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 with allylic diphosphates to produce prenyl diphosphates whose chain lengths are absolutely determined by each enzyme. To investigate the mechanism of the consecutive reaction and the determination of the ultimate chain length, a random mutational approach was planned. A geranylgeranyl-diphosphate synthase gene from Sulfolobus acidocaldarius was randomly mutagenized by NaNO 2 treatment to construct a library of mutated geranylgeranyl-diphosphate synthase genes on a yeast expression vector. The library was screened for suppression of a pet phenotype of yeast C296-LH 3 , which is deficient in hexaprenyl-diphosphate synthase. Five mutants that could grow on a YEPG plate, which contained only glycerol as an energy source instead of glucose, were selected from ϳ1,400 mutants. All selected mutated enzymes catalyzed the formation of polyprenyl diphosphates with prenyl chains longer than geranylgeranyl diphosphate. Especially mutants 1, 3, and 5 showed the strongest elongation activity to produce large amounts of geranylfarnesyl diphosphate with a concomitant amount of hexaprenyl diphosphate. Sequence analysis revealed that each mutant contained a few amino acid substitutions and that the mutation of Phe-77, which is located on the fifth amino acid upstream from the first aspartate-rich consensus motif, is the most effective for elongating the ultimate product. Amino acid alignment of known prenyltransferases around this position and our previous observations on farnesyl-diphosphate synthase (Ohnuma, S.-i., Nakazawa, T., Hemmi, H., Hallberg, A.-M., Koyama, T., Ogura, K., and Nishino, T. (1996) J. Biol. Chem. 271, 10087-10095) clearly indicate that the amino acid at the position of all prenyltransferases must regulate the chain elongation.Prenyltransferases, which are essential enzymes in isoprenoid biosynthesis, catalyze the consecutive condensation of isopentenyl diphosphate (IPP) 1 with allylic diphosphates to synthesize linear prenyl diphosphates with various chain lengths. These enzymes are classified according to the products with the longest chain length and the geometry of the double bonds that are formed by the condensations. So far, a number of prenyltransferases have been found in various organisms and characterized (1). For example, farnesyl-diphosphate synthase, which is a key enzyme in the biosynthesis of steroids, prenylquinones, farnesylated proteins, and dolichols, catalyzes the condensations of IPP with dimethylallyl diphosphate (DMAPP; C 5 ) and with geranyl diphosphate (GPP; C 10 ) to give farnesyl diphosphate (FPP; C 15 ) as an ultimate product. Geranylgeranyl-diphosphate (GGPP; C 20 ) synthase, whose product is a precursor of carotenoids, geranylgeranylated proteins, chlorophylls, and ether-linked lipids of archaebacteria, utilizes DMAPP, GPP, and FPP as allylic substrates to give an amphipathic molecule containing four isoprene units, GGPP (Fig.

Coesite occurs in garnets from quartz schists and pelitic schists in the Makbal Complex of Kyrgyzian TienShan. The thick quartz schists interbedded with pelitic schists are the transitional facies after coesite schists. Quartz -pseudomorph after coesite also appears widely in pelitic schists and quartz schists, but is rare in eclogitic rocks. The growth zoning of garnets and Na -Ca amphiboles in eclogites and the garnet -omphacite geothermometry indicate that the metamorphic P -T paths of eclogites are different from each. Ultra -high (UHP) pressure metamorphic rocks of the Makbal Complex are mainly composed of pelitic and quartz schists. All eclogite lenses are included in the host UHPM rocks, although most of them do not contain UHP metamorphic (UHPM) evidences. The K -Ar ages of phengite in a host pelitic schist and of paragonite in an eclogite are approximately 500 Ma, while those of biotite and phengite in a biotite -bearing mica schist and of winchite in a winchite schist are approximately 769 -717 Ma and 881 Ma, respectively. The evidence indicates that this complex is a tectonic mélange. These geological relationships suggest that the ascending substances must be pelitic and quartz schists. The UHPM rocks in the ascending material capture many exotic blocks as xenoliths during intrusion into the upper crust.

To isolate Arabidopsis cDNAs that encode signal transducers and components involved in the regulation of raeiosis, a trans-complementation analysis was performed using a Schizosaccharomyces pombe meiosis-defective mutant in which the genes for pheromone receptors were disabled. One cDNA obtained in this screening encodes a polypeptide, named AML1, that shows significant similarity to S. pombe Mei2 protein and has three putative RNA-recognition motifs like as Mei2. Mei2 is involved in the regulation of meiosis in fission yeast. Northern blot analysis showed that the AML1 gene is expressed in each organ. The possible functions of AML1 are discussed.

Prenyltransferases (prenyl diphosphate synthases), which are a broad group of enzymes that catalyze the consecutive condensation of homoallylic diphosphate of isopentenyl diphosphates (IPP, C5) with allylic diphosphates to synthesize prenyl diphosphates of various chain lengths, have highly conserved regions in their amino acid sequences. Based on the above information, three prenyltransferase homologue genes were cloned from a thermophilic cyanobacterium, Synechococcus elongatus. Through analyses of the reaction products of the enzymes encoded by these genes, it was revealed that one encodes a thermolabile geranylgeranyl (C20) diphosphate synthase, another encodes a farnesyl (C15) diphosphate synthase whose optimal reaction temperature is 60 degrees C, and the third one encodes a prenyltransferase whose optimal reaction temperature is 75 degrees C. The last enzyme could catalyze the synthesis of five prenyl diphosphates of farnesyl, geranylgeranyl, geranylfarnesyl (C25), hexaprenyl (C30), and heptaprenyl (C35) diphosphates from dimethylallyl (C5) diphosphate, geranyl (C10) diphosphate, or farnesyl diphosphate as the allylic substrates. The product specificity of this novel kind of enzyme varied according to the ratio of the allylic and homoallylic substrates. The situations of these three S. elongatus enzymes in a phylogenetic tree of prenyltransferases are discussed in comparison with a mesophilic cyanobacterium of Synechocystis PCC6803, whose complete genome has been reported by Kaneko et al. (1996).