1887

Abstract

To investigate the mechanism of cold adaptation of isocitrate lyase (ICL; EC 4.1.3.1) from the psychrophilic bacterium , Gln207 and Gln217 of this enzyme were substituted by His and Lys, respectively, by site-directed mutagenesis. His184 and Lys194 of ICL from , corresponding to the two Gln residues of ICL, are highly conserved in the ICLs of many organisms and are known to be essential for catalytic function. The mutated ICLs (-Q207H and -Q217K, respectively) and wild-type enzymes of and (-WT and -WT) with His-tagged peptides were overexpressed in cells and purified to homogeneity. Thermolabile -WT and mutated ICLs were susceptible to digestion with trypsin, while relatively thermostable -WT was resistant to trypsin digestion, suggesting that the thermostability and resistance to tryptic digestion of the ICLs are related. -Q207H and -Q217K showed specific activities similar to -WT at temperatures between 30 °C and 40 °C, but their activities between 10 °C and 25 °C were decreased, indicating that the two Gln residues of the ICL play important roles in its cold adaptation. Phylogenetic analysis of ICLs from various organisms revealed that the ICL can be categorized in a novel group, subfamily 3, together with several eubacterial ICLs.

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2004-10-01
2019-11-12
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References

  1. Aghajanian, S., Hovsepyan, M., Geoghegan, K. F., Chrunyk, B. A. & Engel, P. C. ( 2003; ). A thermally sensitive loop in clostridial glutamate dehydrogenase detected by limited proteolysis. J Biol Chem 278, 1067–1074.[CrossRef]
    [Google Scholar]
  2. Alvarez, M., Zeelen, J. P., Mainfroid, V., Rentier-Delrue, F., Martial, J. A., Wyns, L., Wierenga, R. K. & Maes, D. ( 1998; ). Triose-phosphate isomerase (TIM) of the psychrophilic bacterium Vibrio marinus. Kinetic and structural properties. J Biol Chem 273, 2199–2206.[CrossRef]
    [Google Scholar]
  3. Andrade, M. A., Chacon, P., Merelo, J. J. & Moran, F. ( 1993; ). Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Eng 6, 383–390.[CrossRef]
    [Google Scholar]
  4. Bentahir, M., Feller, G., Aittaleb, M. & 8 other authors ( 2000; ). Directed evolution study of temperature adaptation in a psychrophilic enzyme. J Mol Biol 297, 1015–1026.[CrossRef]
    [Google Scholar]
  5. Britton, K. L., Langridge, S. J., Baker, P. J., Weeradechapon, K., Sedelnikova, S. E., Lucas, J. R., Rice, D. W. & Turner, G. ( 2000; ). The crystal structure and active site location of isocitrate lyase from the fungus Aspergillus nidulans. Structure 8, 349–362.[CrossRef]
    [Google Scholar]
  6. Britton, K. L., Abeysinghe, I. S., Baker, P. J. & 8 other authors ( 2001; ). The structure and domain organization of Escherichia coli isocitrate lyase. Acta Crystallogr Sect D Biol Crystallogr 57, 1209–1218.[CrossRef]
    [Google Scholar]
  7. Cozzone, A. J. ( 1998; ). Regulation of acetate metabolism by protein phosphorylation in Escherichia coli. Annu Rev Microbiol 52, 127–164.[CrossRef]
    [Google Scholar]
  8. Davail, S., Feller, G., Narinx, E. & Gerday, C. ( 1994; ). Cold adaptation of proteins. Purification, characterization, and sequence of the heat-labile subtilisin from the antarctic psychrophile Bacillus TA41. J Biol Chem 269, 17448–17453.
    [Google Scholar]
  9. Diehl, P. & McFadden, B. A. ( 1993; ). Site-directed mutagenesis of lysine 193 in Escherichia coli isocitrate lyase by use of unique restriction enzyme site elimination. J Bacteriol 175, 2263–2270.
    [Google Scholar]
  10. Diehl, P. & McFadden, B. A. ( 1994; ). The importance of four histidine residues in isocitrate lyase from Escherichia coli. J Bacteriol 176, 927–931.
