Farnesyl diphosphate (FPP) synthase[2.5.1.10] catalyzes the condensation of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate(DMAPP) and with geranyl diphophate (GPP) to produce E,E-FPP as a final product and does not catalyze a condensation beyond FPP. Recently, it was observed that in Bacillus stearothermophilus FPP synthase, a replacement of tyrosine with histidine at 81 position which is located on the fifth amino acid before aspartate-rich motif, caused the mutated FPP synthas to catalyze geranylgeranyl diphosphaye (C_<20>) synthesis. Then we constructed the mutated FPP synthase, in which tyrosine-81 was substituted with glycine and studied the substrate specificity of this enzyme using the substrate analogs (1-28) as depicted in Fig 1. The results showed that the reactivities of the all analogs with the mutant enzyme (Y81G) were higuer than those of the wild enzyme as shown in Table 1 and 2. In the case of analogs having hydrocarbon as side chain (3-9), the reactivities were higher than those of the wild enzyme. The number of IPP-incoporation also was bigger than that of the wild enzyme. These results were same to those of the natural substrates as reported before. It has been reported that the substrate specificity of FPP synthase of Bacillus stearothermophilus is more stringent than that of FPP synthase of pig liver. Namely, the wild enzyme can hardly accept the analogs having oxygen atom in their chain. On the other hand, FPP synthase of pig liver can accept them. The mutant enzyme can also accept these analogs (14-19) as substrate better than the wild enzyme as shown in Table 1. This observation showed that the substrate specificity of the mutant enzyme is changed and becomes similar to that of FPP synthase of pig liver. And this fact suggest that this mutant enzume may be used for organic synthesis better than the wild enzyme because of accepting analogs having hetero atom such as oxygen atom. The analogs (20-21), which have no methyl group at 3-position of DMAPP analog, were tested to show that these analogs can not be accepted by both of the wild and the mutant enzyme. This finding was also found in the reaction of FPP synthase of mammmalian FPP synthase. This fact strongly indicates that the methyl group at 3-position of GPP is very crucial for the substrate recognition of.FPP synthase including the mutant enzyme.
