Protein Engineering, Vol. 14, No. 2, 85-91,
February 2001
© 2001 Oxford University Press
Adaptation of a thermophilic enzyme, 3-isopropylmalate dehydrogenase, to low temperatures
1 Department of Life Science, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226-8503, 2 Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Horinouchi 1432–1, Hachioji, Tokyo 192-0392 and 4 Institute of Applied Biochemistry, University of Tsukuba, Tennoudai 1–1–1, Tsukuba 305-8572, Japan
Random mutagenesis coupled with screening of the active enzyme at a low temperature was applied to isolate cold-adapted mutants of a thermophilic enzyme. Four mutant enzymes with enhanced specific activities (up to 4.1-fold at 40°C) at a moderate temperature were isolated from randomly mutated Thermus thermophilus 3-isopropylmalate dehydrogenase. Kinetic analysis revealed two types of cold-adapted mutants, i.e. kcat-improved and Km-improved types. The kcat-improved mutants showed less temperature-dependent catalytic properties, resulting in improvement of kcat (up to 7.5-fold at 40°C) at lower temperatures with increased Km values mainly for NAD. The Km-improved enzyme showed higher affinities toward the substrate and the coenzyme without significant change in kcat at the temperatures investigated (30–70°C). In kcat-improved mutants, replacement of a residue was found near the binding pocket for the adenine portion of NAD. Two of the mutants retained thermal stability indistinguishable from the wild-type enzyme. Extreme thermal stability of the thermophilic enzyme is not necessarily decreased to improve the catalytic function at lower temperatures. The present strategy provides a powerful tool for obtaining active mutant enzymes at lower temperatures. The results also indicate that it is possible to obtain cold-adapted mutant enzymes with high thermal stability.
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