Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Feb;178(3):723–727. doi: 10.1128/jb.178.3.723-727.1996

Further stabilization of 3-isopropylmalate dehydrogenase of an extreme thermophile, Thermus thermophilus, by a suppressor mutation method.

T Kotsuka 1, S Akanuma 1, M Tomuro 1, A Yamagishi 1, T Oshima 1
PMCID: PMC177718  PMID: 8550506

Abstract

We succeeded in further improvement of the stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme thermophile, Thermus thermophilus, by a suppressor mutation method. We previously constructed a chimeric IPMDH consisting of portions of thermophile and mesophile enzymes. The chimeric enzyme is less thermostable than the thermophile enzyme. The gene encoding the chimeric enzyme was subjected to random mutagenesis and integrated into the genome of a leuB-deficient mutant of T. thermophilus. The transformants were screened at 76 degrees C in minimum medium, and three independent stabilized mutants were obtained. The leuB genes from these three mutants were cloned and analyzed. The sequence analyses revealed Ala-172-->Val substitution in all of the mutants. The thermal stability of the thermophile IPMDH was improved by introducing the amino acid substitution.

Full Text

The Full Text of this article is available as a PDF (216.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ahern T. J., Casal J. I., Petsko G. A., Klibanov A. M. Control of oligomeric enzyme thermostability by protein engineering. Proc Natl Acad Sci U S A. 1987 Feb;84(3):675–679. doi: 10.1073/pnas.84.3.675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andreadis A., Rosenthal E. R. The nucleotide sequence of leuB from Salmonella typhimurium. Biochim Biophys Acta. 1992 Jan 6;1129(2):228–230. doi: 10.1016/0167-4781(92)90493-j. [DOI] [PubMed] [Google Scholar]
  3. Bini F., De Rossi E., Barbierato L., Riccardi G. Molecular cloning and sequencing of the beta-isopropylmalate dehydrogenase gene from the cyanobacterium Spirulina platensis. J Gen Microbiol. 1992 Mar;138(3):493–498. doi: 10.1099/00221287-138-3-493. [DOI] [PubMed] [Google Scholar]
  4. Das G., Hickey D. R., McLendon D., McLendon G., Sherman F. Dramatic thermostabilization of yeast iso-1-cytochrome c by an asparagine----isoleucine replacement at position 57. Proc Natl Acad Sci U S A. 1989 Jan;86(2):496–499. doi: 10.1073/pnas.86.2.496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Davidow L. S., Kaczmarek F. S., DeZeeuw J. R., Conlon S. W., Lauth M. R., Pereira D. A., Franke A. E. The Yarrowia lipolytica LEU2 gene. Curr Genet. 1987;11(5):377–383. doi: 10.1007/BF00378180. [DOI] [PubMed] [Google Scholar]
  6. Hamasawa K., Kobayashi Y., Harada S., Yoda K., Yamasaki M., Tamura G. Molecular cloning and nucleotide sequence of the 3-isopropylmalate dehydrogenase gene of Candida utilis. J Gen Microbiol. 1987 Apr;133(4):1089–1097. doi: 10.1099/00221287-133-4-1089. [DOI] [PubMed] [Google Scholar]
  7. Hecht M. H., Hehir K. M., Nelson H. C., Sturtevant J. M., Sauer R. T. Increasing and decreasing protein stability: effects of revertant substitutions on the thermal denaturation of phage lambda repressor. J Cell Biochem. 1985;29(3):217–224. doi: 10.1002/jcb.240290306. [DOI] [PubMed] [Google Scholar]
  8. Imada K., Sato M., Tanaka N., Katsube Y., Matsuura Y., Oshima T. Three-dimensional structure of a highly thermostable enzyme, 3-isopropylmalate dehydrogenase of Thermus thermophilus at 2.2 A resolution. J Mol Biol. 1991 Dec 5;222(3):725–738. doi: 10.1016/0022-2836(91)90508-4. [DOI] [PubMed] [Google Scholar]
