Adaptation of Isopropylmalate Dehydrogenase to Thermophilic and Mesophilic Temperatures
Shunji SUZUKI, Satoshi AKANUMA, Masatada TAMAKOSHI, Akihiko YAMAGISHI, and Tairo OSHIMA*
Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0355, Japan
We have constructed an integration-host vector system using Thermus therrnophilus, an extreme thermophile, as the host. Using this genetic manipulation system, any desired gene from a mesophile can be integrated into an operon in the chromosomal DNA of the thermophile and expressed in the thermophile cells. Under the conditions in which the translation product of the integrated mesophile gene is essential for the growth of the host thermophile cells, the transformant cells are temperature sensitive, and are able to grow only in a limited temperature range. However, stabilized mutants which produce stabilized protein coded by the mesophilic gene, evolved spontaneously after prolonged incubation at higher temperatures. Isopropylmalate dehydrogenases from a variety of mesophiles were stabilized by this "laboratory evolution" techniques. The laboratory evolution is complementary to the theoretical design in making a robust enzyme, and often induced unexpected mutations which are hard to think out. Recently we succeeded to construct a genetic system for the opposite direction; using an E. coli mutant, it is now possible to integrate a gene from T. thermophilus into leucine operon of E. coli chromosome. In our experiments, leuB gene (coding for isopropylmalate dehydrogenase) from T. thermophilus was integrated in the mesophile chromosome. Since the enzymatic activity of the thermophile enzyme is much lower than that of the E. coli counterpart at the mesophilic temperature, the growth of the transformant was poor at first. However prolonged incubation at the mesophilic temperature produced mutants which are able to grow rapidly. The leuB gene and the dehydrogenase were isolated from the mutants and their chemical structures were analyzed. Isopropylmalate dehydrogenases from the mutants are more active at 40ーC than that of the wild type enzyme from the thermophile. Some of the adapted enzymes were as stable as that of the thermophile enzyme; that is, the stability was not changed by the adaptation to the mesophilic temperature. The present study suggests that it is not necessary to trade the stability to heat with the efficient activity at the mesophilic temperature, and that it is possible to create an enzyme with unusual resistance to heat and high catalytic activity at the ambient temperature.
