Genomic organization and expression analysis of a farnesyl diphosphate synthase gene (FPPS2) in apples (Malus domestica Borkh.)

A farnesyl diphosphate synthase gene (FPPS2), which contains 11 introns and 12 exons, was isolated from the apple cultivar “White Winter Pearmain”. When it was compared to our previously reported FPPS1, its each intron size was different, its each exon size was the same as that of FPPS1 gene, 30 nucleotide differences were found in its coding sequence. Based on these nucleotide differences, specific primers were designed to perform expression analysis; the results showed that it expressed in both fruit and leaf, its expression level was obviously lower than that of FPPS1 gene in fruit which was stored at 4 °C for 5 weeks. This is the first report concerning two FPPS genes and their expression comparison in apples.     

Expression of farnesyl pyrophosphate synthase is increased in diabetic cardiomyopathy

Farnesyl pyrophosphate synthase (FPPS)-catalyzed isoprenoid intermediates are involved in diabetic cardiomyopathy. This study investigated the specific role of FPPS in the development of diabetic cardiomyopathy. We demonstrated that FPPS expression was elevated in both in vivo and in vitro models of diabetic cardiomyopathy. FPPS inhibition decreased the expression of proteins related to cardiac fibrosis and cardiomyocytic hypertrophy, including collagen I, collagen III, connective tissue growth factor, natriuretic factor, brain natriuretic peptide, and β-myosin heavy chain. Furthermore, FPPS inhibition and knockdown prevented phosphorylated c-Jun N-terminal kinase 1/2 (JNK1/2) activation in vitro. In addition, a JNK1/2 inhibitor downregulated high-glucose-induced responses to diabetic cardiomyopathy. Finally, immunofluorescence revealed that cardiomyocytic size was elevated by high glucose and was decreased by zoledronate, small-interfering farnesyl pyrophosphate synthase (siFPPS), and a JNK1/2 inhibitor. Taken together, our findings indicate that FPPS and JNK1/2 may be part of a signaling pathway that plays an important role in diabetic cardiomyopathy.     

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

Farnesyl pyrophosphate (FPP) synthase catalyzes the biosynthesis of FPP, which is the precursors of sesquiterpenoids such as floral scent volatiles, from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). cDNA encoding wintersweet (Chimonanthus praecox L.) FPP synthase was isolated by the RT-PCR and RACE methods. The deduced amino acid sequence showed a high identity to plant FPP synthases. Expression of the gene in Escherichia coli yielded FPPS activity that catalyzed the synthesis of FPP as a main product. Tissue-specific and developmental analyses of the mRNA levels of CpFPPS and volatile sesquiterpenoids levels in C. praecox flowers revealed that the FPPS may play a regulatory role in floral volatile sesquiterpenoids of wintersweet.     

Evidence for trans-trans and cis-cis farnesyl pyrophosphate synthesis in Gossipium hirsutum

A protein fraction capable of catalysing the formation of all four geometrical isomers of farnesyl pyrophosphate has been isolated from cotton roots. Using neryl pyrophosphate and isopentenyl pyrophosphate as substrates the product was found to be cis-cis farnesyl pyrophosphate and possibly trans-cis farnesyl pyrophosphate. Geranyl pyrophosphate and isopentenyl pyrophosphate as substrates yielded trans-trans and possible cis-trans farnesyl pyrophosphate. During purification of the active protein fraction, the ratio of utilization of geranyl pyrophosphate and neryl pyrophosphate did not remain constant, indicating that two enzymes may be involved, one specific for cis C₁₀-substrate and the other for trans C₁₀-substrate.     

Studies on isoprenoid biosynthesis with bacterial intact cells

For the study on the regulation of isoprenoid biosynthesis with intact cells, some strains of bacteria capable of growing on mevalonate as a sole carbon source were isolated from soil. Many of them incorporated [¹⁴C]-mevalonate, [¹⁴C]isopentenyl- and [¹⁴C]farnesyl pyrophosphates into the cells. However, radioactivity was found in their degradation products but not in isoprenoids. Addition of [¹⁴C]isopentenyl pyrophosphate, farnesyl pyrophosphate and Mg²⁺ ions in combination to the culture of a strain of Arthrobacter gave rise to ¹⁴C-incorporation into isoprenoids. Radioactivity was found in polyprenol, its pyrophosphate, monophosphate and fatty acid esters. The reactions of isopentenyl- and farnesyl pyrophosphates syntheses seemed to be rate-limiting steps.     