  9. Imai R., Sekiguchi T., Nosoh Y., Tsuda K. The nucleotide sequence of 3-isopropylmalate dehydrogenase gene from Bacillus subtilis. Nucleic Acids Res. 1987 Jun 25;15(12):4988–4988. doi: 10.1093/nar/15.12.4988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kagawa Y., Nojima H., Nukiwa N., Ishizuka M., Nakajima T., Yasuhara T., Tanaka T., Oshima T. High guanine plus cytosine content in the third letter of codons of an extreme thermophile. DNA sequence of the isopropylmalate dehydrogenase of Thermus thermophilus. J Biol Chem. 1984 Mar 10;259(5):2956–2960. [PubMed] [Google Scholar]
  11. Kellis J. T., Jr, Nyberg K., Fersht A. R. Energetics of complementary side-chain packing in a protein hydrophobic core. Biochemistry. 1989 May 30;28(11):4914–4922. doi: 10.1021/bi00437a058. [DOI] [PubMed] [Google Scholar]
  12. Kirino H., Aoki M., Aoshima M., Hayashi Y., Ohba M., Yamagishi A., Wakagi T., Oshima T. Hydrophobic interaction at the subunit interface contributes to the thermostability of 3-isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophilus. Eur J Biochem. 1994 Feb 15;220(1):275–281. doi: 10.1111/j.1432-1033.1994.tb18623.x. [DOI] [PubMed] [Google Scholar]
  13. Kirino H., Oshima T. Molecular cloning and nucleotide sequence of 3-isopropylmalate dehydrogenase gene (leuB) from an extreme thermophile, Thermus aquaticus YT-1. J Biochem. 1991 Jun;109(6):852–857. doi: 10.1093/oxfordjournals.jbchem.a123470. [DOI] [PubMed] [Google Scholar]
  14. Koyama Y., Hoshino T., Tomizuka N., Furukawa K. Genetic transformation of the extreme thermophile Thermus thermophilus and of other Thermus spp. J Bacteriol. 1986 Apr;166(1):338–340. doi: 10.1128/jb.166.1.338-340.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kuroki R., Taniyama Y., Seko C., Nakamura H., Kikuchi M., Ikehara M. Design and creation of a Ca2+ binding site in human lysozyme to enhance structural stability. Proc Natl Acad Sci U S A. 1989 Sep;86(18):6903–6907. doi: 10.1073/pnas.86.18.6903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Matsumura M., Becktel W. J., Levitt M., Matthews B. W. Stabilization of phage T4 lysozyme by engineered disulfide bonds. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6562–6566. doi: 10.1073/pnas.86.17.6562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Matsumura M., Becktel W. J., Matthews B. W. Hydrophobic stabilization in T4 lysozyme determined directly by multiple substitutions of Ile 3. Nature. 1988 Aug 4;334(6181):406–410. doi: 10.1038/334406a0. [DOI] [PubMed] [Google Scholar]
  18. Matthews B. W., Nicholson H., Becktel W. J. Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6663–6667. doi: 10.1073/pnas.84.19.6663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nicholson H., Becktel W. J., Matthews B. W. Enhanced protein thermostability from designed mutations that interact with alpha-helix dipoles. Nature. 1988 Dec 15;336(6200):651–656. doi: 10.1038/336651a0. [DOI] [PubMed] [Google Scholar]
  20. Numata K., Muro M., Akutsu N., Nosoh Y., Yamagishi A., Oshima T. Thermal stability of chimeric isopropylmalate dehydrogenase genes constructed from a thermophile and a mesophile. Protein Eng. 1995 Jan;8(1):39–43. doi: 10.1093/protein/8.1.39. [DOI] [PubMed] [Google Scholar]
  21. Onodera K., Sakurai M., Moriyama H., Tanaka N., Numata K., Oshima T., Sato M., Katsube Y. Three-dimensional structures of chimeric enzymes between Bacillus subtilis and Thermus thermophilus 3-isopropylmalate dehydrogenases. Protein Eng. 1994 Apr;7(4):453–459. doi: 10.1093/protein/7.4.453. [DOI] [PubMed] [Google Scholar]
