Studies on interdomain interaction of 3-isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophilus, by constructing chimeric enzymes.
In our previous study, we showed that a chimeric isopropylmalate dehydrogenase, 2T2M6T, between an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, isopropylmalate dehydrogenases (the name roughly denotes the primary structure; the first 20% from the N-terminal is coded by the thermophile leuB gene, next 20% by mesophile, and the rest by the thermophile gene) denatured in two steps with a stable intermediate, suggesting that in the chimera some of the interdomain interaction was lost by amino acid substitutions in the "2M" part. To identify the residues involved in the interdomain interactions, the first and the second halves of the 2M part of the chimera were substituted with the corresponding sequence of the thermophile enzyme. Both chimeras, 3T1M6T and 2T1M7T, apparently showed one transition in the thermal denaturation without any stable intermediate state, suggesting that the cooperativity of the conformational stability was at least partly restored by the substitutions. The present study also suggested involvement of one or more basic residues in the unusual stability of the thermophile enzyme.
Numata
K
K
Department of Life Science, Tokyo Institute of Technology, Yokohama, Japan.
Hayashiiwasaki
Y
Y
Yutani
K
K
Oshima
T
T
D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000201:enzymology*
D011993:Recombinant Fusion Proteins; D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase