Isoprenoids comprise a family of more than 55000 natural products with great structural variety derived from five-carbon isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). Allylic diphosphates such as farnesyl diphosphate (FPP) synthesized from DMAPP and IPP serve as outlet points for a great variety of products. A group of prenyltransferases catalyzing chain elongation of FPP to designated lengths by consecutive condensation reactions with specific numbers of IPP are classified as cis and trans types according to the stereochemistry of the double bonds formed by IPP condensation. The complete kinetics of the multistep IPP condensation reactions by both types of enzymes has been determined using steady-state and pre-steady-state approaches. Because their crystal structures were determined in conjunction with biochemical studies, a more thorough understanding of their catalytic mechanisms, protein conformational changes, and product chain-length determination mechanisms has been gained recently. Since these prenyltransferases play important roles, potent inhibitors have been identified and their cocrystal structures have been determined for drug development. In this review, the current knowledge of these prenyltransferases that synthesize prenyl oligomers or polymers is summarized.

The polyisoprenoid alcohols (dolichols and polyprenols) are found in all living organism, from bacteria to mammals. In animal and yeast cells polyisoprenoids are derived from the cytoplasmic mevalonate (MVA) pathway while in plants two biosynthetic pathways, the MVA and the plastidial methylerythritol phosphate (MEP) pathway provide precursors for polyisoprenoid biosynthesis. The key enzymes of polyisoprenoid synthesis are cis-prenyltransferases (CTPs), responsible for construction of the long hydrocarbon skeleton. CPTs elongate a short all-trans precursor, oligoprenyl diphosphate, by sequential addition of the desired number of isopentenyl diphosphate molecules which results in formation of a stretch of cis units. Several genes encoding CPT have been cloned from bacteria, plants and mammals. Interestingly, in Arabidopsis, the tissue-specific expression of ten putative cis-prenyltransferases was observed. In contrast to polyisoprenoid phosphates serving as cofactors in the biosynthesis of glycoproteins, glucosyl phosphatidyl inositol (GPI) anchor or bacterial peptidoglycan, the biological importance of polyprenols and dolichols still remains a question of debate besides their function of reservoir of substrates for kinase. These extremely hydrophobic superlipids are postulated to be involved in intracellular traffic of proteins and in cellular defense against adverse environmental conditions. Recent publications show a direct link between the dolichol biosynthetic pathway and congenital disorders of glycosylation (CDG). These discoveries highlighting the cellular significance of polyisoprenoids simultaneously establish the background for future pharmacological interventions. Our mini-review summarizes the results of recent studies on polyisoprenoids.

Plants accumulate a family of hydrophobic polymers known as polyprenols, yet how they are synthesized, where they reside in the cell, and what role they serve is largely unknown. Using Arabidopsis thaliana as a model, we present evidence for the involvement of a plastidial cis-prenyltransferase (AtCPT7) in polyprenol synthesis. Gene inactivation and RNAi-mediated knockdown of AtCPT7 eliminated leaf polyprenols, while its overexpression increased their content. Complementation tests in the polyprenol-deficient yeast rer2 mutant and enzyme assays with recombinant AtCPT7 confirmed that the enzyme synthesizes polyprenols of approximately 55 carbons in length using geranylgeranyl diphosphate (GGPP) and isopentenyl diphosphate as substrates. Immunodetection and in vivo localization of AtCPT7 fluorescent protein fusions showed that AtCPT7 resides in the stroma of mesophyll chloroplasts. The enzymatic products of AtCPT7 accumulate in thylakoid membranes, and in their absence, thylakoids adopt an increasingly

Isoprenoids form an extensive group of natural products involved in a number of important biological processes. Their biosynthesis proceeds through sequential 1'-4 condensations of isopentenyl diphosphate (C5) with an allylic acceptor, the first of which is dimethylallyl diphosphate (C5). The reactions leading to the production of geranyl diphosphate (C10), farnesyl diphosphate (C15) and geranylgeranyl diphosphate (C20), which are the precursors of mono-, sesqui- and diterpenes, respectively, are catalyzed by a group of highly conserved enzymes known as short-chain isoprenyl diphosphate synthases, or prenyltransferases. In recent years, the sequences of many new prenyltransferases have become available, including those of several plant and animal geranyl diphosphate synthases, revealing novel mechanisms of product chain-length selectivity and an intricate evolutionary path from a putative common ancestor. Finally, there is considerable interest in designing inhibitors specific to short-chain prenyltransferases, for the purpose of developing new drugs or pesticides that target the isoprenoid biosynthetic pathway.

NphB is a soluble prenyltransferase from Streptomyces sp. strain CL190 that attaches a geranyl group to a 1,3,6,8-tetrahydroxynaphthalene-derived polyketide during the biosynthesis of anti-oxidant naphterpin. Here we report multiple chemoenzymatic syntheses of various prenylated compounds from aromatic substrates including flavonoids using two prenyltransferases NphB and SCO7190, a NphB homolog from Streptomyces coelicolor A3(2), as biocatalysts. NphB catalyzes carbon-carbon-based and carbon-oxygen-based geranylation of a diverse collection of hydroxyl-containing aromatic acceptors. Thus, this simple method using the prenyltransferases can be used to explore novel prenylated aromatic compounds with biological activities. Kinetic studies with NphB reveal that the prenylation reaction follows a sequential ordered mechanism.

Terpene synthases are responsible for synthesis of a large number of terpenes in plants using substrates provided by two distinct metabolic pathways, the mevalonate-dependent pathway that is located in cytosol and has been suggested to be responsible for synthesis of sesquiterpenes (C15), and 2-C-methyl-D-erythritol-4-phosphate pathway located in plastids and suggested to be responsible for the synthesis of hemi- (C5), mono- (C10), and diterpenes (C20). Recent advances in characterization of genes and enzymes responsible for substrate and end product biosynthesis as well as efforts in metabolic engineering have demonstrated existence of a number of multi-substrate terpene synthases. This review summarizes the progress in the characterization of such multi-substrate terpene synthases and suggests that the presence of multi-substrate use might have been significantly underestimated. Multi-substrate use could lead to important changes in terpene product profiles upon substrate profile changes under perturbation of metabolism in stressed plants as well as under certain developmental stages. We therefore argue that multi-substrate use can be significant under physiological conditions and can result in complicate modifications in terpene profiles.