  22. Pantoliano M. W., Whitlow M., Wood J. F., Rollence M. L., Finzel B. C., Gilliland G. L., Poulos T. L., Bryan P. N. The engineering of binding affinity at metal ion binding sites for the stabilization of proteins: subtilisin as a test case. Biochemistry. 1988 Nov 1;27(22):8311–8317. doi: 10.1021/bi00422a004. [DOI] [PubMed] [Google Scholar]
  23. Sakai Y., Tani Y. Directed mutagenesis in an asporogenous methylotrophic yeast: cloning, sequencing, and one-step gene disruption of the 3-isopropylmalate dehydrogenase gene (LEU2) of Candida boidinii to derive doubly auxotrophic marker strains. J Bacteriol. 1992 Sep;174(18):5988–5993. doi: 10.1128/jb.174.18.5988-5993.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sakaki Y., Oshima T. Isolation and characterization of a bacteriophage infectious to an extreme thermophile, Thermus thermophilus HB8. J Virol. 1975 Jun;15(6):1449–1453. doi: 10.1128/jvi.15.6.1449-1453.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sandberg W. S., Terwilliger T. C. Influence of interior packing and hydrophobicity on the stability of a protein. Science. 1989 Jul 7;245(4913):54–57. doi: 10.1126/science.2787053. [DOI] [PubMed] [Google Scholar]
  26. Sekiguchi T., Suda M., Ishii T., Nosoh Y., Tsuda K. The nucleotide sequence of 3-isopropylmalate dehydrogenase gene from Bacillus caldotenax. Nucleic Acids Res. 1987 Jan 26;15(2):853–853. doi: 10.1093/nar/15.2.853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shortle D., Lin B. Genetic analysis of staphylococcal nuclease: identification of three intragenic "global" suppressors of nuclease-minus mutations. Genetics. 1985 Aug;110(4):539–555. doi: 10.1093/genetics/110.4.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Suzuki Y., Ito N., Yuuki T., Yamagata H., Udaka S. Amino acid residues stabilizing a Bacillus alpha-amylase against irreversible thermoinactivation. J Biol Chem. 1989 Nov 15;264(32):18933–18938. [PubMed] [Google Scholar]
  29. Takada T., Akanuma S., Kotsuka T., Tamakoshi M., Yamagishi A., Oshima T. Recombination-Deficient Mutants of an Extreme Thermophile, Thermus thermophilus. Appl Environ Microbiol. 1993 Aug;59(8):2737–2739. doi: 10.1128/aem.59.8.2737-2739.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Takagi M., Kobayashi N., Sugimoto M., Fujii T., Watari J., Yano K. Nucleotide sequencing analysis of a LEU gene of Candida maltosa which complements leuB mutation of Escherichia coli and leu2 mutation of Saccharomyces cerevisiae. Curr Genet. 1987;11(6-7):451–457. doi: 10.1007/BF00384606. [DOI] [PubMed] [Google Scholar]
  31. Tanaka T., Kawano N., Oshima T. Cloning of 3-isopropylmalate dehydrogenase gene of an extreme thermophile and partial purification of the gene product. J Biochem. 1981 Feb;89(2):677–682. doi: 10.1093/oxfordjournals.jbchem.a133245. [DOI] [PubMed] [Google Scholar]
  32. Yamada T., Akutsu N., Miyazaki K., Kakinuma K., Yoshida M., Oshima T. Purification, catalytic properties, and thermal stability of threo-Ds-3-isopropylmalate dehydrogenase coded by leuB gene from an extreme thermophile, Thermus thermophilus strain HB8. J Biochem. 1990 Sep;108(3):449–456. doi: 10.1093/oxfordjournals.jbchem.a123220. [DOI] [PubMed] [Google Scholar]
  33. Yutani K., Ogasahara K., Tsujita T., Sugino Y. Dependence of conformational stability on hydrophobicity of the amino acid residue in a series of variant proteins substituted at a unique position of tryptophan synthase alpha subunit. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4441–4444. doi: 10.1073/pnas.84.13.4441. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES