Duplication and soldier-specific expression of geranylgeranyl diphosphate synthase genes in a nasute termite Nasutitermes takasagoensis.

In the evolutionarily-derived termite subfamily Nasutitermitinae (family Termitidae), soldiers defend their nestmates by discharging polycyclic diterpenes from a head projection called the "nasus." The diterpenes are synthesised in the frontal gland from the precursor geranylgeranyl diphosphate (GGPP), which is generally used for post-translational modification of proteins in animals. In this study, we constructed a comprehensive gene catalogue to search for genes involved in the diterpene biosynthesis by assembling RNA sequencing reads of Nasutitermes takasagoensis, identifying eight gene copies for GGPP synthase (GGPPS). The number of gene copies is much larger in contrast to other related insects. Gene cloning by reverse transcription-PCR and rapid amplification of cDNA ends confirmed that seven GGPPS genes (NtGGPPS1 to NtGGPPS7) have highly variable untranslated regions. Molecular phylogenetic analysis showed that the NtGGPPS7 gene was grouped with homologs obtained from ancestral termites that have only a single copy of the gene, and the NtGGPPS6 gene was grouped with homologs obtained from a basal lineage of termitids, in which soldiers do not synthesise diterpenes. As the sister group to this clade, furthermore, a monophyletic clade included all the other NtGGPPS genes (NtGGPPS1 to NtGGPPS5). Expression analyses revealed that NtGGPPS7 gene was expressed in all the examined castes and tissues, whereas all the other genes were expressed only in the soldier head. These results suggest that gene duplication followed by subfunctionalisation of the GGPPS genes might have accompanied the evolution of chemical defence in the nasute termite lineage.

Salicylic acid as an effective elicitor for improved taxol production in endophytic fungus Pestalotiopsis microspora.

Salicylic acid (SA) is an effective elicitor to increase taxol production in Pestalotiopsis microspora. Addition of SA at the concentration of 300 µM yielded taxol 625.47 µg L-1, 45- fold higher than that of the control. Elicitation of the role of SA in the fungal taxol biosynthetic pathway revealed that SA enhanced reactive oxygen species and lipid peroxidation of unsaturated fatty acids of P. microspora mycelia. This oxidative process stimulates isoprene biosynthetic pathway by triggering expression of the geranylgeranyl pyrophosphate synthase gene leading to improved biosynthesis of taxol in P. microspora.

Plasmodium falciparum parasites overexpressing farnesyl diphosphate synthase/geranylgeranyl diphosphate synthase are more resistant to risedronate.

Farnesyl diphosphate synthase/geranylgeranyl diphosphate synthase (FPPS/GGPPS) is a key enzyme in the synthesis of isoprenic chains. Risedronate, a bisphosphonate containing nitrogen (N-BP), is a potent inhibitor of blood stage Plasmodium. Here, we show that P. falciparum parasites overexpressing FPPS/GGPPS are more resistant to risedronate, suggesting that this enzyme is an important target, and bisphosphonate analogues can be used as potential antimalarial drugs.

Plasmodium falciparum parasites overexpressing farnesyl diphosphate synthase/geranylgeranyl diphosphate synthase are more resistant to risedronate

Farnesyl diphosphate synthase/geranylgeranyl diphosphate synthase (FPPS/GGPPS) is a key enzyme in the synthesis of isoprenic chains. Risedronate, a bisphosphonate containing nitrogen (N-BP), is a potent inhibitor of blood stage Plasmodium. Here, we show that P. falciparum parasites overexpressing FPPS/GGPPS are more resistant to risedronate, suggesting that this enzyme is an important target, and bisphosphonate analogues can be used as potential antimalarial drugs.

Efficient production of lycopene in Saccharomyces cerevisiae by expression of synthetic crt genes from a plasmid harboring the ADH2 promoter.

Lycopene is an effective antioxidant proposed as a possible treatment for some cancers and other degenerative human conditions. This study aims at generation of a yeast strain (Saccharomyces cerevisiae) of efficient productivity of lycopene by overexpressing synthetic genes derived from crtE, crtB and crtI genes of Erwinia uredovora. These synthetic genes were constructed in accordance with the preferred codon usage in S. cerevisiae but with no changes in amino acid sequences of the gene products. S. cerevisiae cells were transformed with these synthetic crt genes, whose expression was regulated by the ADH2 promoter, which is de-repressed upon glucose depletion. The RT-PCR and Western blotting analyses indicated that the synthetic crt genes were efficiently transcribed and translated in crt-transformed S. cerevisiae cells. The highest level of lycopene in one of the transformed lines was 3.3mglycopene/g dry cell weight, which is higher than the previously reported levels of lycopene in other microorganisms transformed with the three genes. These results suggest the excellence of using the synthetic crt genes and the ADH2 promoter in generation of recombinant S. cerevisiae that produces a high level of lycopene. The level of ergosterol was reversely correlated to that of lycopene in crt-transformed S. cerevisiae cells, suggesting that two pathways for lycopene and ergosterol syntheses compete for the use of farnesyl diphosphate.