    [Google Scholar]
  11. Feller, G. & Gerday, C. ( 1997; ). Psychrophilic enzymes: molecular basis of cold adaptation. Cell. Mol Life Sci 53, 830–841.[CrossRef]
    [Google Scholar]
  12. Fields, P. A. & Somero, G. N. ( 1998; ). Hot spots in cold adaptation: localized increases in conformational flexibility in lactate dehydrogenase A4 orthologs of antarctic notothenioid fishes. Proc Natl Acad Sci U S A 95, 11476–11481.[CrossRef]
    [Google Scholar]
  13. Gerday, C., Aittaleb, M., Arpigny, J. L., Baise, E., Chessa, J. P., Garsoux, G., Petrescu, I. & Feller, G. ( 1997; ). Psychrophilic enzymes: a thermodynamic challenge. Biochim Biophys Acta 1342, 119–131.[CrossRef]
    [Google Scholar]
  14. Gerike, U., Danson, M. J. & Hough, D. W. ( 2001; ). Cold-active citrate synthase: mutagenesis of active-site residues. Protein Eng 14, 655–661.[CrossRef]
    [Google Scholar]
  15. Kim, S. Y., Hwang, K. Y., Kim, S. H., Sung, H. C., Han, Y. S. & Cho, Y. ( 1999; ). Structural basis for cold adaptation. Sequence, biochemical properties, and crystal structure of malate dehydrogenase from a psychrophile Aquaspirillium arcticum. J Biol Chem 274, 11761–11767.[CrossRef]
    [Google Scholar]
  16. Ko, Y. H. & McFadden, B. A. ( 1990; ). Alkylation of isocitrate lyase from Escherichia coli by 3-bromopyruvate. Arch Biochem Biophys 278, 373–380.[CrossRef]
    [Google Scholar]
  17. Ko, Y. H., Vanni, P., Munske, G. R. & McFadden, B. A. ( 1991; ). Substrate-decreased modification by diethyl pyrocarbonate of two histidines in isocitrate lyase from Escherichia coli. Biochemistry 30, 7451–7456.[CrossRef]
    [Google Scholar]
  18. Ko, Y. H., Cremo, C. R. & McFadden, B. A. ( 1992; ). Vanadate-dependent photomodification of serine 319 and 321 in the active site of isocitrate lyase from Escherichia coli. J Biol Chem 267, 91–95.
    [Google Scholar]
  19. Kornberg, H. L. ( 1966; ). The role and control of the glyoxylate cycle in Escherichia coli. Biochem J 99, 1–11.
    [Google Scholar]
  20. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef]
    [Google Scholar]
  21. Lonhienne, T., Gerday, C. & Feller, G. ( 2000; ). Psychrophilic enzymes: revisiting the thermodynamic parameters of activation may explain local flexibility. Biochim Biophys Acta 1543, 1–10.[CrossRef]
    [Google Scholar]
  22. Lönn, A., Gárdonyi, M., van Zyl, W., Hahn-Hägerdal, B. & Otero, R. C. ( 2002; ). Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis. Gene cloning and protein characterization. Eur J Biochem 269, 157–163.[CrossRef]
    [Google Scholar]
  23. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. ( 1951; ). Protein measurement with the Folin phenol reagent. J Biol Chem 193, 265–275.
    [Google Scholar]
  24. Matsuoka, M. & McFadden, B. A. ( 1988; ). Isolation, hyperexpression, and sequencing of the aceA gene encoding isocitrate lyase in Escherichia coli. J Bacteriol 170, 4528–4536.