Terpenes are the largest class of small-molecule natural products on earth, and the most abundant by mass. Here, we summarize recent developments in elucidating the structure and function of the proteins involved in their biosynthesis. There are six main building blocks or modules (alpha, beta, gamma, delta, epsilon, and zeta) that make up the structures of these enzymes: the alphaalpha and alphadelta head-to-tail trans-prenyl transferases that produce trans-isoprenoid diphosphates from C(5) precursors; the epsilon head-to-head prenyl transferases that convert these diphosphates into the tri- and tetraterpene precursors of sterols, hopanoids, and carotenoids; the betagamma di- and triterpene synthases; the zeta head-to-tail cis-prenyl transferases that produce the cis-isoprenoid diphosphates involved in bacterial cell wall biosynthesis; and finally the alpha, alphabeta, and alphabetagamma terpene synthases that produce plant terpenes, with many of these modular enzymes having originated from ancestral alpha and beta domain proteins. We also review progress in determining the structure and function of the two 4Fe-4S reductases involved in formation of the C(5) diphosphates in many bacteria, where again, highly modular structures are found.

The isoprenoid biosynthetic pathway provides intermediates for the synthesis of a multitude of natural products which serve numerous biochemical functions in plants: sterols (isoprenoids with a C30 backbone) are essential components of membranes; carotenoids (C40) and chlorophylls (which contain a C20 isoprenoid side-chain) act as photosynthetic pigments; plastoquinone, phylloquinone and ubiquinone (all of which contain long isoprenoid side-chains) participate in electron transport chains; gibberellins (C20), brassinosteroids (C30) and abscisic acid (C15) are phytohormones derived from isoprenoid intermediates; prenylation of proteins (with C15 or C20 isoprenoid moieties) may mediate subcellular targeting and regulation of activity; and several monoterpenes (C10), sesquiterpenes (C15) and diterpenes (C20) have been demonstrated to be involved in plant defense. Here we present a comprehensive analysis of genes coding for enzymes involved in the metabolism of isoprenoid-derived compounds in Arabidopsis thaliana. By combining homology and sequence motif searches with knowledge regarding the phylogenetic distribution of pathways of isoprenoid metabolism across species, candidate genes for these pathways in A. thaliana were obtained. A detailed analysis of the vicinity of chromosome loci for genes of isoprenoid metabolism in A. thaliana provided evidence for the clustering of genes involved in common pathways. Multiple sequence alignments were used to estimate the number of genes in gene families and sequence relationship trees were utilized to classify their individual members. The integration of all these datasets allows the generation of a knowledge-based metabolic map of isoprenoid metabolic pathways in A. thaliana and provides a substantial improvement of the currently available gene annotation.

Some plant terpenes such as sterols and carotenes are part of primary metabolism and found essentially in all plants. However, the majority of the terpenes found in plants are classified as 'secondary' compounds, those chemicals whose synthesis has evolved in plants as a result of selection for increased fitness via better adaptation to the local ecological niche of each species. Thousands of such terpenes have been found in the plant kingdom, but each species is capable of synthesizing only a small fraction of this total. In plants, a family of terpene synthases (TPSs) is responsible for the synthesis of the various terpene molecules from two isomeric 5-carbon precursor 'building blocks', leading to 5-carbon isoprene, 10-carbon monoterpenes, 15-carbon sesquiterpenes and 20-carbon diterpenes. The bryophyte Physcomitrella patens has a single TPS gene, copalyl synthase/kaurene synthase (CPS/KS), encoding a bifunctional enzyme producing ent-kaurene, which is a precursor of gibberellins. The genome of the lycophyte Selaginella moellendorffii contains 18 TPS genes, and the genomes of some model angiosperms and gymnosperms contain 40-152 TPS genes, not all of them functional and most of the functional ones having lost activity in either the CPS- or KS-type domains. TPS genes are generally divided into seven clades, with some plant lineages having a majority of their TPS genes in one or two clades, indicating lineage-specific expansion of specific types of genes. Evolutionary plasticity is evident in the TPS family, with closely related enzymes differing in their product profiles, subcellular localization, or the in planta substrates they use.

Isoprenyl diphosphate synthases catalyze addition of allylic diphosphate primers to the isoprene unit in isopentenyl diphosphate to produce polyisoprenoid diphosphates with well defined chain lengths. Phylogenetic correlations suggest that the synthases which catalyze formation of isoprenoid diphosphates with (E) double bonds have evolved from a common ancestor. X-ray crystallographic studies of farnesyl diphosphate synthase in conjunction with site-directed mutagenesis have provided important new information about the residues involved in binding and catalysis and the source of chain length selectivity for the enzymes that catalyze chain elongation.

This work suggests that, in Catharanthus roseus, the inactive small subunit of heteromeric GPP synthase (GPPS) interacts with bifunctional G(G)PPS and redirects the metabolic flux, thus acting as primary regulator of monoterpene indole alkaloid biosynthesis, whereas homomeric GPPS could be involved in ubiquinone formation.Catharanthus roseus is the sole source of two most important monoterpene indole alkaloid (MIA) anti-cancer agents: vinblastine and vincristine. MIAs possess a terpene and an indole moiety derived from terpenoid and shikimate pathways, respectively. Geranyl diphosphate (GPP), the entry point to the formation of terpene moiety, is a product of the condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) by GPP synthase (GPPS). Here, we report three genes encoding proteins with sequence similarity to large subunit (CrGPPS.LSU) and small subunit (CrGPPS.SSU) of heteromeric GPPSs, and a homomeric GPPSs. CrGPPS.LSU is a bifunctional enzyme producing both GPP and geranyl geranyl diphosphate (GGPP), CrGPPS.SSU is inactive, whereas CrGPPS is a homomeric enzyme forming GPP. Co-expression of both subunits in Escherichia coli resulted in heteromeric enzyme with enhanced activity producing only GPP. While CrGPPS.LSU and CrGPPS showed higher expression in older and younger leaves, respectively, CrGPPS.SSU showed an increasing trend and decreased gradually. Methyl jasmonate (MeJA) treatment of leaves significantly induced the expression of only CrGPPS.SSU. GFP localization indicated that CrGPPS.SSU is plastidial whereas CrGPPS is mitochondrial. Transient overexpression of AmGPPS.SSU in C. roseus leaves resulted in increased vindoline, immediate monomeric precursor of vinblastine and vincristine. Although C. roseus has both heteromeric and homomeric GPPS enzymes, our results implicate the involvement of only heteromeric GPPS with CrGPPS.SSU regulating the GPP flux for MIA biosynthesis.

