecular Biology, Baltimore, United States - Maryland
Jaenicke, R., Schurig, H., Beaucamp, N., and Ostendorp, R. (1996) Adv. Protein Chem. 48, 181-269
Russel, R. J., and Tylor, G. L. (1995) Curr. Opin. Biotechnol. 6, 370-374
Rice, D. W., Yip, K. S., Stillman, T. J., Britton, K. L., Fuentes, A., Connerton, I., Pasquo, A., Scandura, R., and Engel, P. C. (1996) FEMS Microbiol. Rev. 18, 105-117
Pfeil, W., Gesierich, U., Kleeman, G. R., and Sterner, R. (1997) J. Mol. Biol. 272, 591-596
Peters, J., Baumeister, W., and Lupas, A. (1996) J. Mol. Biol. 257, 1031-1041
Hennig, M., Sterner, R., Kirschner, K., and Jansonius, J. N. (1997) Biochemistry 36, 6009-6016
Lim, J. H., Yu, Y. G., Han, Y. S., Cho, S., Ahn, B. Y., Kim, S. H., and Cho, Y. (1997) J. Mol. Biol. 270, 259-274
Huber, R., Langworthy, T. A., König, H., Thomm, M., Woese, C. R., Sleytr, U. B., and Stetter, K. O. (1986) Arch. Microbiol. 144, 324-333
Fahey, R. C., Kalb, E., and Harris, J. I. (1971) Biochem. J. 124, 77P
Rieder, S. V., and Rose, I. A. (1959) J. Biol. Chem. 234, 1007-1010
Farber, G. K., and Petsko, G. A. (1990) Trends Biochem. Sci. 15, 228-234
Banner, D. W., Bloomer, A. C., Petsko, G. A., Philips, D. C., Pogson, C. I., Wilson, I. A., Corran, P. H., Furth, A. J., Milman, J. D., Offord, R. E., Priddle, J. D., and Waley, S. G. (1975) Nature 255, 609-614
Lolis, E., Alber, T., Davenport, R. C., Rose, D., Hartman, F. C., and Petsko, G. A. (1990) Biochemistry 29, 6609-6618
Wierenga, R. K., Noble, M. E. M., Vriend, G., Nauche, S., and Hol, W. G. J. (1991) J. Mol. Biol. 220, 995-1015
Noble, M. E. M., Zeelen, J. P., Wierenga, R. K., Mainfroid, V., Goraj, K., Gohimont, A.-C., and Martial, J. A. (1993) Acta Crystallogr. Sec. D 49, 403-417
Mande, S. C., Mainfroid, V., Kalk, K. H., Goraj, K., Martial, J. A., and Hol, W. G. J. (1994) Protein Sci. 3, 810-821
Delboni, L. F., Mande, S. C., Rentier-Delrue, F., Mainfroid, V., Turley, S., Vellieux, M. D., Martial, J. A., and Hol, W. G. J. (1995) Protein Sci. 4, 2594-2604
Velanker, S. S., Ray, S. S., Gokhale, R. S., Hemalatha Balaram, S. S., and Murthy, M. R. N. (1997) Structure 5, 751-761
Alvarez, M., Zeelen, J. P., Mainfroid, V., Rentier-Delrue, F., Martial, J. A., Wyns, L., Wierenga, R. K., and Maes, D. (1998) J. Biol. Chem. 273, 2199-2206
Maldonado, E., Soriano-Garcia, M., Moreno, A., Cabrera, N., Garza-Ramos, G., Tuena, de Gomez-Puyou, M., Gomez-Puyou, A., Perez-Monfort, R. (1998) J. Mol. Biol. 283, 193-203
Williams, J. C., Zeelen, J. Ph., Neubauer, G., Vriend, G., Backmann, J., Michels, P. A. M., Lambeir, A.-M., and Wierenga, R. K. (1999) Protein Eng. 12, 243-250
Rentier-Delrue, F., Mande, S. C., Moyens, S., Terpstra, P., Mainfroid, V., Goraj, K., Hol, W. G. J., and Martial, J. A. (1993) J. Mol. Biol. 229, 85-93
Schurig, H., Beaucamp, N., Ostendorp, R., Jaenicke, R., Adler, E., and Knowles, J. R. (1995) EMBO J. 14, 442-451
Beaucamp, N., Hofmann, A., Kellerer, B., and Jaenicke, R. (1997) Protein Sci. 6, 2159-2165
Kaneko, T., Tanaka, A., Sato, S., Kotani, H., Sazuka, T., Miyajima, N., Sugiura, M., and Tabata, S. (1995) DNA Res. 2, 153-166
Pfeil, W. (1998) Protein Stability and Folding: A Collection of Thermodynamics Data, Springer, Berlin, Heidelberg
