| Research Abstract |
In order to develop molecular design for making proteins heat stable based on konwledge obtained from biochemical and molecular biological studies on thermophiles, stabilization of 3-isopropylmalate dehydrogenase have been attempted. The stabilized mutants were produced by (1) theoretical design, and (2) evolutionary molecular engineering method.
The three dimensional structure of 3-isopropylmalate dehydrogenase of an extreme thermophile, Thermus thermophilus, was determined at 1.9A resolution. Based on the structure, mutations have been designed to improve the stabilities of mesophile and chimeric isopropylmalate dehydrogenases. For instance, the structure of the thermophile enzyme suggested importance of hydrophobic interactions at the subunit-subunit interface. Residues at the interface of the Escherichia coli enzyme were replaced with more hydrophobic ones, and the mutant showed a higher denaturation temperature than that of the wild type enzyme. The second approach to prepare stabilized proteins is evolutionary molecular engineering. An integration host-vector system was constructed using T.thermophilus as the host. A gene coding a chimeric enzyme was integrated into the chromosome of the host. The transformant thus obtained could not grow at higher temperature than 70ﾟC in the absence of leucine. However, after a prolonged incubation at 76 or 79ﾟC,stabilized mutants appeared which produce stabilized isopropylmalate dehydrogenase. The genes for the stabilized enzyme were analyzed. A result from the present study suggested that a protein can be stabilized if an empty gap inside the molecule is filled by substituting with larger amino acid residue (s). Based on this suggestion, the heat stable enzyme of the thermophile was further stabilized by sitedirected mutagenesis.
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