Myrcene, which accounts for 30-50% of the essential oil in hop (Humulus lupulus L.) trichomes, derives from geranyl diphosphate (GPP), the common precursor of monoterpenes. Full-length sequences of heterodimeric GPP synthase small subunit (GPPS.SSU, belonging to the SSU I subfamily) and large subunit (LSU) cDNAs were mined from a hop trichome cDNA library. The SSU was inactive, whereas the LSU produced GPP, farnesyl diphosphate, and geranylgeranyl diphosphate (GGPP) from dimethylallyl diphosphate and isopentenyl diphosphate in vitro. Coexpression of both subunits in Escherichia coli yielded a heterodimeric enzyme exhibiting altered ratios of GPP and GGPP synthase activities and greatly enhanced catalytic efficiency. Transcript analysis suggested that the heterodimeric geranyl(geranyl)diphosphate synthase [G(G)PPS] is involved in myrcene biosynthesis in hop trichomes. The critical role of the conserved CxxxC motif (where

Geranyl diphosphate (GPP), the universal precursor of monoterpenes, is formed from isopentenyl diphosphate and dimethylallyl diphosphate by the action of geranyl diphosphate synthase, one of the key branchpoint enzymes of terpene biosynthesis. Three types of GPP synthase can be distinguished in plants based on sequence similarity and subunit architecture, but until now individual species have been reported to contain only one of these types. Here we show that the conifer, Norway spruce (Picea abies), contains two different types of GPP synthase belonging to two separate groups of homodimeric proteins. One enzyme, designated PaIDS2 (P. abies isoprenyl diphosphate synthase 2), has high sequence similarity to other gymnosperm GPP synthases. It produces solely GPP in in vitro assays after expression in Escherichia coli and likely participates in monoterpene biosynthesis accompanying induced oleoresin formation, based on dramatic increases in transcript level after methyl jasmonate application. The other enzyme, designated PaIDS3, has highest similarity to the previously reported Arabidopsis thaliana GPP synthase and several other angiosperm sequences, and is not associated with induced oleoresin formation in Norway spruce. In vitro assay of this protein and one encoded by a similar gene sequence from Quercus robur gave substantial amounts of the larger prenyl diphosphates, FPP and GGPP, in addition to GPP. Hence these proteins may not be involved in monoterpene formation and could conceivably form products in addition to GPP in planta.

The precursor of all monoterpenes is the C10 acyclic intermediate geranyl diphosphate (GPP), which is formed from the C5 compounds isopentenyl diphosphate and dimethylallyl diphosphate by GPP synthase (GPPS). We have discovered that Antirrhinum majus (snapdragon) and Clarkia breweri, two species whose floral scent is rich in monoterpenes, both possess a heterodimeric GPPS like that previously reported from Mentha piperita (peppermint). The A. majus and C. breweri cDNAs encode proteins with 53% and 45% amino acid sequence identity, respectively, to the M. piperita GPPS small subunit (GPPS.SSU). Expression of these cDNAs in Escherichia coli yielded no detectable prenyltransferase activity. However, when each of these cDNAs was coexpressed with the M. piperita GPPS large subunit (GPPS.LSU), which shares functional motifs and a high level of amino acid sequence identity with geranylgeranyl diphosphate synthases (GGPPS), active GPPS was obtained. Using a homology-based cloning strategy, a GPPS.LSU cDNA also was isolated from A. majus. Its coexpression in E. coli with A. majus GPPS.SSU yielded a functional heterodimer that catalyzed the synthesis of GPP as a main product. The expression in E. coli of A. majus GPPS.LSU by itself yielded active GGPPS, indicating that in contrast with M. piperita GPPS.LSU, A. majus GPPS.LSU is a functional GGPPS on its own. Analyses of tissue-specific, developmental, and rhythmic changes in the mRNA and protein levels of GPPS.SSU in A. majus flowers revealed that these levels correlate closely with monoterpene emission, whereas GPPS.LSU mRNA levels did not, indicating that the levels of GPPS.SSU, but not GPPS.LSU, might play a key role in regulating the formation of GPPS and, thus, monoterpene biosynthesis.

Geranyl diphosphate (GPP) is the direct precursor of all monoterpenoids and is the prenyl source of many meroterpenoids, such as geranylated coumarins. GPP synthase (GPPS) localized in plastids is responsible for providing the substrate for monoterpene synthases and prenyltransferases for synthesis of aromatic substances that are also present in plastids, but GPPS activity in Lithospermum erythrorhizon localizes to the cytosol, in which GPP is utilized for the biosynthesis of naphthoquinone pigments, which are shikonin derivatives. This study describes the identification of the cytosol-localized GPPS gene, LeGPPS, through EST- and homology-based approaches followed by functional analyses. The deduced amino acid sequence of the unique LeGPPS showed greater similarity to that of farnesyl diphosphate synthase (FPPS), which generally localizes to the cytosol, than to plastid-localized conventional GPPS. Biochemical characterization revealed that recombinant LeGPPS predominantly produces GPP along with a trace amount of FPP. LeGPPS expression was mainly detected in root bark, in which shikonin derivatives are produced, and in shikonin-producing cultured cells. The GFP fusion protein in onion (Allium cepa) cells localized to the cytosol. Site-directed mutagenesis of LeGPPS and another FPPS homolog identified in this study, LeFPPS1, showed that the His residue at position 100 of LeGPPS, adjacent to the first Asp-rich motif, contributes to substrate preference and product specificity, leading to GPP formation. These results suggest that LeGPPS, which is involved in shikonin biosynthesis, is recruited from cytosolic FPPS and that point mutation(s) result in the acquisition of GPPS activity.

