| Research Abstract |
An efficient system for obtaining a large amount of a thermostable farnesyl diphosphate (FPP) synthase mutant, in which a site-directed mutation was introduced, was established. This system includes 1.Introduction of a site-directed mutagenesis, 2.Overprotection of the mutated enzyme in Escherichiacoli, 3.Purification by heat treatment followed by two kinds of chromatographies.
The mutants, R295V,D296G,and H297L,which have replacements of Arg-295 with Val, Asp-296 with Gly, and His-297 with Leu, respectively, showed similar catalytic activities to that of the wild-type enzyme. However, their Km values for isopentenyl diphosphate (IPP) are approximately twice that of the wild-type. C289F,which has a replacement of Cys-289 with Phe showed a Km value for IPP 10 times larger than that of the wild-type. The mutant enzymes D224A,D224E,D225I and D228A,in which one of the Asp residues of DDXXD motif in region VI had been replaced, showed much lower activities than that of the wild-type.
Detailed examination of the substrate specificities of the FPP synthase mutants described above will be carried out in order to find new enzymes that catalyze new C-C bond forming reactions that cannot be catalyzed the wild-type enzyme.
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