Gebbia, J. A., Backenson, P. B., Coleman, J. L., Anda, P., and Benach, J. L. (1997) Gene (Amst.) 188, 221-228
Leyva-Vazquez, M. A., and Setlow, P. (1994) J. Bacteriol. 176, 3903-3910
Yuan, P. M., Talent, J. M., and Gracy, R. W. (1981) Mech. Ageing Dev. 17, 151-162
Tyler-Cross, R., and Schirch, V. (1991) J. Biol. Chem. 266, 22549-22556
Tomazic, S. J., and Klibanov, A. M. (1988) J. Biol. Chem. 263, 3086-3091
Hayes, C. S., and Setlow, P., (1997) J. Bacteriol. 179, 6020-6027
Chen, H., Ford, C., and Reilly, P. J. (1994) Biochem. J. 301, 275-281
Kossiakoff, A. A. (1988) Science 240, 191-194
Stephenson, R. C., and Clarke, S. (1989) J. Biol. Chem. 264, 6164-6170
Wright, H. T. (1991) Protein Eng. 4, 283-294
Tomizawa, H., Yamada, H., Hashimoto, Y., and Imoto, T. (1995) Protein Eng. 8, 1023-1028
Gracy, R. W., Lu, H. S., Yuan, P. M., and Talent, J. M. (1983) in Altered Proteins and Ageing (Adelman, R. C., and Roth, G. S., eds) pp 27-34, CRC Press, Inc., Boca Raton, FL
Casal, J. I., Ahern, T. J., Davenport, R. C., Petsko, G. A., and Klibanov, A. M. (1987) Biochemistry 26, 1258-1264
L'Hoir, C., Renard, A., and Martial, J. A. (1990) Gene (Amst.) 89, 47-52
Rosenberg, A. H., Lade, B. N., Chui, D. S., Lin, S. W., Dunn, J. J., and Studier, F. W. (1987) Gene (Amst.) 56, 125-135
Ochman, H., Medhora, M. M., Garza, D., and Hartl, D. L. (1990) in PCR Protocols: A Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J.) pp. 219-227, Academic Press, Inc., San Diego
Studier, F. W., and Moffatt, B. A. (1986) J. Mol. Biol. 189, 113-130
Misset, O., and Opperdoes, F. (1984) Eur. J. Biochem. 144, 475-483
Otwinowski, Z. (1993) DENZO: An Oscillation Data Processing Program for Macromolecule Crystallography, Yale University, New Haven, CT
Collaborative Computational Project 4 (1994) Acta Crystallogr. Sec. D 50, 760-763
Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjelgaard, M. (1991) Acta Crystallogr. Sec. A 47, 110-119
Brünger, A. T. (1992) X-PLOR: A System for X-ray Crystallography and NMR, Version 3.1, Yale University Press, New Haven, CT
Brünger, A. T., Adams, P. D., Clore, M. G., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sec. D 54, 905-921
Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 24, 946-950
Vriend, G. (1990) J. Mol. Graphics 8, 52-56
Ahern, T. J., Casal, J. I., Petsko, G. A., and Klibanov, A. M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 675-679
Wouters, J. (1998) Protein Sci. 7, 2472-2475
McCarthy, D. J., Lolis, E., Komives, E. A., and Petsko, G. A. (1994) Biochemistry 33, 2815-2823
Lodi, P. J., Chang, L. C., Knowles, J. R., and Komives, E. A. (1994) Biochemistry 33, 2809-2814
Kamal, M., Hoog, J. O., Kaiser, R., Shafqat, J., Razzaki, T., Zaidi, Z. H., and Jornvall, H. (1995) FEBS Lett. 374, 363-366
Shimizu, T., Imai, M., Araki., S., Kishida, K., Terasawa, Y., and Hachimori, A. (1997) Int. J. Biochem. Cell Biol. 29, 303-310
Baumann, C., Xirasagar, S., and Gollnick, P. (1997) J. Biol. Chem. 272, 19863-19869
Zavodszky, P., Kardos, J., Svingor, A., and Petsko, G. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7406-7411
Jaenicke, R. (1991) Eur. J. Biochem. 202, 715-728
Grättinger, M., Dankesreiter, A., Schurig, H., Jaenicke, R. (1998) J. Mol. Biol. 280, 525-533