Catharanthus roseus is the sole source of two of the most important anticancer monoterpene indole alkaloids (MIAs), vinblastine and vincristine and their precursors, vindoline and catharanthine. The MIAs are produced from the condensation of precursors derived from indole and terpene secoiridoid pathways. It has been previously reported that the terpene moiety limits MIA biosynthesis in C. roseus. Here, to overcome this limitation and enhance MIAs levels in C. roseus, bifunctional geranyl(geranyl) diphosphate synthase [G(G)PPS] and geraniol synthase (GES) that provide precursors for early steps of terpene moiety (secologanin) formation, were overexpressed transiently by agroinfiltration and stably by Agrobacterium-mediated transformation. Both transient and stable overexpression of G(G)PPS and co-expression of G(G)PPS+GES significantly enhanced the accumulation of secologanin, which in turn elevated the levels of monomeric MIAs. In addition, transgenic C. roseus plants exhibited increased levels of root alkaloid ajmalicine. The dimeric alkaloid vinblastine was enhanced only in G(G)PPS but not in G(G)PPS+GES transgenic lines that correlated with transcript levels of peroxidase-1 (PRX1) involved in coupling of vindoline and catharanthine into 3',4'-anhydrovinblastine, the immediate precursor of vinblastine. Moreover, first generation (T1) lines exhibited comparable transcript and metabolite levels to that of T0 lines. In addition, transgenic lines displayed normal growth similar to wild-type plants indicating that the bifunctional G(G)PPS enhanced flux toward both primary and secondary metabolism. These results revealed that improved availability of early precursors for terpene moiety biosynthesis enhanced production of MIAs in C. roseus at the whole plant level. This is the first report demonstrating enhanced accumulation of monomeric and dimeric MIAs including root MIA ajmalicine in C. roseus through transgenic approaches.

We collected and completely sequenced 28,469 full-length complementary DNA clones from Oryza sativa L. ssp. japonica cv. Nipponbare. Through homology searches of publicly available sequence data, we assigned tentative protein functions to 21,596 clones (75.86%). Mapping of the cDNA clones to genomic DNA revealed that there are 19,000 to 20,500 transcription units in the rice genome. Protein informatics analysis against the InterPro database revealed the existence of proteins presented in rice but not in Arabidopsis. Sixty-four percent of our cDNAs are homologous to Arabidopsis proteins.

Sorghum, an African grass related to sugar cane and maize, is grown for food, feed, fibre and fuel. We present an initial analysis of the approximately 730-megabase Sorghum bicolor (L.) Moench genome, placing approximately 98% of genes in their chromosomal context using whole-genome shotgun sequence validated by genetic, physical and syntenic information. Genetic recombination is largely confined to about one-third of the sorghum genome with gene order and density similar to those of rice. Retrotransposon accumulation in recombinationally recalcitrant heterochromatin explains the approximately 75% larger genome size of sorghum compared with rice. Although gene and repetitive DNA distributions have been preserved since palaeopolyploidization approximately 70 million years ago, most duplicated gene sets lost one member before the sorghum-rice divergence. Concerted evolution makes one duplicated chromosomal segment appear to be only a few million years old. About 24% of genes are grass-specific and 7% are sorghum-specific. Recent gene and microRNA duplications may contribute to sorghum's drought tolerance.

Farnesyl diphosphate (FPP) synthase (FPS) catalyses the synthesis of FPP, the major substrate used by cytosolic and mitochondrial branches of the isoprenoid pathway. Arabidopsis contains two farnesyl diphosphate synthase genes, FPS1 and FPS2, that encode isozymes FPS1L (mitochondrial), FPS1S and FPS2 (both cytosolic). Here we show that simultaneous knockout of both FPS genes is lethal for Arabidopsis, and embryo development is arrested at the pre-globular stage, demonstrating that FPP-derived isoprenoid metabolism is essential. In addition, lack of FPS enzyme activity severely impairs male genetic transmission. In contrast, no major developmental and metabolic defects were observed in fps1 and fps2 single knockout mutants, demonstrating the redundancy of the genes. The levels of sterols and ubiquinone, the major mitochondrial isoprenoid, are only slightly reduced in the single mutants. Although one functional FPS gene is sufficient to support isoprenoid biosynthesis for normal growth and development, the functions of FPS1 and FPS2 during development are not completely redundant. FPS1 activity has a predominant role during most of the plant life cycle, and FPS2 appears to have a major role in seeds and during the early stages of seedling development. Lack of FPS2 activity in seeds, but not of FPS1 activity, is associated with a marked reduction in sitosterol content and positive feedback regulation of 3-hydroxy-3-methylglutaryl CoA reductase activity that renders seeds hypersensitive to the 3-hydroxy-3-methylglutaryl CoA reductase inhibitor mevastatin.

We have cloned a new geranylgeranyl pyrophosphate (GGPP) synthase gene, designated GGPS6, from Arabidopsis thaliana genomic DNA. Nucleotide sequence analysis revealed that the GGPS6 gene contains an open reading frame coding for a protein of 343 amino acid residues with a calculated molecular mass of 37,507 Da. Also, the gene is not interrupted by an intron. The predicted amino acid sequence of the GGPS6 gene shows significant homology (34.0-57.7%) with other GGPP synthases from Arabidopsis. The GGPS6 protein contains a N-terminal signal peptide which is thought to function as an organelle targeting sequence. In fact, the GGPS6-GFP fusion protein was found to be localized exclusively in mitochondria when expressed in tobacco BY-2 cells. In vitro analysis of the enzyme activity as well as genetic complementation analysis with Erwinia uredovora crt gene cluster expressed in Escherichia coli showed that the GGPS6 gene most certainly encodes a GGPP synthase catalyzing the conversion of farnesyl pyrophosphate to GGPP.

