Farnesyl diphosphate synthase. Altering the catalytic site to select for geranyl diphosphate activity.

We previously reported the isolation of a partial-length human fetal-liver cDNA encoding farnesyl diphosphate (FPP) synthase (EC 2.5.1.10) and the expression of an active FPP synthase fusion protein in Escherichia coli. The expressed human FPP synthase fusion protein has now been purified to apparent homogeneity by using two chromatographic steps. The purification scheme allowed the preparation of 1.8 mg of homogeneous protein from 149 mg of crude extract in a 64% yield with a 52-fold enrichment. A single band with a subunit molecular mass of 39 kDa was observed by Coomassie Blue staining after SDS/PAGE. A molecular mass of 78-80 kDa was calculated for the native form of the fusion protein by h.p.l.c. on a SEC-250 column, suggesting that the active fusion protein is a dimer. The purified fusion protein has FPP synthase condensation activities in the presence of both substrates, isopentenyl diphosphate and geranyl diphosphate. Enzyme activity was inhibited by a bisubstrate analogue of isopentenyl diphosphate and dimethylallyl diphosphate, and a small amount of higher prenyltransferase was observed. Michaelis constants for isopentenyl diphosphate and geranyl diphosphate were 0.55 and 0.43 microM respectively, and Vmax for synthesis of farnesyl diphosphate from these substrates was 1.08 mumol/min per mg. These results suggest that the structure and catalytic properties of the expressed FPP synthase fusion protein are virtually identical with those of the native human liver enzyme.

Farnesyl diphosphate synthase catalyzes the sequential head-to-tail condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate. In plants the presence of farnesyl diphosphate synthase isozymes offers the possibility of differential regulation. Three full-length cDNAs encoding putative isoprenoid synthases, FDS-1, FDS-2, and FDS-5, with greater than 89% similarity were isolated from a Big Sagebrush Artemisia tridentata cDNA library using a three-step polymerase chain reaction protocol. One of the open reading frames, FDS-5, encoded a protein with an N-terminal amino acid extension that was identified as a plastidial targeting peptide. Recombinant histidine-tagged versions of three proteins were purified, and their enzymatic properties were characterized. FDS-1 and FDS-2 synthesized farnesyl diphosphate as the final chain elongation product, but their kinetic behavior varied. FDS-1 prefers geranyl diphosphate over dimethylallyl diphosphate as an allylic substrate and is active at acidic pH values compared with FDS-2. In contrast, FDS-5 synthesized two irregular monoterpenoids, chrysanthemyl diphosphate and lavandulyl diphosphate, when incubated with dimethylallyl diphosphate and an additional product, the regular monoterpene geranyl diphosphate, when incubated with isopentenyl diphosphate and dimethylallyl diphosphate. Specific cellular functions are proposed for each of the three enzymes, and a scenario for evolution of isoprenyl synthases in plants is presented.

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.

Substrate analogues for isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where the C3 methyl groups were replaced by chlorine, were synthesized and evaluated as substrates for avian farnesyl diphosphate synthase (FPPase). The IPP analogue (3-ClIPP) was a cosubstrate when incubated with dimethylallyl diphosphate (DMAPP) or geranyl diphosphate (GPP) to give the corresponding chlorinated analogues of geranyl diphosphate (3-ClGPP) and farnesyl diphosphate (3-ClFPP), respectively. No products were detected in incubations of 3-ClIPP with 3-ClDMAPP. Incubation of IPP with 3-ClDMAPP gave 11-ClFPP as the sole product. Values of K(M)(3-ClIPP) (with DMAPP) and K(M)(3-ClDMAPP) (with IPP) were similar to those for IPP and DMAPP; however, values of k(cat) for both analogues were substantially lower. These results are consistent with a dissociative electrophilic alkylation mechanism where the rate-limiting step changes from heterolytic cleavage of the carbon-oxygen bond in the allylic substrate to alkylation of the double bond of the homoallylic substrate.