Correlation between time-dependent inhibition of human farnesyl pyrophosphate synthase and blockade of mevalonate pathway by nitrogen-containing bisphosphonates in cultured cells

A class of drugs successfully used for treatment of metabolic bone diseases is the nitrogen-containing bisphosphonates (N-BPs), which act by inhibiting the vital enzyme, farnesyl pyrophosphate synthase (FPPS), of the mevalonate pathway. Inhibition of FPPS by N-BPs results in the intracellular accumulation of isopentenyl pyrophosphate (IPP) and consequently induces the biosynthesis of a cytotoxic ATP analog (ApppI). Previous cell-free data has reported that N-BPs inhibit FPPS by time-dependent manner as a result of the conformational change. This associated conformational change can be measured as an isomerization constant (K_isom) and reflects the binding differences of the N-BPs to FPPS. In the present study, we tested the biological relevance of the calculated K_isom values of zoledronic acid, risedronate and five experimental N-BP analogs in the cell culture model. We used IPP/ApppI formation as a surrogate marker for blocking of FPPS in the mevalonate pathway.As a result, a correlation between the time-dependent inhibition of FPPS and IPP/ApppI formation by N-BPs was observed. This outcome indicates that the time-dependent inhibition of FPPS enzyme is a biologically significant mechanism and further supports the use of the K_isom calculations for evaluation of the overall potency of the novel FPPS inhibitors. Additionally, data illustrates that IPP/ApppI analysis is a useful method to monitor the intracellular action of drugs and drug candidates based on FPPS inhibition.     

Biosynthesis of abscisic acid from farnesol derivatives in Cercospora rosicola

(E,E)?[1?¹⁴C]Farnesyl phosphate and (E,E)?[1?¹⁴C]farnesyl pyrophosphate were both converted to abscisic acid by Cercospora rosicola resuspensions. (E,E)?[1?¹⁴C]Farnesol, (E,Z)?[1?¹⁴C]farnesol, (E,Z)?[1?¹⁴C]farnesyl pyrophosphate, (E,E)?[1?¹⁴C]farnesic acid, and (E,Z)?[1?¹⁴C]farnesic acid were not converted to abscisic acid by the fungus. These findings provide information on the sequence of the reactions involved in converting farnesyl pyrophosphate to abscisic acid. Specifically, they suggest that the transformations involving the three terminal carbons in the side chain occur after one or more changes elsewhere in the molecule.     

New insights into molecular recognition of 1,1-bisphosphonic acids by farnesyl diphosphate synthase

As part of our project pointed at the search of new antiparasitic agents against American trypanosomiasis (Chagas disease) and toxoplasmosis a series of 2-alkylaminoethyl-1-hydroxy-1,1-bisphosphonic acids has been designed, synthesized and biologically evaluated against the etiologic agents of these parasitic diseases, Trypanosoma cruzi and Toxoplasma gondii, respectively, and also towards their target enzymes, T. cruzi and T. gondii farnesyl pyrophosphate synthase (FPPS), respectively. Surprisingly, while most pharmacologically active bisphosphonates have a hydroxyl group at the C-1 position, the additional presence of an amino group at C-3 resulted in decreased activity towards either T. cruzi cells or TcFPPS. Density functional theory calculations justify this unexpected behavior. Although these compounds were devoid of activity against T. cruzi cells and TcFPPS, they were efficient growth inhibitors of tachyzoites of T. gondii. This activity was associated with a potent inhibition of the enzymatic activity of TgFPPS. Compound 28 arises as a main example of this family of compounds exhibiting an ED₅₀ value of 4.7 μM against tachyzoites of T. gondii and an IC₅₀ of 0.051 μM against TgFPPS.     

