A short prenyl chain elongating enzyme, farnesyl diphosphate (FPP, C_<15>) synthase (FPS) catalyzes the sequential head-to-tail condensation of isopentenyl diphosphate (IPP, C_5) with an allylic primer substrate, dimethylallyl- (DMAPP) or geranyl diphosphate (GPP) to form FPP [1]. The genes encoding for the enzyme FPS have been cloned and sequenced from a wide variety of organisms [2]. The enzymes have several conserved regions in their amino acid sequences, which include two aspartate-rich motifs. It has been reported that the fifth amino acid before the first aspartate-rich motif (FARM) regulates the ultimate prenyl chain length of the product [3,5-7]. We constructed several kind of mutated FPSs of Bacillus stearothermophilus, in which tyrosine (Y)-81 was substituted with arginine (Y81R), with aspartate (Y81D), or with serine (Y81S). In order to study on the substrate specificity of these mutant enzymes with respect to the artificial allylic substrate homologs that have a hydrophilic group at ω-position, we examined the reactivity as allylic substrates of methoxy- or propoxygeranyl diphosphate (or 3-methylpent-2-en-4-ynyl diphosphate) with IPP for wild and mutated FPSs. The enzymatic reaction of methoxy- or propoxyGPP with IPP by use of wild-type B. stearothermophilus FPS gave methoxy- or propoxyFPP. On the other hand, the reaction of methoxy- or propoxyGPP with IPP by use of the mutated FPS, Y81R (Y81D or Y81S) gave methoxy- or propoxygeranylgeraniol as the major product, which could be given only a trace amount when the wild-type enzyme was employed. From a viewpoint of synthetic application of enzymatic reaction, these enzymatic reactions are interesting for the syntheses of biologically active substances.