A short-chain prenyl diphosphate synthase in an Escherichia coli mutant that lacked the gene coding for farnesyl diphosphate synthase, ispA, was separated from other prenyl diphosphate synthases by DEAE-Toyopearl column chromatography. The purified enzyme catalyzed the condensation of isopentenyl diphosphate with dimethylallyl diphosphate to form farnesyl diphosphate and geranylgeranyl diphosphate.

Farnesyl diphosphate (FPP)/geranylgeranyl diphosphate (GGPP) synthase, a bifunctional enzyme that synthesizes C15 and C20 isoprenoid diphosphates from isopentenyl diphosphate and dimethylallyl diphosphate, was purified to homogeneity from the archaebacterium Methanobacterium thermoautotrophicum. The only activities detected from synthesis of FPP and GGPP copurified through (NH4)2SO4 precipitation and four chromatographic steps. The pure enzyme was a 79-kDa homodimer that catalyzed the sequential addition of isopentenyl diphosphate to dimethylallyl diphosphate, geranyl diphosphate, and FPP by a non-processive mechanism which allowed substantial amounts of FPP to accumulate during turnover, creating a pool for further elongation to GGPP or for synthesis of squalene. The bifunctional enzyme required Mg2+ or Mn2+ and was optimally active at 65 degrees C. Catalysis of chain elongation in M. thermoautotrophicum differs from related reactions in eubacteria and eukaryotes, where distinct FPP synthases and GGPP synthases are found.

Molecular cloning and expression of Chimonanthus praecox farnesyl pyrophosphate synthase gene and its possible involvement in the biosynthesis of floral volatile sesquiterpenoids

IntroductionCurcuma wenyujin (Zingiberaceae), a medicinally significant species within the Curcuma genus, is highly regarded in traditional Chinese medicine for its rich terpenoid constituents. These specialized metabolites serve as the principal bioactive components underpinning the plant's therapeutic effects. As key enzymes in terpenoid biosynthesis, trans-prenyltransferases (trans-PTs) play crucial roles in regulating metabolic flux.MethodsTrans-PTs in C. wenyujin were identified through a comprehensive transcriptome-wide analysis. The identified CwPTs were classified by constructing a phylogenetic tree. Their transcriptional responses to abscisic acid (ABA), methyl jasmonate (MeJA), and gibberellic acid (GA) were assessed. The catalytic functions of CwFPS1 and CwGGPS1 were characterized through in vitro enzyme assays and heterologous expression in Escherichia coli.ResultsEight trans-CwPTs were identified in C. wenyujin. Among them, five CwPTs function as geranylgeranyl pyrophosphate synthases (GGPS), two as solanesyl pyrophosphate synthases (SPS), and one as farnesyl pyrophosphate synthase (FPS). Expression profile assay showed more significant changes in the transcription levels of CwPTs in response to ABA and MeJA than to GA. CwFPS1 catalyzed the biosynthesis of farnesyl pyrophosphate (FPP) through the sequential condensation of dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP), as well as geranyl pyrophosphate (GPP) and IPP. CwGGPS1 catalyzed the condensation of GPP or FPP with IPP to generate geranylgeranyl pyrophosphate (GGPP) in vitro. Furthermore, these functions were confirmed in Escherichia coli.DiscussionOur research establishes a molecular foundation for understanding terpenoid biosynthesis in C. wenyujin. Future sequencing of the C. wenyujin genome will facilitate the development of additional CwPTs.