Tripterygium wilfordii is known to contain various types of bioactive diterpenoids that exhibit many remarkable activities. Many studies have recently been targeted toward the elucidation of the diterpenoids biosynthetic pathways in attempts to obtain these compounds with a view to solving the dilemma of low yield in plants. However, the short-chain prenyltransferases (SC-PTSs) responsible for the formation of geranylgeranyl diphosphate (GGPP), a crucial precursor for synthesizing the skeleton structures of diterpenoids, have not been characterized in depth. Here, T. wilfordii transcriptome data were used to identify eight putative GGPPSs, including two small subunits of geranyl diphosphate synthase (GPPS.SSU). Of them, GGPPS1, GGPPS7, GGPPS8, GPPS.SSU II and GPPS.SSU were translocated mainly into chloroplasts, and GGPPS8 exhibited the optimal catalytic efficiency with respect to catalyzing the formation of GGPP. In addition, the expression pattern of GGPPS8 was similar to that of downstream terpene synthase genes that are directly correlated with triptolide production in roots, indicating that GGPPS8 was most likely to participate in triptolide biosynthesis in roots among the studied enzymes. GPPS.SSU was inactive alone but interacted with GGPPS1, GGPPS7 and GGPPS8 to change the product from GGPP to GPP. These findings implicate that these candidate genes can be regulated to shift the metabolic flux toward diterpenoid formation, increasing the yields of bioactive diterpenoids in plants.

A novel prenyltransferase, farnesylgeranyl diphosphate (FGPP) synthase (EC 2.5.1.X), which synthesizes C25-prenyl diphosphate, was found in the haloalkaliphilic archaeon Natronobacterium pharaonis. It was separated from geranylgeranyl diphosphate (GGPP) synthase (EC 2.5.1.29), which synthesizes C20-prenyl diphosphate, a major prenyltransferase in this organism. The highest activity of FGPP synthase was observed when GGPP was used as the allylic substrate. FGPP synthase may synthesize a precursor for the C25 moiety of C20, C25 diether lipids using a longer allylic diphosphate, such as GGPP synthesized by GGPP synthase, rather than dimethylallyl diphosphate, which is the product of isopentenyl diphosphate isomerase.

Prenyltransferases catalyse sequential condensations of isopentenyl diphosphate with allylic diphosphates. Previously, we reported the presence of farnesylgeranyl diphosphate (FGPP) synthase activity synthesizing C25 isoprenyl diphosphate in Natronobacterium pharaonis which is a haloalkaliphilic archaeon having C20-C25 diether lipids in addition to C20-C20 diether lipids commonly occurring in archaea [Tachibana, A. (1994) FEBS Lett. 341, 291-294]. Recently, it was found that a newly isolated aerobic hyperthermophilic archaeon, Aeropyrum pernix, had only C25-C25 diether lipids, not the usual C20-containing lipids [Morii, H., Yagi, H., Akutsu, H., Nomura, N., Sako, Y. & Koga, Y. (1999) Biochim. Biophys. Acta 1436, 426-436]. In this report, we describe the isoloation from A. pernix of the novel prenyltransferase gene, fgs, encoding FGPP synthase. The protein encoded by fgs was expressed in Escherichia coli as a glutathione S-transferase fusion protein and produced FGPP as a final product. Phylogenetic analysis of fgs with other prenyltransferases revealed that the short-chain prenyltransferase family is divided into three subfamilies: bacterial subfamily I, eukaryotic subfamily II, and archaeal subfamily III. fgs is clearly contained within the archaeal geranylgeranyl diphosphate (GGPP) synthase group (subfamily III), suggesting that FGPP synthase evolved from an archaeal GGPP synthase with an alteration in product specificity.

The gene of (all-E) geranylfarnesyl diphosphate synthase that is responsible for the biosynthesis of methanophenazine, an electron carrier utilized for methanogenesis, was cloned from a methanogenic archaeon Methanosarcina mazei Go1. The properties of the recombinant enzyme and the results of phylogenetic analysis suggest that the enzyme is closely related to (all-E) prenyl diphosphate synthases that are responsible for the biosynthesis of respiratory quinones, rather than to the enzymes involved in the biosynthesis of archaeal membrane lipids, including (all-E) geranylfarnesyl diphosphate synthase from a thermophilic archaeon.

Isoprenyl diphosphate synthases (IDSs) catalyze some of the most basic steps in terpene biosynthesis by producing the prenyl diphosphate precursors of each of the various terpenoid classes. Most plants investigated have distinct enzymes that produce the short-chain all-trans (E) prenyl diphosphates geranyl diphosphate (GDP, C10 ), farnesyl diphosphate (FDP, C15 ) or geranylgeranyl diphosphate (GGDP, C20 ). In the genome of Arabidopsis thaliana, 15 trans-product-forming IDSs are present. Ten of these have recently been shown to produce GGDP by genetic complementation of a carotenoid pathway engineered into Escherichia coli. When verifying the product pattern of IDSs producing GGDP by a new LC-MS/MS procedure, we found that five of these IDSs produce geranylfarnesyl diphosphate (GFDP, C25 ) instead of GGDP as their major product in enzyme assays performed in vitro. Over-expression of one of the GFDP synthases in A. thaliana confirmed the production of GFDP in vivo. Enzyme assays with A. thaliana protein extracts from roots but not other organs showed formation of GFDP. Furthermore, GFDP itself was detected in root extracts. Subcellular localization studies in leaves indicated that four of the GFDP synthases were targeted to the plastoglobules of the chloroplast and one was targeted to the mitochondria. Sequence comparison and mutational studies showed that the size of the R group of the 5th amino acid residue N-terminal to the first aspartate-rich motif is responsible for C25 versus C20 product formation, with smaller R groups (Ala and Ser) resulting in GGDP (C20 ) as a product and a larger R group (Met) resulting in GFDP (C25 ).

