1887

Abstract

To elucidate determinants of differences in thermostability between mesophilic and psychrophilic monomeric isocitrate dehydrogenases (IDHs) from (IDH) and (IDH), respectively, chimeric enzymes derived from the two IDHs were constructed based on the recently resolved three-dimensional structure of IDH, and several characteristics of the two wild-type and six chimeric IDHs were examined. These characteristics were then compared with those of dimeric IDH from (IDH). All recombinant enzymes with a (His)-tag attached to the N-terminal were overexpressed in the cells and purified by Ni-affinity chromatography. The catalytic activity ( ) and catalytic efficiency ( / ) of the wild-type IDH and IDH were higher than those of IDH, implying that an improved catalytic rate more than compensates for the loss of a catalytic site in the former two IDHs due to monomerization. Analyses of the thermostability and kinetic parameters of the chimeric enzymes indicated that region 2, corresponding to domain II, and particularly region 3 located in the C-terminal part of domain I, are involved in the thermolability of IDH, and that the corresponding two regions of IDH are important for exhibiting higher catalytic activity and affinity for isocitrate than IDH. The relationships between the stability, catalytic activity and structural characteristics of IDH and IDH are discussed.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27667-0
2005-04-01
2019-10-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/4/mic1511083.html?itemId=/content/journal/micro/10.1099/mic.0.27667-0&mimeType=html&fmt=ahah

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. Barrera, C. R. & Jurtshuk, P. ( 1970; ). Characterization of the highly active isocitrate (NADP+) dehydrogenase of Azotobacter vinelandii. Biochim Biophys Acta 220, 416–429.[CrossRef]
    [Google Scholar]
  4. Bentahir, M., Feller, G., Aittaleb, M., Lamotte-Brasseur, J., Himri, T., Chessa, J. P. & Gerday, C. ( 2000; ). Structural, kinetic, and calorimetric characterization of the cold-active phosphoglycerate kinase from the Antarctic Pseudomonas sp. TACII18. J Biol Chem 275, 11147–11153.[CrossRef]
    [Google Scholar]
  5. Ceccarelli, C., Grodsky, N. B., Ariyaratne, N., Colman, R. F. & Bahnson, B. J. ( 2002; ). Crystal structure of porcine mitochondrial NADP+-dependent isocitrate dehydrogenase complexed with Mn2+ and isocitrate: insights into the enzyme mechanism. J Biol Chem 277, 43454–43462.[CrossRef]
    [Google Scholar]
  6. Chen, R. & Jeong, S. S. ( 2000; ). Functional prediction: identification of protein orthologs and paralogs. Protein Sci 9, 2344–2353.[CrossRef]
    [Google Scholar]
  7. Chen, R. & Yang, H. ( 2000; ). A highly specific monomeric isocitrate dehydrogenase from Corynebacterium glutamicum. Arch Biochem Biophys 383, 238–245.[CrossRef]
    [Google Scholar]
  8. Dean, A. M. & Golding, G. B. ( 1997; ). Protein engineering reveals ancient adaptive replacements in isocitrate dehydrogenase. Proc Natl Acad Sci U S A 94, 3104–3109.[CrossRef]
    [Google Scholar]
  9. de Backer, M., McSweeney, S., Rasmussen, H. B., Riise, B. W., Lindley, P. & Hough, E. ( 2002; ). The 1·9 Å crystal structure of heat-labile shrimp alkaline phosphatase. J Mol Biol 318, 1265–1274.[CrossRef]
    [Google Scholar]
  10. Eikmanns, B. J., Rittmann, D. & Sahm, H. ( 1995; ). Cloning, sequence analysis, expression, and inactivation of the Corynebacterium glutamicum icd gene encoding isocitrate dehydrogenase and biochemical characterization of the enzyme. J Bacteriol 177, 774–782.
    [Google Scholar]
  11. Fields, P. A. ( 2001; ). Protein function at thermal extremes: balancing stability and flexibility. Comp Biochem Physiol A129, 417–431.
    [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. Fukunaga, N., Imagawa, S., Sahara, T., Ishii, A. & Suzuki, M. ( 1992; ). Purification and characterization of monomeric isocitrate dehydrogenase with NADP+-specificity from Vibrio parahaemolyticus Y-4. J Biochem 112, 849–855.