The diatom Rhizosolenia setigera Brightwell produces highly branched isoprenoid (HBI) hydrocarbons that are ubiquitously present in marine environments. The hydrocarbon composition of R. setigera varies between C25 and C30 HBIs depending on the life cycle stage with regard to auxosporulation. To better understand how these hydrocarbons are biosynthesized, we characterized the farnesyl pyrophosphate (FPP) synthase (FPPS) enzyme of R. setigera. An isolated 1465-bp cDNA clone contained an open reading frame spanning 1299-bp encoding a protein with 432 amino acid residues. Expression of the RsFPPS cDNA coding region in Escherichia coli produced a protein that exhibited FPPS activity in vitro. A reduction in HBI content from diatoms treated with an FPPS inhibitor, risedronate, suggested that RsFPPS supplies precursors for HBI biosynthesis. Product analysis by gas chromatography-mass spectrometry also revealed that RsFPPS produced small amounts of the cis-isomers of geranyl pyrophosphate and FPP, candidate precursors for the cis-isomers of HBIs previously characterized. Furthermore, RsFPPS gene expression at various life stages of R. setigera in relation to auxosporulation were also analyzed. Herein, we present data on the possible role of RsFPPS in HBI biosynthesis, and it is to our knowledge the first instance that an FPPS was cloned and characterized from a diatom.

Prenyltransferase (EC 2.5.1.1) has been purified to homogeneity from the supernatant fraction of yeast by ammonium sulfate fractionation, diethylaminoethyl-cellulose and hydroxylapatite chromatography, and column isoelectric focusing techniques. The active enzyme from isoelectric focusing columns emerged as a single symmetrical peak with specific activities 15- to 35-fold higher than previously reported preparations. The enzyme was found to be homogeneous by continuous polyacrylamide gel electrophoresis at pH 8.4 and discontinuous polyacrylamide gel electrophoresis at pH 6.9 as well as sodium dodecyl sulfate polyacrylamide electrophoresis at pH 7.0. By means of gel chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was shown to be a dimer with a molecular weight of 84,000 plus or minus 10%. The isoelectric point of the enzyme was determined to be 5.3. The enzyme synthesizes farnesyl and geranylgeranyl pyrophosphates from dimethylallyl, geranyl, and farnesyl pyrophosphates. Michaelis constants for the enzyme were 4, 8, and 14 mu M for isopentenyl, dimethylallyl, and geranyl pyrophosphates, respectively.

The chain elongation reaction catalyzed by polyprenyl diphosphate synthases is the fundamental building reaction in the isoprenoid pathway. During chain elongation, the hydrocarbon moiety in an allylic isoprenoid diphosphate is added to the carbon–carbon double bond of isopentenyl diphosphate (IPP). The chain elongation enzymes can be divided into two genetically different families depending on whether the stereochemistry of the newly formed double bond during each cycle of chain elongation is E or Z. Farnesyl diphosphate (FPP) synthase, a member of the E-double bond family, is the best studied of the chain elongation enzymes and serves as a paradigm for understanding the reactions catalyzed by E-polyprenyl diphosphate synthases. The mechanism for chain elongation is a stereoselective electrophilic alkylation of the carbon–carbon double bond in IPP by the allylic substrate. X-ray structures of avian and E. coli FPP synthases have provided important insights about the mechanism for chain elongation and a structural basis for understanding the stereochemistry of the reaction.

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).

Negative ion fast atom bombardment mass spectrometry of isoprenoid diphosphates and related analogs

A variety of rat tissues were screened at low stringency with a rat farnesyl pyrophosphate (FPP) synthetase cDNA. In testis, an FPP synthetase-related RNA was detected that was larger than the liver FPP synthetase mRNA and was present at very high levels comparable with liver FPP synthetase RNA levels obtained from rats fed diets supplemented with cholestyramine and mevinolin. Sequence analysis of testis cDNA clones, together with primer extension and S1 nuclease experiments, indicated that testis FPP synthetase transcripts contain an extended 5' untranslated region. The 5' extension contained one or two out-of-frame upstream ATGs, depending on the site of transcription initiation. Protein in vitro translation studies indicated that the extended 5' untranslated region may play a role in regulating the translation of the FPP synthetase polypeptide in rat testis. Southern blot analysis with a probe containing both testis and liver 5' untranslated sequences provided evidence that both liver and testis transcripts derive from the same gene. The data suggest that an upstream testis-specific promoter results in the abundant production of FPP synthetase transcripts that are translated at low efficiency; another promoter functions in liver and other somatic tissues and directs the regulated synthesis of shorter discrete transcripts.