Terpenoids are the largest and most diverse class of plant-specialized metabolites, which function in diverse physiological processes during plant development. In the biosynthesis of plant terpenoids, short-chain prenyltransferases (SC-PTs), together with terpene synthases (TPSs), play critical roles in determining terpenoid diversity. SC-PTs biosynthesize prenyl pyrophosphates with different chain lengths, and these compounds are the direct precursors of terpenoids. Arabidopsis thaliana possesses a subgroup of SC-PTs whose functions are not clearly known. In this study, we focus on 10 geranylgeranyl pyrophosphate synthase-like [GGPPSL] proteins, which are commonly thought to produce GGPP [C20]. We found that a subset of members of the Arabidopsis GGPPSL gene family have undergone neo-functionalization: GGPPSL6, 7, 9, and 10 mainly have geranylfarnesyl pyrophosphate synthase activity (C25; renamed AtGFPPS1, 2, 3, and 4), and GGPPSL8 produces even longer chain prenyl pyrophosphate (>/= C30; renamed polyprenyl pyrophosphate synthase 2, AtPPPS2). By solving the crystal structures of AtGFPPS2, AtPPPS2, and AtGGPPS11, we reveal the product chain-length determination mechanism of SC-PTs and interpret it as a

Plant sesterterpenoids, an important class of terpenoids, are widely distributed in various plants, including food crops. However, little is known about their biosynthesis. Here, we cloned and functionally characterized a plant geranylfarnesyl diphosphate synthase (Lc-GFDPS), the enzyme producing the C25 prenyl diphosphate precursor to all sesterterpenoids, from the glandular trichomes of the woody plant Leucosceptrum canum. GFDPS catalyzed the formation of GFDP after expression in Escherichia coli. Overexpressing GFDPS in Arabidopsis thaliana also gave an extract catalyzing GFDP formation. GFDPS was strongly expressed in glandular trichomes, and its transcript profile was completely in accordance with the sesterterpenoid accumulation pattern. GFDPS is localized to the plastids, and inhibitor studies indicated its use of isoprenyl diphosphate substrates supplied by the 2-C-methyl-D-erythritol 4-phosphate pathway. Application of a jasmonate defense hormone induced GFDPS transcript and sesterterpenoid accumulation, while reducing feeding and growth of the generalist insect Spodoptera exigua, suggesting that these C25 terpenoids play a defensive role. Phylogenetic analysis suggested that GFDPS probably evolved from plant geranylgeranyl diphosphate synthase under the influence of positive selection. The isolation of GFDPS provides a model for investigating sesterterpenoid formation in other species and a tool for manipulating the formation of this group in plants and other organisms.

The structural gene for thermostable farnesyl diphosphate synthase from Bacillus stearothermophilus was cloned, sequenced, and overexpressed in Escherichia coli cells. A 1,260-nucleotide sequence of the cloned fragment was determined. This sequence specifies an open reading frame of 891 nucleotides for farnesyl diphosphate synthase. The deduced amino acid sequence shows a 42% similarity with that of E. coli FPP synthase [Fujisaki et al. (1990) J. Biochem. 108, 995-1000]. Comparison with prenyltransferases from a wide range of organisms, from bacteria to human, revealed the presence of seven highly conserved regions. In contrast to thermolabile prenyltransferases, which have four to six cysteine residues, the thermostable farnesyl diphosphate synthase carries only two cysteine residues. This enzyme is also unique in that some of the amino acids that are fully conserved in equivalents from other sources are replaced by functionally different amino acids. Construction of an overproducing strain provided a sufficient supply of this enzyme and it was purified to homogeneity. The purified recombinant enzyme is immunochemically identical with the native B. stearothermophilus enzyme, and it is not inactivated even after treatment at 65 degrees C for 70 min.

Isoprenyl diphosphate synthases are ubiquitous enzymes that catalyze the basic chain-elongation reaction in the isoprene biosynthetic pathway. Pairwise sequence comparisons were made for 6 farnesyl diphosphate synthases, 6 geranylgeranyl diphosphate synthases, and a hexaprenyl diphosphate synthase. Five regions with highly conserved residues, two of which contain aspartate-rich DDXX(XX)D motifs found in many prenyltransferases, were identified. A consensus secondary structure for the group, consisting mostly of alpha-helices, was predicted for the multiply aligned sequences from amino acid compositions, computer assignments of local structure, and hydropathy indices. Progressive sequence alignments suggest that the 13 isoprenyl diphosphate synthases evolved from a common ancestor into 3 distinct clusters. The most distant separation is between yeast hexaprenyl diphosphate synthetase and the other enzymes. Except for the chromoplastic geranylgeranyl diphosphate synthase from Capsicum annuum, the remaining farnesyl and geranylgeranyl diphosphate synthases segregate into prokaryotic/archaebacterial and eukaryotic families.

Farnesyl diphosphate synthases have been shown to possess seven highly conserved regions (I-VII) in their amino acid sequences [Koyama et al. (1993) J. Biochem. (Tokyo) 113, 355-363]. Site-directed mutants of farnesyl diphosphate synthase from Bacillus stearothermophilus were made to evaluate the roles of the conserved aspartic acids in region VI and lysines in regions I, V, and VI. The aspartate at position 224 was changed to alanine or glutamate (mutants designated as D224A and D224E, respectively); aspartates at positions 225 and 228 were changed to isoleucine and alanine (D225I, D228A); lysine at position 238 was changed to either alanine or arginine (K238A, K238R). The lysines at positions 47 and 183 were changed to isoleucine and alanine (K471, K183A), respectively. Kinetic analyses of the wild-type and mutant enzymes indicated that the mutagenesis of Asp-224 and Asp-225 resulted in a decrease of Kcat values of approximately 10(4)- to 10(5)-fold compared to the wild type. On the other hand, D228A showed a Kcat value approximately one-tenth of that of the wild type, and the k(m) value for isopentenyl diphosphate increased approximately 10-fold. Both K471 and K183A showed k(m) values for isopentenyl diphosphate 20-fold larger and kcat values 70-fold smaller than the wild type. These results suggest that the two conserved lysines in regions I and V contribute to the binding of isopentenyl diphosphate and that the first and the second aspartates in region VI are involved in catalytic function. Aspartate-228 is also important for the binding of isopentenyl diphosphate rather than for catalytic reaction.

