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  • br Introduction A great many isoprenoids with varieties of


    Introduction A great many isoprenoids with varieties of structural diversity, such as steroids, carotenoids, prenyl quinones, and natural rubber, occur in nature. All of these biosynthetic precursors are constructed by the action of prenyl chain elongating enzymes [1], [2], [3], [4]. These enzymes can be divided roughly into two major families, E- and Z-prenyl chain elongating enzyme, each of which are further classified into short-chain-, medium-chain-, and long-chain prenyl diphosphate synthases as shown in Scheme 1[3], [4]. Geranyl diphosphate-, farnesyl diphosphate- and geranylgeranyl diphosphate synthases are included in groups of E-short prenyl chain elongating enzymes (-I) [5], [6], [7], [8]. Hexaprenyl diphosphate- and heptaprenyl diphosphate synthases are in E-medium prenyl chain elongating enzymes (-II) [9], [10], [11], [12]. Moreover, E-long prenyl chain elongating enzymes (-III) include octaprenyl diphosphate- and solanesyl diphosphate synthases [13], [14]. Among the Z-prenyl chain elongating enzymes (Z)-farnesyl diphosphate synthase is in the Z-short prenyl chain HC-030031 enzyme (-I) [15], [16]. Decaprenyl diphosphate- and undecaprenyl diphosphate synthases are included in a group of Z-medium prenyl chain elongating enzymes (-II) [17], [18], [19]. Furthermore, dehydrodolichyl diphosphate- and natural rubber synthases are included in the Z-long prenyl chain elongating enzymes (-III) [20], [21], [22]. (E)-Farnesyl diphosphate synthase (FPP synthase) [EC], which has been widely studied for many years, catalyzes the stereospecific condensation of isopentenyl diphosphate (IPP, 1) with dimethylallyl diphosphate (DMAPP, 2) via geranyl diphosphate (GPP, 3) to give (all-E)-farnesyl diphosphate (FPP, 4) as the final product as shown in Scheme 2. Tarshis et al. have reported the detailed research on recombinant avian FPS [23], [24]. They have reported that the benzyl groups of F112 and F113 in the avian enzyme have played the especially important role for chain length determination. Furthermore, they reported that longer-chain prenyl diphosphates were afforded by mutated enzyme which replaced the phenylalanine by alanine and/or serine. On the other hand, we considered that tyrosin-81 of Bacillus stearothermophilus FPS is equivalent to this portion (F112, F113) of avian enzyme. As shown in Scheme 3, we constructed two kinds of mutated type FPP synthases of B. stearothermophilus in which tyrosine-81 was substituted with aspartate (Y81D), and with serine (Y81S), and recently reported the substrate specificities of wild and the mutated farnesyl diphosphate synthases with GPP homologs [25].
    Results and discussion To investigate the reactivities of MOMODMAPP and propoxyGPP, we examined substrate specificities of wild and mutated FPP synthases of B. stearothermophilus.
    Conclusion Allylic substrate homologs such as MOMODMAPP and propoxyGPP were examined for the reactivity as artificial substrates for farnesyl diphosphate synthase as illustrated in Scheme 4. The wild type FPP synthase reaction of 2a with 1 gave 4a as a single condensation product. On the other hand, the reaction with the mutated FPP synthase (Y81D or Y81S) gave 4b as a double condensation product. However, according to the optimized calculation of MOMOFOH, the molecular length of the optimized structure of 4b–OH resembles to that of GGOH (Scheme 5). At the FPP synthase reaction of 3a and 1, when the wild type enzyme was used, 5a was obtained, and when the mutated enzyme Y81D was used, 5a and 5b were the products.
    Acknowledgments We thank Dr. H. Hemmi and Dr. T. Nakayama of Tohoku University for providing the E. coli DH5α expression vectors for the FPP synthase mutants. This work was supported in part by the Feasibility Study for Science and Technology Incubation Program in Advanced Regions (to M.N.) from the Japan Science and Technology Agency (JST).