78(P-4) 遺伝子組換え体、短鎖プレニル二リン酸合成酵素を用いたエポキシアルコールの不斉合成(ポスター発表の部)

Short prenyl chain elongating enzymes, farnesyl diphosphate (FPP. C_<15>) synthase (FPS), and geranylgeranyl diphosphate (GGPP, C_<20>) synthase (GGPS) catalyze the sequential head-to-tail condensation of isopentenyl diphosphate (IPP, C_5) with an allylic primer substrate to form FPP and GGPP, respectively [1]. The genes encoding for these enzymes have been cloned and sequenced [2]. These 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 product [3,6]. We constructed mutated FPS of Bacillus stearothermophilus, in which tyrosine (Y)-81 was substituted with arginine (Y81R), with aspartate (Y81D), and with serine (Y81S), and mutated GGPS of Sulfolobus acidocaldarius, in which phenylalanine (F)-77 was substituted with glycine (F77G) and with serine (F77S), respectively. In order to synthesize some chiral epoxyalcohols, we examined the reaction of (R,S)-epoxygeranyl diphosphate (epoxyGPP) with 3-alkyl group homologs of IPP by use of these wild and mutated enzymes. The enzymatic reaction of racemic epoxyGPP with IPP by use of wild-type B. stearothermophilus FPS gave (S)-epoxyFPP, which was hydrolyzed with phosphatase to epoxyfarnesol (ee 30% of S-enantiomer by HPLC using chiral column). The reaction of epoxyGPP with 3-ethyIIPP gave 3-ethyl-epoxyGOH (yield: 142.6%). The reaction of epoxyGPP with IPP using a mutated FPS Y81R (or Y81D) gave epoxygeranylgeraniol, which could be given only a trace amount by use of the wild-type enzyme. From a viewpoint of synthetic application of enzymatic reaction, these reactions are interesting for the syntheses of biologically active substances such as pheromones and hormones.