    [Google Scholar]
  25. Miyazaki, K., Wintrode, P. L., Grayling, R. A., Rubingh, D. N. & Arnold, F. H. ( 2000; ). Directed evolution study of temperature adaptation in a psychrophilic enzyme. J Mol Biol 297, 1015–1026.[CrossRef]
    [Google Scholar]
  26. Rehman, A. & McFadden, B. A. ( 1996; ). The consequences of replacing histidine 356 in isocitrate lyase from Escherichia coli. Arch Biochem Biophys 336, 309–315.[CrossRef]
    [Google Scholar]
  27. Rehman, A. & McFadden, B. A. ( 1997a; ). Serine 319 and 321 are functional in isocitrate lyase from Escherichia coli. Curr Microbiol 34, 205–211.[CrossRef]
    [Google Scholar]
  28. Rehman, A. & McFadden, B. A. ( 1997b; ). Lysine 194 is functional in isocitrate lyase from Escherichia coli. Curr Microbiol 35, 14–17.[CrossRef]
    [Google Scholar]
  29. Rehman, A. & McFadden, B. A. ( 1997c; ). Cysteine 195 has a critical functional role in catalysis by isocitrate lyase from Escherichia coli. Curr Microbiol 35, 267–269.[CrossRef]
    [Google Scholar]
  30. Russell, R. J., Gerike, U., Danson, M. J., Hough, D. W. & Taylor, G. L. ( 1998; ). Structural adaptations of the cold-active citrate synthase from an Antarctic bacterium. Structure 6, 351–361.[CrossRef]
    [Google Scholar]
  31. Serrano, J. A. & Bonete, M. J. ( 2001; ). Sequencing, phylogenetic and transcriptional analysis of the glyoxylate bypass operon (ace) in the halophilic archaeon Haloferax volcanii. Biochim Biophys Acta 1520, 154–162.[CrossRef]
    [Google Scholar]
  32. Sharma, V., Sharma, S., Hoener zu Bentrup, K., McKinney, J. D., Russell, D. G., Jacobs, W. R., Jr & Sacchettini, J. C. ( 2000; ). Structure of isocitrate lyase, a persistence factor of Mycobacterium tuberculosis. Nat Struct Biol 7, 663–668.[CrossRef]
    [Google Scholar]
  33. Sheridan, P. P., Panasik, N., Coombs, J. M. & Brenchley, J. E. ( 2000; ). Approaches for deciphering the structural basis of low temperature enzyme activity. Biochim Biophys Acta 1543, 417–433.[CrossRef]
    [Google Scholar]
  34. Somero, G. N. ( 1995; ). Proteins and temperature. Annu Rev Physiol 57, 43–68.[CrossRef]
    [Google Scholar]
  35. Suzuki, T., Yasugi, M., Arisaka, F., Yamagishi, A. & Oshima, T. ( 2001; ). Adaptation of a thermophilic enzyme, 3-isopropylmalate dehydrogenase, to low temperatures. Protein Eng 14, 85–91.[CrossRef]
    [Google Scholar]
  36. Taguchi, S., Ozaki, A., Nonaka, T., Mitsui, Y. & Momose, H. ( 1999; ). A cold-adapted protease engineered by experimental evolution system. J Biochem (Tokyo) 126, 689–693.[CrossRef]
    [Google Scholar]
  37. Thomas, T. & Cavicchioli, R. ( 2000; ). Effect of temperature on stability and activity of elongation factor 2 proteins from Antarctic and thermophilic methanogens. J Bacteriol 182, 1328–1332.[CrossRef]
    [Google Scholar]
  38. Vanni, P., Giachetti, E., Pinzauti, G. & McFadden, B. A. ( 1990; ). Comparative structure, function and regulation of isocitrate lyase, an important assimilatory enzyme. Comp Biochem Physiol B 95, 431–458.
    [Google Scholar]
  39. Watanabe, S., Takada, Y. & Fukunaga, N. ( 2001; ). Purification and characterization of a cold-adapted isocitrate lyase and a malate synthase from Colwellia maris, a psychrophilic bacterium. Biosci Biotechnol Biochem 65, 1095–1103.[CrossRef]
    [Google Scholar]
  40. Watanabe, S., Yamaoka, N., Takada, Y. & Fukunaga, N. ( 2002a; ). The cold-inducible icl gene encoding thermolabile isocitrate lyase of a psychrophilic bacterium, Colwellia maris. Microbiology 148, 2579–2589.
    [Google Scholar]
  41. Watanabe, S., Yamaoka, N., Fukunaga, N. & Takada, Y. ( 2002b; ). Purification and characterization of a cold-adapted isocitrate lyase, and expression analysis of the cold-inducible isocitrate lyase gene from a psychrophilic bacterium, Colwellia psychrerythraea. Extremophiles 6, 397–405.[CrossRef]
    [Google Scholar]
  42. Wetlaufer, D. B. ( 1962; ). Ultraviolet spectra of proteins and amino acids. Adv Protein Chem 17, 304.
    [Google Scholar]
  43. Wintrode, P. L., Miyazaki, K. & Arnold, F. H. ( 2000; ). Cold adaptation of a mesophilic subtilisin-like protease by laboratory evolution. J Biol Chem 275, 31635–31640.[CrossRef]
    [Google Scholar]
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