GGPPS1 predicts the biological character of hepatocellular carcinoma in patients with cirrhosis.

BACKGROUND: Hepatocellular carcinoma (HCC) has been associated with diabetes and obesity, but a possible connection with the metabolic syndrome (MetS) and its potential interaction with hepatitis and cirrhosis are open to discussion. Our previous investigations have shown that GGPPS1 plays a critical role during hyperinsulinism. In this report, the expression and distribution of GGPPS1 in liver cancer, and its clinical significance were investigated. METHODS: 70 patients with hepatocellular carcinoma (HCC) were included in this study. Three different types of tissues from each HCC patient were assembled immediately after surgical resection: tumor-free tissue >5 cm far from tumor edge (TF), adjacent nonmalignant tissue within 2 cm (AT), and tissue from the tumor (TT). Normal liver tissues from 10 liver transplant donors served as healthy control (HC) while 10 patients with liver cirrhosis as cirrhosis control (CC). The expression and distribution of GGPPS1 were detected by immunohistochemistry, western blots, or real-time PCR. The relationship between the expression of GGPPS1 and clinic pathologic index were analyzed. RESULTS: We found that GGPPS1 was intensified mainly in the cytoplasm of liver tumor cells. Both the expression of GGPPS1 mRNA and protein were upregulated in TT comparing to AT or TF. Meanwhile, HCC patients with cirrhosis had relative higher expression of GGPPS1. In addition, many pathologic characters show close correlation with GGPPS1, such as tumor stage, vessel invasion, and early recurrence. CONCLUSION: GGPPS1 may play a critical role during the development of HCC from cirrhosis and is of clinical significance for predicting biological character of HCC.

Cloning, expression and characterization of an insect geranylgeranyl diphosphate synthase from Choristoneura fumiferana.

Geranylgeranyl diphosphate synthase (GGPPS) catalyzes the condensation of the non-allylic diphosphate, isopentenyl diphosphate (IPP; C5), with allylic diphosphates to generate the C20 prenyl chain (GGPP) used for protein prenylation and diterpenoid biosynthesis. Here, we cloned the cDNA of a GGPPS from the spruce budworm, Choristoneura fumiferana, and characterized the corresponding recombinant protein (rCfGGPPS). As shown for other type-III GGPPSs, rCfGGPPS preferred farnesyl diphosphate (FPP; C15) over other allylic substrates for coupling with IPP. Unexpectedly, rCfGGPPS displayed inhibition by its FPP substrate at low IPP concentration, suggesting the existence of a mechanism that may regulate intracellular FPP pools. rCfGGPPS was also inhibited by its product, GGPP, in a competitive manner with respect to FPP, as reported for human and bovine brain GGPPSs. A homology model of CfGGPPS was prepared and compared to human and yeast GGPPSs. Consistent with its enzymological properties, CfGGPPS displayed a larger active site cavity that can accommodate the binding of FPP and GGPP in the region normally occupied by IPP and the allylic isoprenoid tail, and the binding of GGPP in an alternate orientation seen for GGPP binding to the human protein. To begin exploring the role of CfGGPPS in protein prenylation, its transcripts were quantified by qPCR in whole insects, along with those of other genes involved in this pathway. CfGGPPS was expressed throughout insect development and the abundance of its transcripts covaried with that of other prenylation-related genes. Our qPCR results suggest that geranylgeranylation is the predominant form of prenylation in whole C. fumiferana.

AuaA, a membrane-bound farnesyltransferase from Stigmatella aurantiaca, catalyzes the prenylation of 2-methyl-4-hydroxyquinoline in the biosynthesis of aurachins.

Aurachins are quinoline alkaloids isolated from the myxobacterium Stigmatella aurantiaca. They are substituted with an isoprenoid side chain and act as potent inhibitors in the electron transport chain. A biosynthetic gene cluster that contains at least five genes (auaA-auaE) has been identified for aurachin biosynthesis. In this study, auaA, the gene encoding a putative prenyltransferase of 326 amino acids, was cloned and overexpressed in Escherichia coli. Biochemical investigations showed that AuaA catalyzes the prenylation of 2-methyl-4-hydroxyquinoline in the presence of farnesyl diphosphate (FPP), thereby resulting in the formation of aurachin D. The hydroxyl group at position C4 of the quinoline ring is essential for an acceptance by AuaA; this was concluded by testing 18 quinoline derivatives or analogues with AuaA and FPP. (1) H NMR and HR-EI-MS analyses of six isolated enzyme products revealed the presence of a farnesyl moiety at position C3 of the quinoline ring. K(M) values of 43 and 270 µM were determined for FPP and 2-methyl-4-hydroxyquinoline, respectively. Like other known membrane-bound prenyltransferases, the reaction catalyzed by AuaA is dependent on the presence of metal ions such as Mg(2+) , Mn(2+) and Co(2+) , although no typical (N/D)DXXD binding motif was found in the sequence.