Partial purification and properties of prenyltransferase from Pisum sativum

Prenyltransferase (EC 2.5.1.1; assayed as farnesyl pyrophosphate synthetase)was purified 106-fold from an homogenate of 3-day-old seedlings of Pisum sativum. Some of the properties of the purified enzyme were determined and these differed in several significant respects from those reported for preparations from other sources, e.g. the apparent MW was 96000 ± 4000 and the preparation could be dissociated into two subunits of MW 45000 ± 3000. The total activity of the extractable enzyme went through a sharp maximum (in the range 1 to 28 days) 3 days after germination. Farnesyl pyrophosphate was formed in cell-free extracts of peas from either isopentenyl pyrophosphate alone, or this together with geranyl pyrophosphate (optimum yields 1.2 and 10% respectively). Use of [1-¹⁴C]- and [4-¹⁴C]-isopentenyl pyrophosphates as the sole substrates and degradation of the products showed that the crude extracts contained a pool of the biogenetic equivalent of 3,3-dimethylallyl pyrophosphate. No analogous pool of geranyl pyrophosphate could be detected.     

Farnesyl diphosphate synthase localizes to the cytoplasm of Trypanosoma cruzi and T. brucei

The farnesyl diphosphate synthase (FPPS) has previously been characterized in trypanosomes as an essential enzyme for their survival and as the target for bisphosphonates, drugs that are effective both in vitro and in vivo against these parasites. Enzymes from the isoprenoid pathway have been assigned to different compartments in eukaryotes, including trypanosomatids. We here report that FPPS localizes to the cytoplasm of both Trypanosoma cruzi and T. brucei, and is not present in other organelles such as the mitochondria and glycosomes.     

Mechanism of cis-prenyltransferase reaction probed by substrate analogues

Undecaprenyl pyrophosphate synthase (UPPS) is a cis-type prenyltransferases which catalyzes condensation reactions of farnesyl diphosphate (FPP) with eight isopentenyl pyrophosphate (IPP) units to generate C₅₅ product. In this study, we used two analogues of FPP, 2-fluoro-FPP and [1,1-²H₂]FPP, to probe the reaction mechanism of Escherichia coli UPPS. The reaction rate of 2-fluoro-FPP with IPP under single-turnover condition is similar to that of FPP, consistent with the mechanism without forming a farnesyl carbocation intermediate. Moreover, the deuterium secondary KIE of 0.985 ± 0.022 measured for UPPS reaction using [1,1-²H₂]FPP supports the associative transition state. Unlike the sequential mechanism used by trans-prenyltransferases, our data demonstrate E. coli UPPS utilizes the concerted mechanism.     

Syntheses and characterization of non-bisphosphonate quinoline derivatives as new FPPS inhibitors

Background

Farnesyl pyrophosphate synthase (FPPS) is a key regulatory enzyme in the biosynthesis of cholesterol and in the post-translational modification of signaling proteins. It has been reported that non-bisphosphonate FPPS inhibitors targeting its allosteric binding pocket are potentially important for the development of promising anti-cancer drugs.

Methods

The following methods were used: organic syntheses of non-bisphosphonate quinoline derivatives, enzyme inhibition studies, fluorescence titration assays, synergistic effect studies of quinoline derivatives with zoledronate, ITC studies for the binding of FPPS with quinoline derivatives, NMR-based HAP binding assays, molecular modeling studies, fluorescence imaging assay and MTT assays.

Results

We report our syntheses of a series of quinoline derivatives as new FPPS inhibitors possibly targeting the allosteric site of the enzyme. Compound 6b showed potent inhibition to FPPS without significant hydroxyapatite binding affinity. The compound showed synergistic inhibitory effect with active-site inhibitor zoledronate. ITC experiment confirmed the good binding effect of compound 6b to FPPS, and further indicated the binding ratio of 1:1. Molecular modeling studies showed that 6b could possibly bind to the allosteric binding pocket of the enzyme. The fluorescence microscopy indicated that these compounds could get into cancer cells.