Living gymnosperms comprise only a little more than 1000 species, but represent four of the five main lineages of seed plants, including cycads, ginkgos, gnetophytes and conifers. This group has huge ecological and economic value, and has drawn great interest from the scientific community. Here we review recent advances in our understanding of gymnosperm evolution and biogeography, including phylogenetic relationships at different taxonomic levels, patterns of species diversification, roles of vicariance and dispersal in development of intercontinental disjunctions, modes of molecular evolution in different genomes and lineages, and mechanisms underlying the formation of large nuclear genomes. It is particularly interesting that increasing evidence supports a sister relationship between Gnetales and Pinaceae (the Gnepine hypothesis) and the contribution of recent radiations to present species diversity, and that expansion of retrotransposons is responsible for the large and complex nuclear genome of gymnosperms. In addition, multiple coniferous genera such as Picea very likely originated in North America and migrated into the Old World, further indicating that the center of diversity is not necessarily the place of origin. The Bering Land Bridge acted as an important pathway for dispersal of gymnosperms in the Northern Hemisphere. Moreover, the genome sequences of conifers provide an unprecedented opportunity and an important platform for the evolutionary studies of gymnosperms, and will also shed new light on evolution of many important gene families and biological pathways in seed plants. (c) 2014 Elsevier Inc.

Bark beetle infestation and associated fungal infection are a serious disease problem in conifer species. Conifers have evolved elaborate, constitutive and inducible, terpene-based defense mechanisms to deter insect pests and their symbiotic fungal pathogens. This process involves the secretion of oleoresin, a complex mixture of monoterpenes, sesquiterpenes and diterpenoid acids. Induced oleoresinosis in grand fir (Abies grandis) provides a model system for studying the regulation of defensive terpene biosynthesis and for identifying relevant genes. The ecological relationships between conifers, beetle pests, beetle predators and fungal pathogens present several possible avenues for manipulating oleoresin composition to improve tree resistance. Possible examples include chemically disguising the host, adding toxins and altering the levels of pheromone precursors, attractants for predators or hormone mimics to disrupt insect development. Strategies and prospects for generating transgenic conifers with increased defense capability are discussed.

Insects select their hosts, but trees cannot select which herbivores will feed upon them. Thus, as long-lived stationary organisms, conifers must resist the onslaught of varying and multiple attackers over their lifetime. Arguably, the greatest threats to conifers are herbivorous insects and their associated pathogens. Insects such as bark beetles, stem- and wood-boring insects, shoot-feeding weevils, and foliage-feeding budworms and sawflies are among the most devastating pests of conifer forests. Conifer trees produce a great diversity of compounds, such as an enormous array of terpenoids and phenolics, that may impart resistance to a variety of herbivores and microorganisms. Insects have evolved to specialize in resistance to these chemicals -- choosing, feeding upon, and colonizing hosts they perceive to be best suited to reproduction. This review focuses on the plant-insect interactions mediated by conifer-produced terpenoids. To understand the role of terpenoids in conifer-insect interactions, we must understand how conifers produce the wide diversity of terpenoids, as well as understand how these specific compounds affect insect behaviour and physiology. This review examines what chemicals are produced, the genes and proteins involved in their biosynthesis, how they work, and how they are regulated. It also examines how insects and their associated pathogens interact with, elicit, and are affected by conifer-produced terpenoids.

Defense-related terpenoid biosynthesis in conifers is a dynamic process closely associated with specialized anatomical structures that allows conifers to cope with attack from many potential pests and pathogens. The constitutive and inducible terpenoid defense of conifers involves several hundred different monoterpenes, sesquiterpenes and diterpenes. Changing arrays of these many compounds are formed from the general isoprenoid pathway by activities of large gene families for two classes of enzymes, the terpene synthases and the cytochrome P450-dependent monooxygenases of the CYP720B group. Extensive studies have been conducted on the genomics, proteomics and molecular biochemical characterization of these enzymes. Many of the conifer terpene synthases are multi-product enzymes, and the P450 enzymes of the CYP720B group are promiscuous in catalyzing multiple oxidations, along homologous series of diterpenoids, from a broad spectrum of substrates. The terpene synthases and CYP720B genes respond to authentic or simulated insect attack with increased transcript levels, protein abundance and enzyme activity. The constitutive and induced oleoresin terpenoids for conifer defense accumulate in preformed cortical resin ducts and in xylem trauma-associated resin ducts. Formation of these resin ducts de novo in the cambium zone and developing xylem, following insect attack or treatment of trees with methyl jasmonate, is a unique feature of the induced defense of long-lived conifer trees.

Wounding of Norway spruce by inoculation with sterile agar, or agar containing the pathogenic fungus Ceratocystis polonica, induced traumatic resin duct formation in the stem. Visible anatomical responses occurred in the cambium 6-9 d post-inoculation. Near the inoculation site cellular proliferation, polyphenolic accumulation, and lignification were induced as a wound reaction to seal the damaged area. Five centimetres from the inoculation site cells in the cambial zone swelled and divided to form clusters. By 18 d post-inoculation, these cells began to differentiate into resin duct epithelial cells surrounding incipient schizogenous lumens. Mature axial traumatic ducts appeared by 36 d as a row of ducts in the xylem centripetal to the cambium. The ducts formed an interconnected network continuous with radial resin ducts. Parenchyma cells surrounding the ducts accumulated polyphenols that disappeared as the cells differentiated into tracheids. These polyphenols appeared to contain fewer sugar residues compared to those accumulating in the secondary phloem, as indicated by the periodic acid-Schiff's staining. The epithelial cells did not accumulate polyphenols but contained immunologically detectable phenylalanine ammonia lyase (EC 4.3.1.5), indicating synthesis of phenolics as a possible resin component. These findings may represent a defense mechanism in Norway spruce against the pathogenic fungus Ceratocystis polonica.