Isolation and functional analysis of two Cistus creticus cDNAs encoding geranylgeranyl diphosphate synthase.

Cistus creticus ssp. creticus is an indigenous shrub of the Mediterranean area. The glandular trichomes covering its leaf surfaces secrete a resin called "ladanum", which among others contains a number of specific labdane-type diterpenes that exhibit antibacterial and antifungal action as well as in vitro and in vivo cytotoxic and cytostatic activity against human cancer cell lines. In view of the properties and possible future exploitation of these metabolites, it was deemed necessary to study the geranylgeranyl diphosphate synthase enzyme (GGDPS, EC 2.5.1.30), a short chain prenyltransferase responsible for the synthesis of the precursor molecule of all diterpenes. In this work, we present the cloning, functional characterisation and expression profile at the gene and protein levels of two differentially expressed C. creticus full-length cDNAs, CcGGDPS1 and CcGGDPS2. Heterologous yeast cell expression system showed that these cDNAs exhibited GGDPS enzyme activity. Gene and protein expression analyses suggest that this enzyme is developmentally and tissue-regulated showing maximum expression in trichomes and smallest leaves (0.5-1.0cm). This work is the first attempt to study the terpenoid biosynthesis at the molecular level in C. creticus ssp. creticus.

Inhibition of geranylgeranyl diphosphate synthase induces apoptosis through multiple mechanisms and displays synergy with inhibition of other isoprenoid biosynthetic enzymes.

Inhibitors of isoprenoid synthesis are widely used for treatment of human diseases, including hypercholesterolemia and osteoporosis, and they have the potential to be useful for treatment of cancer. Statin drugs inhibit the enzyme HMG-CoA reductase, whereas nitrogenous bisphosphonates have more recently been shown to inhibit farnesyl disphosphate synthase. In addition, our laboratory has recently developed several potent and specific bisphosphonate inhibitors of geranylgeranyl diphosphate synthase, including digeranyl bisphosphonate. Because all three enzymes fall in the same biosynthetic pathway and many of the biological effects are due to depletion of downstream products, we hypothesized that simultaneous inhibition of these enzymes would result in synergistic growth inhibition. In this study, we show that inhibition of geranylgeranyl diphosphate synthase induces apoptosis in K562 leukemia cells. This induction of apoptosis is in part dependent upon both geranylgeranyl diphosphate depletion and accumulation of farnesyl diphosphate. Combinations of either lovastatin or zoledronate with digeranyl bisphosphonate synergistically inhibited growth and induced apoptosis. These combinations also potently inhibited cellular geranylgeranylation. These results support the potential for combinations of multiple inhibitors of isoprene biosynthesis to inhibit cancer cell growth or metastasis at clinically achievable concentrations.

Cloning and expression of a geranylgeranyl diphosphate synthase gene: insights into the synthesis of termite defence secretion.

In Nasutitermes takasagoensis, a termite in which soldiers perform specialized chemical defence, Nts19-1 gene is highly expressed exclusively in soldier head. In this study, two types of transcripts for this gene were obtained, and the full-length cDNAs were determined by rapid amplification of cDNA ends (RACE). These transcripts were putative homologues of the geranylgeranyl diphosphate (GGPP) synthase gene, involved in the condensation of dimethylallyl diphosphate with isopentenyl diphosphate in the isoprenoid biosynthetic pathway. The genes were thus termed NtGGPPS1. GGPP is a precursor of diterpenes in plants. In situ hybridization localized NtGGPPS1 expression to the epidermal secretory cells of the frontal gland reservoir where many kinds of diterpenes are produced, suggesting that NtGGPPS1 is involved in the biosynthesis of defence secretion.

Geranylgeranyl-pyrophosphate (GGPP) synthase is down-regulated during differentiation of osteoblastic cell line MC3T3-E1.

Isoprenylation of geranylgeranyl-pyrophosphate (GGPP) is critical for activation of small GTPases. We examined the roles of GGPP synthase (GGPPS) during the differentiation induced by the cell-to-cell contact in osteoblastic cell line MC3T3-E1 cells. We found that (1) both mRNA and protein expression of GGPPS was reduced with decrement of its activity during the differentiation, (2) GGOH, which is converted to GGPP in the cells, inhibited differentiation. These results suggest that the decrement of GGPP is critical for the cell-to-cell contact-induced differentiation, in which the down-regulation of GGPPS might be involved.