Conclusions

Our results showed that quinoline derivative 6b could become a new lead compound for further optimization for cancer treatment.

General significance

The traditional FPPS active-site inhibitors bisphosphonates show poor membrane permeability to tumor cells, due to their strong polarity. The development of new non-bisphosphonate FPPS inhibitors with good cell membrane permeability is potentially important.     

Molecular regulation of santalol biosynthesis in Santalum album L.

Santalum album L. commonly known as East-Indian sandal or chandan is a hemiparasitic tree of family santalaceae. Santalol is a bioprospecting molecule present in sandalwood and any effort towards metabolic engineering of this important moiety would require knowledge on gene regulation. Santalol is a sesquiterpene synthesized through mevalonate or non-mevalonate pathways. First step of santalol biosynthesis involves head to tail condensation of isopentenyl pyrophosphate (IPP) with its allylic co-substrate dimethyl allyl pyrophosphate (DMAPP) to produce geranyl pyrophosphate (GPP; C10 — a monoterpene). GPP upon one additional condensation with IPP produces farnesyl pyrophosphate (FPP; C15 — an open chain sesquiterpene). Both the reactions are catalyzed by farnesyl diphosphate synthase (FDS). Santalene synthase (SS), a terpene cyclase catalyzes cyclization of open ring FPP into a mixture of cyclic sesquiterpenes such as α-santalene, epi-β-santalene, β-santalene and exo bergamotene, the main constituents of sandal oil. The objective of the present work was to generate a comprehensive knowledge on the genes involved in santalol production and study their molecular regulation. To achieve this, sequences encoding farnesyl diphosphate synthase and santalene synthase were isolated from sandalwood using suppression subtraction hybridization and 2D gel electrophoresis technology. Functional characterization of both the genes was done through enzyme assays and tissue-specific expression of both the genes was studied. To our knowledge, this is the first report on studies on molecular regulation, and tissue-specific expression of the genes involved in santalol biosynthesis.     

Characterization of two monoterpene synthases involved in floral scent formation in Hedychium coronarium

Hedychium coronarium, a perennial herb belonging to the family Zingiberaceae, is cultivated as a garden plant or cut flower as well as for medicine and aromatic oil. Its flowers emit a fresh and inviting scent, which is mainly because of monoterpenes present in the profile of the floral volatiles. However, fragrance produced as a result of monoterpenes has not been well studied. In the present study, two novel terpene synthase (TPS) genes (HcTPS7 and HcTPS8) were isolated to study the biosynthesis of monoterpenes in H. coronarium. In vitro characterization showed that the recombinant HcTPS7 was capable of generating sabinene as its main product, in addition to nine sub-products from geranyl diphosphate (GPP). Recombinant HcTPS8 almost specifically catalyzed the formation of linalool from GPP, while it converted farnesyl diphosphate (FPP) to α-bergamotene, cis-α-bisabolene, β-farnesene and other ten sesquiterpenes. Subcellular localization experiments revealed that HcTPS7 and HcTPS8 were located in plastids. Real-time PCR analyses showed that HcTPS7 and HcTPS8 genes were highly expressed in petals and sepals, but were almost undetectable in vegetative organs. The changes of their expression levels in petals were positively correlated with the emission patterns of sabinene and linalool, respectively, during flower development. The results indicated that HcTPS7 and HcTPS8 were involved in the biosynthesis of sabinene and linalool in H. coronarium flowers. Results on these two TPSs first characterized from H. coronarium provide new insights into molecular mechanisms of terpene biosynthesis in this species and also lay the basis for biotechnological modification of floral scent profile in Hedychium.     

