Isoprenoids, which include over 23,000 known metabolites, are the most chemically diverse family of naturally occurring compounds. The essential and major biosynthetic step in all isoprenoid metabolism is the elongation of isoprene units by prenyltransferases ( Fig. 1) (1). These enzymes, which consecutively mediate alkylation of isopentenyl diphosphate (IPP, 1 by allylic diphosphates, are classified according to the chain length of the final product and the stereochemistry of double bond formed by the condensations. So far, a number of prenyltransferases have been determined from various organisms.For example, farnesyl diphosphate (FPP) synthase (EC 2.5.1.1) catalyzes the sequential condensations of two molecules of IPP (C-5) with dimethylallyl diphosphate (DMAPP, C-5) to give a C-15 compound with E-stereochemistry. The product, FPP, occupies a central point leading to several branches of the pathway for the synthesis of important classes of compounds, including sterols, farnesylated proteins, hemes, respiratory quinones, sesquiterpenes, and dolichols. On the other hand, geranylgeranyl diphosphate (GGPP, C-20) synthase (EC 2.5.1.29) catalyzes the condensation of IPP to give (all-E)-GGPP, which plays as a precursor for carotenoids, chlorophylls, geranylgeranylated proteins, and archaebacterial membrane lipids.These prenyltransferases catalyze the same sort of condensation and have a similarity in amino acid sequences (2, 3). However, every enzyme does not catalyze a further condensation of IPP than the general ultimate product. Until now it has been left in question how the consecutive condensations precisely stop at a destined step.Recently, our group succeeded in converting FPP synthase from Bacillus stearothermophilus to GGPP synthase using chemical random mutagenesis followed by an in vivo color selection (4). From the analysis of the mutations in the FPP synthases whose product specificities had become the same as GGPP synthase, we defined three amino acids that could determine the final chain length; leucine at position 34, tyrosine at position 81, and valine at position 157. In particular, the mutated enzyme that has a substitution of histidine for tyrosine at position 81, which is situated at the fifth amino acid before the first aspartate rich consensus motif, the most effectively produces GGPP. Moreover, our group also showed that, in the case of Sulfolobus acidocaldarius GGPP synthase, the amino acid at the same position also determines the chain length of the product, GGPP (5). Thus, in this paper, we precisely analyze the role of the amino acid at position 81 of B. stearothermophilus FPP synthase on chain length determination.

Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to produce prenyl diphosphates whose chain lengths are absolutely determined by each enzyme. In order to investigate the mechanisms of the consecutive reaction and of the determination of ultimate chain length, a random mutational approach was planned. The farnesyl diphosphate (FPP) synthase gene of Bacillus stearothermophilus was subjected to random mutagenesis by NaNO2 treatment to construct libraries of mutated FPP synthase genes on a high-copy plasmid. From the libraries, the mutants that showed the activity of geranylgeranyl diphosphate (GGPP) synthase were selected by the red-white screening method (Ohnuma, S.-i., Suzuki, M., and Nishino, T. (1994) J. Biol. Chem. 268, 14792-14797), which utilized carotenoid synthetic genes, phytoene synthase, and phytoene desaturase, to visualize the formation of GGPP in vivo. Eleven red positive clones were identified from about 24,300 mutants, and four (mutant 1, 2, 3, and 4) of them were analyzed for the enzyme activities. Results of in vitro assays demonstrated that all these mutants produced (all-E)-GGPP although the amounts were different. Each mutant was found to contain a few amino acid substitutions: mutant 1, Y81H and L275S; mutant 2, L34V and R59Q; mutant 3, V157A and H182Y; mutant 4, Y81H, P239R, and A265T. Site-directed mutagenesis showed that Y81H, L34V, or V157A was essential for the expression of the activity of GGPP synthase. Especially, the replacement of tyrosine 81 by histidine is the most effective because the production ratios of GGPP to FPP in mutant 1 and 4 are the largest. Based on prediction of the secondary structure, it is revealed that the tyrosine 81 situates on a point 11 approximately 12 A apart from the first DDXXD motif, whose distance is similar to the length of hydrocarbon moiety of FPP. These data might suggest that the aromatic ring of tyrosine 81 blocks the chain elongation longer than FPP. Comparisons of kinetic parameters of the mutated and wild type enzymes revealed several phenomena that may relate with the change of the ultimate chain length. They are a decrease of the total reaction rate, increase of Kmfor dimethylallyl diphosphate, decrease of Vmax for dimethylallyl diphosphate, and allylic substrate dependence of Km for IPP.

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The effect of gamma-amino butyric acid (GABA)-enriched soybean on blood pressure was investigated in male spontaneously hypertensive rats. Ten-week-old rats were given diets containing graded levels of GABA-enriched soybean powder for 8 weeks. The systolic blood pressure in rats fed 0.15% GABA diet was significantly lower at 1st week and maintained lower values for 4 weeks as compared with 0% GABA controls. No effect on blood pressure was found in those of 0.03 and 0.3% GABA. The results suggest that there exist appropriate dietary GABA level to get the blood pressure lowering effect.

Radially polarized intense terahertz (THz) radiation behind a thin foil irradiated by ultrahigh-contrast ultrashort relativistic laser pulse is recorded by a single-shot THz time-domain spectroscopy system. As the thickness of the target is reduced from 30 to 2 µm, the duration of the THz emission increases from 5 to over 20 ps and the radiation energy increases dramatically, reaching ∼10.5mJ per pulse, corresponding to a laser-to-THz radiation energy conversion efficiency of 1.7%. The efficient THz emission can be attributed to reflection (deceleration and acceleration) of the laser-driven hot electrons by the target-rear sheath electric field. The experimental results are consistent with that of a simple model as well as particle-in-cell simulation.