Abstract
A mutant strain of Thermus thermophilus which contains deletions in the 3'-terminal region of its leuB gene showed a temperature-sensitive growth phenotype in the absence of leucine. Three phenotypically thermostable mutants were isolated from the temperature-sensitive strain by spontaneous evolution. Each pseudorevertant carried a tandem sequence duplication in the 3' region of its leuB gene. The mutated 3-isopropylmalate dehydrogenases encoded by the leuB genes from the pseudorevertants were more thermostable than the enzyme from the temperature-sensitive strain. Structural analyses suggested that the decreased thermostability of the enzyme from the temperature-sensitive strain was caused by reducing hydrophobic and electrostatic interactions in the carboxyl-terminal region and that the recovered stability of the enzymes from the pseudorevertants was due to the restoration of the hydrophobic interaction. Our results indicate that tandem sequence duplications are the general genetic way to alter protein characteristics in evolution.
Full Text
The Full Text of this article is available as a PDF (231.3 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Hall B. G. Changes in the substrate specificities of an enzyme during directed evolution of new functions. Biochemistry. 1981 Jul 7;20(14):4042–4049. doi: 10.1021/bi00517a015. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Kless H., Vermaas W. Tandem sequence duplications functionally complement deletions in the D1 protein of photosystem II. J Biol Chem. 1995 Jul 14;270(28):16536–16541. doi: 10.1074/jbc.270.28.16536. [DOI] [PubMed] [Google Scholar]
- Kotsuka T., Akanuma S., Tomuro M., Yamagishi A., Oshima T. Further stabilization of 3-isopropylmalate dehydrogenase of an extreme thermophile, Thermus thermophilus, by a suppressor mutation method. J Bacteriol. 1996 Feb;178(3):723–727. doi: 10.1128/jb.178.3.723-727.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
- Liao H., McKenzie T., Hageman R. Isolation of a thermostable enzyme variant by cloning and selection in a thermophile. Proc Natl Acad Sci U S A. 1986 Feb;83(3):576–580. doi: 10.1073/pnas.83.3.576. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Tamakoshi M., Yamagishi A., Oshima T. Screening of stable proteins in an extreme thermophile, Thermus thermophilus. Mol Microbiol. 1995 Jun;16(5):1031–1036. doi: 10.1111/j.1365-2958.1995.tb02328.x. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]