Structure and mechanism of the farnesyl diphosphate synthase from Trypanosoma cruzi: implications for drug design

Typanosoma cruzi, the causative agent of Chagas disease, has recently been shown to be sensitive to the action of the bisphosphonates currently used in bone resorption therapy. These compounds target the mevalonate pathway by inhibiting farnesyl diphosphate synthase (farnesyl pyrophosphate synthase, FPPS), the enzyme that condenses the diphosphates of C5 alcohols (isopentenyl and dimethylallyl) to form C10 and C15 diphosphates (geranyl and farnesyl). The structures of the T. cruzi FPPS (TcFPPS) alone and in two complexes with substrates and inhibitors reveal that following binding of the two substrates and three Mg2+ ions, the enzyme undergoes a conformational change consisting of a hinge-like closure of the binding site. In this conformation, it would be possible for the enzyme to bind a bisphosphonate inhibitor that spans the sites usually occupied by dimethylallyl diphosphate (DMAPP) and the homoallyl moiety of isopentenyl diphosphate. This observation may lead to the design of new, more potent anti-trypanosomal bisphosphonates, because existing FPPS inhibitors occupy only the DMAPP site. In addition, the structures provide an important mechanistic insight: after its formation, geranyl diphosphate can swing without leaving the enzyme, from the product site to the substrate site to participate in the synthesis of farnesyl diphosphate.     

Substrate binding mode and reaction mechanism of undecaprenyl pyrophosphate synthase deduced from crystallographic studies

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes eight consecutive condensation reactions of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to form a 55-carbon long-chain product. We previously reported the crystal structure of the apo-enzyme from Escherichia coli and the structure of UPPs in complex with sulfate ions (resembling pyrophosphate of substrate), Mg(2+), and two Triton molecules (product-like). In the present study, FPP substrate was soaked into the UPPs crystals, and the complex structure was solved. Based on the crystal structure, the pyrophosphate head group of FPP is bound to the backbone NHs of Gly29 and Arg30 as well as the side chains of Asn28, Arg30, and Arg39 through hydrogen bonds. His43 is close to the C2 carbon of FPP and may stabilize the farnesyl cation intermediate during catalysis. The hydrocarbon moiety of FPP is bound with hydrophobic amino acids including Leu85, Leu88, and Phe89, located on the alpha3 helix. The binding mode of FPP in cis-type UPPs is apparently different from that of trans-type and many other prenyltransferases which utilize Asprich motifs for substrate binding via Mg(2+). The new structure provides a plausible mechanism for the catalysis of UPPs.     

Functional properties of diapophytoene and related desaturases of C30 and C40 carotenoid biosynthetic pathways

The desaturation reactions of C₃₀ carotenoids from diapophytoene to diaponeurosporene was investigated in vitro and by complementation in Escherichia coli. The expressed diapophytoene desaturase from Staphylococcus aureus inserts three double bonds in an FAD-dependent reaction. The enzyme is inhibited by diphenylamine. In the complementation experiment diapophytoene desaturase was able to convert C₄₀ phytoene to some extend but exhibited a high affinity to ζ-carotene. Comparison to the reaction of a phytoene desaturase from Rhodobacter capsulatus catalyzing a parallel three-step desaturation sequence with the corresponding C₄₀ carotenes revealed that this desaturase can also convert C₃₀ diapophytoene. Other homologous bacterial C₄₀ carotene desaturases could also utilize C₃₀ substrates, including one type of ζ-carotene desaturase which converted diaponeurosporene to diapolycopene. Further complementation experiments including the diapophytoene synthase gene from S. aureus revealed that the C₃₀ carotenogenic pathway is determined by this initial enzyme which is highly homologous to C₄₀ phytoene synthases.     

Expression of a fungal sesquiterpene cyclase gene in transgenic tobacco

The complete coding sequence for the trichodiene synthase gene from Fusarium sporotrichioides was introduced into tobacco (Nicotiana tabacum) under the regulation of the cauliflower mosiac virus 35S promoter. Expression of trichodiene synthase was demonstrated in the leaves of transformed plants. Leaf homogenates incubated with [³H]farnesyl pyrophosphate produced trichodiene as a major product. Trichodiene was detected in the leaves of a transformed plant at a level of 5 to 10 nanograms per gram fresh weight. The introduction of a fungal sesquiterpene cyclase gene into tobacco has resulted in the expression of an active enzyme and the accumulation of low levels of its sesquiterpenoid product.