Plants are sessile organisms which have evolved a wide range of strategies to adjust to their environment. As an example, many species of flowering plants need to be pollinated by an insect. To attract pollinators and seed dispersers and thus to ensure their reproductive and evolutionary success, they release diverse blends of volatile compounds from their flowers. The bewildering array of structures identified in floral scents, which may contain up to 100 different molecules, is dominated by terpenoids, with monoterpenes representing the most abundant components, followed by sesquiterpenes (Knudsen and Gershenzon, 2006). During the last decade, substantial progress has been made in plant volatile research as a result of the development of sensitive methods for dynamic headspace sampling and improvements in the analysis of volatiles by gas chromatography-mass spectrometry. In the context of current research dealing with the terpenoid composition of floral scents, including, for instance, Arabidopsis (Chen et al., 2003), Clarkia (Dudareva et al., 1996), Antirrhinum majus (Dudareva et al., 2003) or Vitis vinifera (Lucker et al., 2004), Nieuwenhuizen et al. (2009) report in this issue a comparative analysis of volatile terpenes released from male and female flowers of Actinidia deliciosa, a dioecious plant for which efficient insect pollination is particularly critical. The kiwifruit flowers of both male and female genotypes are shown to display a similar terpene profile in which two sesquiterpenes, (E,E)-a-farnesene and germacrene D, and one monoterpene, (E)-b-ocimene, are the main constituents. Interestingly, the monoterpene has been found only in petals, suggesting that it plays a specific role. The emission of volatile compounds was observed predominantly during the day when pollinators were at work, but the molecular mechanisms responsible for co-ordinated expression of the corresponding genes and the potential effect of light intensity remain to be investigated. In the next step, Nieuwenhuizen et al. (2009) used a functional genomics approach to identify the terpene synthases (TPS), which could account for the production of these volatile terpenes in both male and female flowers. To date, more than 100 TPS that synthesize mono-, sesqui-, and diterpenes have been isolated and characterized from different plant species. They belong to the terpene synthase gene family, which comprises seven subfamilies designated as Tps-a to Tps-g (Bohlmann et al., 1998; Trapp and Croteau, 2001). One of the most outstanding properties of these enzymes is their ability to make multiple products from a single prenyl diphosphate substrate. As an example, only two sesquiterpene synthases were found to be responsible for the synthesis of the 20 sesquiterpenes identified in the Arabidopsis floral scent (Tholl et al., 2005). However, some TPS also produce single products. Two specialized single-product monoterpene synthases are responsible for the biosynthesis of myrcene and (E)-bocimene, the two major terpenoids of snapdragon floral scent (Dudareva et al., 2003). In A. deliciosa, the authors identified two genes, referred as to AdGDS1 and AdAFS1, which encode two distinct sesquiterpene synthases belonging to subfamilies Tps-a and Tps-f, respectively. The substrates of these enzymes are prenyl diphosphates. Heterologous expression of these cloned Tps indicates that AdGDS1 uses only farnesyl diphosphate (FPP) as a substrate to form (+)-germacrene D as the sole compound while AdFS1 is able to accept both FPP and geranyl diphosphate (GPP) and catalyses the synthesis of afarnesene and (E)b-ocimene, respectively. Interestingly, AdAFS1 belongs to the Tps-f subfamiliy, which comprises only monoterpene synthases. When transiently expressed in Nicotiana benthamiana plants, AdGDS1 and AdAFS induced the formation of the same volatile terpenes as those found for the recombinant proteins in vitro. As the synthesis of terpene compounds is dependent on the availability of substrates for the TPS, the authors addressed the question of their cellular site of synthesis. In plants, all terpenoids arise from the five-carbon precursors isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), which are derived from the two alternative pathways. Sesquiterpene synthesis is expected to occur in the cytosol from FPP synthesized by the mevalonate pathway whereas monoterpene synthesis should take place in the plastids, from GPP arising from the methyl