J. Bacteriol., 02 1996, 723-727, Vol 178, No. 3
Copyright © 1996, American Society for Microbiology

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

T Kotsuka, S Akanuma, M Tomuro, A Yamagishi and T Oshima
Department of Life Science, Tokyo Institute of Technology, Japan.

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.

This article has been cited by other articles:

• Ohkuri, T., Yamagishi, A. (2007). The Effects of Mutations at Position 253 on the Thermostability of the Bacillus subtilis 3-Isopropylmalate Dehydrogenase Subunit Interface. J Biochem 141: 791-797 [Abstract] [Full Text]  
• Ohkuri, T., Yamagishi, A. (2003). Increased thermal stability against irreversible inactivation of 3-isopropylmalate dehydrogenase induced by decreased van der Waals volume at the subunit interface. Protein Eng Des Sel 16: 615-621 [Abstract] [Full Text]  
• Fridjonsson, O., Watzlawick, H., Mattes, R. (2002). Thermoadaptation of {alpha}-Galactosidase AgaB1 in Thermus thermophilus. J. Bacteriol. 184: 3385-3391 [Abstract] [Full Text]  
• Fridjonsson, O., Mattes, R. (2001). Production of Recombinant {alpha}-Galactosidases in Thermus thermophilus. Appl. Environ. Microbiol. 67: 4192-4198 [Abstract] [Full Text]  
• Yasugi, M., Suzuki, T., Yamagishi, A., Oshima, T. (2001). Analysis of the effect of accumulation of amino acid replacements on activity of 3-isopropylmalate dehydrogenase from Thermus thermophilus. Protein Eng Des Sel 14: 601-607 [Abstract] [Full Text]  
• Hirose, R., Suzuki, T., Moriyama, H., Sato, T., Yamagishi, A., Oshima, T., Tanaka, N. (2001). Crystal structures of mutants of Thermus thermophilus IPMDH adapted to low temperatures. Protein Eng Des Sel 14: 81-84 [Abstract] [Full Text]  
• Nurachman, Z., Akanuma, S., Sato, T., Oshima, T., Tanaka, N. (2000). Crystal structures of 3-isopropylmalate dehydrogenases with mutations at the C-terminus: crystallographic analyses of structure-stability relationships. Protein Eng Des Sel 13: 253-258 [Abstract] [Full Text]  
• Haney, P. J., Badger, J. H., Buldak, G. L., Reich, C. I., Woese, C. R., Olsen, G. J. (1999). Thermal adaptation analyzed by comparison of protein sequences from mesophilic and extremely thermophilic Methanococcus species. Proc. Natl. Acad. Sci. USA 96: 3578-3583 [Abstract] [Full Text]  
• Svingor, A., Kardos, J., Hajdu, I., Nemeth, A., Zavodszky, P. (2001). A Better Enzyme to Cope with Cold. COMPARATIVE FLEXIBILITY STUDIES ON PSYCHROTROPHIC, MESOPHILIC, AND THERMOPHILIC IPMDHS. J. Biol. Chem. 276: 28121-28125 [Abstract] [Full Text]