Conifers possess inducible terpenoid defense systems. These systems are associated with the formation of traumatic resin ducts (TRD) and are underpinned by enhanced gene expression and activity of terpene synthases (TPS), enzymes responsible for oleoresin formation. We first determined that Sitka spruce (Picea sitchensis [Bong.] Carriere) had the capacity for TRD formation by mechanically wounding representative trees. We then proceeded to investigate whether the white pine weevil (Pissodes strobi Peck.), a stem-boring insect, can influence the expression of genes encoding monoterpene synthases (mono-tps) in Sitka spruce. We went on to compare this response with the effects of a simulated insect attack by drill wounding. A significant increase in mono-tps transcript level was observed in the leaders of lateral branches of weevil-attacked and mechanically wounded trees. In this study, weevils induced a more rapid enhancement of mono-tps gene expression. A full-length Sitka spruce mono-tps cDNA (PsTPS2) was isolated, expressed in Escherichia coli, and functionally identified as (-)-pinene synthase. The recombinant (-)-pinene synthase catalyzes the formation of (-)-alpha-pinene and (-)-beta-pinene, both of which are known constituents of stem oleoresin in Sitka spruce and increase in abundance after weevil attack. These data suggest that increased (-)-pinene synthase gene expression is an important element of the direct defense system deployed in Sitka spruce after insect attack.

Farnesyl diphosphate synthase (FPS; EC2.5.1.1/EC2. 5.1.10) catalyzes the synthesis of farnesyl diphosphate, and provides precursor for biosynthesis of sesquiterpene and isoprenoids containing more than 15 isoprene units in Ginkgo biloba. Here we report the cloning, characterization and functional analysis of a new cDNA encoding FPS from G. biloba. The full-length cDNA (designated GbFPS) had 1731 bp with an open reading frame of 1170 bp encoding a polypeptide of 390 amino acids. The deduced GbFPS was similar to other known FPSs and contained all the conserved regions of trans-prenyl chain-elongating enzymes. Structural modeling showed that GbFPS had the typical structure of FPS, the most prominent feature of which is the arrangement of 13 core helices around a large central cavity. Southern blot analysis revealed a small FPS gene family in G. biloba. Expression analysis indicated that GbFPS expression was high in roots and leaves, and low in stems. Functional complementation of GbFPS in an FPS-deficient strain confirmed that GbFPS mediates farnesyl diphosphate biosynthesis.

Spruce (Picea spp.) and other conifers employ terpenoid-based oleoresin as part of their defense against herbivores and pathogens. The short-chain isoprenyl diphosphate synthases (IDS) are situated at critical branch points in terpene biosynthesis, producing the precursors of the different terpenoid classes. To determine the role of IDS and to create altered terpene phenotypes for assessing the defensive role of terpenoids, we overexpressed a bifunctional spruce IDS, a geranyl diphosphate and geranylgeranyl diphosphate synthase in white spruce (Picea glauca) saplings. While transcript level (350-fold), enzyme activity level (7-fold), and in planta geranyl diphosphate and geranylgeranyl diphosphate levels (4- to 8-fold) were significantly increased in the needles of transgenic plants, there was no increase in the major monoterpenes and diterpene acids of the resin and no change in primary isoprenoids, such as sterols, chlorophylls, and carotenoids. Instead, large amounts of geranylgeranyl fatty acid esters, known from various gymnosperm and angiosperm plant species, accumulated in needles and were shown to act defensively in reducing the performance of larvae of the nun moth (Lymantria monacha), a conifer pest in Eurasia. These results show the impact of overexpression of an IDS and the defensive role of an unexpected accumulation product of terpenoid biosynthesis with the potential for a broader function in plant protection.

Geranyl diphosphate synthase catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to generate geranyl diphosphate, the essential precursor of monoterpene biosynthesis. Using geranylgeranyl diphosphate synthase from Taxus canadensis as a hybridization probe, four full length cDNA clones, sharing high sequence identity to each other (>69%) and to the Taxus geranylgeranyl diphosphate synthase (>66%), were isolated from a grand fir (Abies grandis) cDNA library. When expressed in Escherichia coli, three of the recombinant enzymes produced geranyl diphosphate and one produced geranylgeranyl diphosphate as the dominant product when supplied with isopentenyl diphosphate and dimethylallyl diphosphate as cosubstrates. One enzyme (AgGPPS2) was confirmed as a specific geranyl diphosphate synthase, in that it accepted only dimethylallyl diphosphate as the allylic cosubstrate and it produced exclusively geranyl diphosphate as product, with a k(cat) of 1.8s(-1). Gel filtration experiments performed on the recombinant geranyl diphosphate synthases, in which the plastidial targeting sequences had been deleted, revealed that these enzymes are homodimers similar to other short-chain prenyltransferases but different from the heterotetrameric geranyl diphosphate synthase of mint.

Geranylgeranyl diphosphate synthase supplies the essential acyclic precursor for Taxol biosynthesis in methyl jasmonate-induced Taxus canadensis suspension cell cultures. A cDNA encoding this prenyltransferase was cloned from an induced T. canadensis cell library. The recombinant enzyme expressed in yeast was confirmed by radiochromatographic analysis to produce geranylgeranyl diphosphate from farnesyl diphosphate and [4-14C]isopentenyl diphosphate and was subjected to preliminary kinetic characterization. The deduced amino acid sequence of this gymnosperm geranylgeranyl diphosphate synthase (393 residues) resembles those of geranylgeranyl diphosphate synthases of angiosperm origin, except for the 90-100 N-terminal residues that correspond to the plastidial transit peptide. The full-length preprotein (42.6 kDa) and two truncated versions, corresponding to putative