    [Google Scholar]
  14. 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]
  15. Hurley, J. H. & Dean, A. M. ( 1994; ). Structure of 3-isopropylmalate dehydrogenase in complex with NAD+: ligand-induced loop closing and mechanism for cofactor specificity. Structure 2, 1007–1016.[CrossRef]
    [Google Scholar]
  16. Hurley, J. H., Thorsness, P. E., Ramalingam, V., Helmers, N. H., Koshland, D. E., Jr & Stroud, R. M. ( 1989; ). Structure of a bacterial enzyme regulated by phosphorylation, isocitrate dehydrogenase. Proc Natl Acad Sci U S A 86, 8635–8639.[CrossRef]
    [Google Scholar]
  17. Hurley, J. H., Dean, A. M., Koshland, D. E., Jr & Stroud, R. M. ( 1991; ). Catalytic mechanism of NADP+-dependent isocitrate dehydrogenase: implications from the structures of magnesium-isocitrate and NADP+ complexes. Biochemistry 30, 8671–8678.[CrossRef]
    [Google Scholar]
  18. Imada, K., Sato, M., Tanaka, N., Katsube, Y., Matsuura, Y. & Oshima, T. ( 1991; ). Three-dimensional structure of a highly thermostable enzyme, 3-isopropylmalate dehydrogenase of Thermus thermophilus at 2·2 Å resolution. J Mol Biol 222, 725–738.[CrossRef]
    [Google Scholar]
  19. Ishii, A., Ochiai, T., Imagawa, S., Fukunaga, N., Sasaki, S., Minowa, O., Mizuno, Y. & Shiokawa, H. ( 1987; ). Isozymes of isocitrate dehydrogenase from an obligately psychrophilic bacterium, Vibrio sp. strain ABE-1: purification, and modulation of activities by growth conditions. J Biochem 102, 1489–1498.
    [Google Scholar]
  20. Ishii, A., Suzuki, M., Sahara, T., Takada, Y., Sasaki, S. & Fukunaga, N. ( 1993; ). Genes encoding two isocitrate dehydrogenase isozymes of a psychrophilic bacterium, Vibrio sp. strain ABE-1. J Bacteriol 175, 6873–6880.
    [Google Scholar]
  21. Kanao, T., Kawamura, M., Fukui, T., Atomi, H. & Imanaka, T. ( 2002; ). Characterization of isocitrate dehydrogenase from the green sulfur bacterium Chlorobium limicola: a carbon dioxide-fixing enzyme in the reductive tricarboxylic acid cycle. Eur J Biochem 269, 1926–1931.[CrossRef]
    [Google Scholar]
  22. 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]
  23. Kirino, H., Aoki, M., Aoshima, M., Hayashi, Y., Ohba, M., Yamagishi, A., Wakagi, T. & Oshima, T. ( 1994; ). Hydrophobic interaction at the subunit interface contributes to the thermostability of 3-isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophilus. Eur J Biochem 220, 275–281.[CrossRef]
    [Google Scholar]
  24. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef]
    [Google Scholar]
  25. LaPorte, D. C., Thorsness, P. E. & Koshland, D. E., Jr ( 1985; ). Compensatory phosphorylation of isocitrate dehydrogenase: a mechanism for adaptation to the intracellular environment. J Biol Chem 260, 10563–10568.
    [Google Scholar]
  26. 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]
  27. Matthews, B. W., Nicholson, H. & Becktel, W. J. ( 1987; ). Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc Natl Acad Sci U S A 84, 6663–6667.[CrossRef]
    [Google Scholar]
  28. 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]
  29. Ochiai, T., Fukunaga, N. & Sasaki, S. ( 1979; ). Purification and some properties of two NADP+-specific isocitrate dehydrogenases from an obligately psychrophilic marine bacterium, Vibrio sp., strain ABE-1. J Biochem 86, 377–384.
    [Google Scholar]
  30. Russell, R. J. M., 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. Sahara, T. ( 2000; ). Studies on structure and function of the bacterial monomeric isocitrate dehydrogenase and regulatory mechanisms of the gene expression. Doctoral dissertation, Hokkaido University, Sapporo, Japan.
  32. Sahara, T., Suzuki, M., Tsuruha, J., Takada, Y. & Fukunaga, N. ( 1999; ). cis-Acting elements responsible for low-temperature-inducible expression of the gene coding for the thermolabile isocitrate dehydrogenase isozyme of a psychrophilic bacterium, Vibrio sp. strain ABE-1. J Bacteriol 181, 2602–2611.
    [Google Scholar]
  33. Sahara, T., Takada, Y., Takeuchi, Y., Yamaoka, N. & Fukunaga, N. ( 2002; ). Cloning, sequencing, and expression of a gene encoding the monomeric isocitrate dehydrogenase of the nitrogen fixing bacterium, Azotobacter vinelandii. Biosci Biotechnol Biochem 66, 489–500.[CrossRef]
    [Google Scholar]
  34. Steen, I. H., Madern, D., Karlstrom, M., Lien, T., Ladenstein, R. & Birkeland, N. K. ( 2001; ). Comparison of isocitrate dehydrogenase from three hyperthermophiles reveals differences in thermostability, cofactor specificity, oligomeric state, and phylogenetic affiliation. J Biol Chem 276, 43924–43931.[CrossRef]
    [Google Scholar]
  35. Stoddard, B. L., Dean, A. & Koshland, D. E., Jr ( 1993; ). Structure of isocitrate dehydrogenase with isocitrate, nicotinamide adenine dinucleotide phosphate, and calcium at 2·5-Å resolution: a pseudo-Michaelis ternary complex. Biochemistry 32, 9310–9316.[CrossRef]
    [Google Scholar]
  36. Suzuki, M., Sahara, T., Tsuruha, J., Takada, Y. & Fukunaga, N. ( 1995; ). Differential expression in Escherichia coli of the Vibrio sp. strain ABE-1 icd-I and icd-II genes encoding structurally different isocitrate dehydrogenase isozymes. J Bacteriol 177, 2138–2142.
    [Google Scholar]
  37. Takada, Y., Ochiai, T., Okuyama, H., Nishi, K. & Sasaki, S. ( 1979; ). An obligately psychrophilic bacterium isolated on the Hokkaido coast. J Gen Appl Microbiol 25, 11–19.[CrossRef]
    [Google Scholar]
  38. Thorsness, P. E. & Koshland, D. E., Jr ( 1987; ). Inactivation of isocitrate dehydrogenase by phosphorylation is mediated by the negative charge of the phosphate. J Biol Chem 262, 10422–10425.
    [Google Scholar]
  39. Wallon, G., Kryger, G., Lovett, S. T., Oshima, T., Ringe, D. & Petsko, G. A. ( 1997a; ). Crystal structures of Escherichia coli and Salmonella typhimurium 3-isopropylmalate dehydrogenase and comparison with their thermophilic counterpart from Thermus thermophilus. J Mol Biol 266, 1016–1031.[CrossRef]
    [Google Scholar]
  40. Wallon, G., Lovett, S. T., Magyar, C., Svingor, A., Szilagyi, A., Zavodszky, P., Ringe, D. & Petsko, G. A. ( 1997b; ). Sequence and homology model of 3-isopropylmalate dehydrogenase from the psychrotrophic bacterium Vibrio sp. I5 suggest reasons for thermal instability. Protein Eng 10, 665–672.[CrossRef]
    [Google Scholar]
  41. 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]
  42. Watanabe, S., Yamaoka, N., Takada, Y. & Fukunaga, N. ( 2002; ). The cold-inducible icl gene encoding thermolabile isocitrate lyase of a psychrophilic bacterium, Colwellia maris. Microbiology 148, 2579–2589.
    [Google Scholar]
  43. Xu, X., Zhao, J., Xu, Z., Peng, B., Huang, Q., Arnold, E. & Ding, J. ( 2004; ). Structures of human cytosolic NADP-dependent isocitrate dehydrogenase reveal a novel self-regulatory mechanism of activity. J Biol Chem 279, 33946–33957.[CrossRef]
    [Google Scholar]
  44. Yasutake, Y., Watanabe, S., Yao, M., Takada, Y., Fukunaga, N. & Tanaka, I. ( 2001; ). Crystallization and preliminary X-ray diffraction studies of monomeric isocitrate dehydrogenase by the MAD method using Mn atoms. Acta Cryst D57, 1682–1685.
    [Google Scholar]
  45. Yasutake, Y., Watanabe, S., Yao, M., Takada, Y., Fukunaga, N. & Tanaka, I. ( 2002; ). Structure of the monomeric isocitrate dehydrogenase: evidence of a protein monomerization by a domain duplication. Structure 10, 1637–1648.[CrossRef]
    [Google Scholar]
  46. Yasutake, Y., Watanabe, S., Yao, M., Takada, Y., Fukunaga, N. & Tanaka, I. ( 2003; ). Crystal structure of the monomeric isocitrate dehydrogenase in the presence of NADP+: insight into the cofactor recognition, catalysis, and evolution. J Biol Chem 278, 36897–36904.[CrossRef]
    [Google Scholar]
  47. Yumoto, I., Kawasaki, K., Iwata, H., Matsuyama, H. & Okuyama, H. ( 1998; ). Assignment of Vibrio sp. strain ABE-1 to Colwellia maris sp. nov., a new psychrophilic bacterium. Int J Syst Bacteriol 48, 1357–1362.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27667-0
Loading
/content/journal/micro/10.1099/mic.0.27667-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error