Farnesyl Pyrophosphate Synthase Is an Essential Enzyme in Trypanosoma brucei

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We report the cloning of a Trypanosoma cruzi gene encoding a solanesyl-diphosphate synthase, TcSPPS. The amino acid sequence (molecular mass ϳ 39 kDa) is homologous to polyprenyl-diphosphate synthases from different organisms, showing the seven conserved motifs and the typical hydrophobic profile. TcSPPS preferred geranylgeranyl diphosphate as the allylic substrate. The final product, as determined by TLC, had nine isoprene units. This suggests that the parasite synthesizes mainly ubiquinone-9 (UQ-9), as described for Trypanosoma brucei and Leishmania major. In fact, that was the length of the ubiquinone extracted from epimastigotes, as determined by high-performance liquid chromatography. Expression of TcSPPS was able to complement an Escherichia coli ispB mutant. A punctuated pattern in the cytoplasm of the parasite was detected by immunofluorescence analysis with a specific polyclonal antibody against TcSPPS. An overlapping fluorescence pattern was observed using an antibody directed against the glycosomal marker pyruvate phosphate dikinase, suggesting that this step of the isoprenoid biosynthetic pathway is located in the glycosomes. Co-localization in glycosomes was confirmed by immunogold electron microscopy and subcellular fractionation. Because UQ has a central role in energy production and in reoxidation of reduction equivalents, TcSPPS is promising as a new chemotherapeutic target.

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

A Solanesyl-diphosphate Synthase Localizes in Glycosomes of Trypanosoma cruzi

Ferella

Montalvetti

Rohloff

et al. 2006

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“…Both of these genes are single copy. RNA interference experiments in T. brucei show that the FPPS gene is essential (9), as it appears to be in many organisms (10,11).…”

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confidence: 99%

“…Genes encoding FPPS and GGPPS, also referred as short chain prenyltransferases, have been cloned from various species, including rats (2), humans (3,4), Saccharomyces cerevisiae (5), and plants (6,7). In protist parasites, the FPPS gene has been cloned from Trypanosoma cruzi (8) and Trypanosoma brucei (9). Both of these genes are single copy.…”

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confidence: 99%

The Farnesyl-diphosphate/Geranylgeranyl-diphosphate Synthase of Toxoplasma gondii Is a Bifunctional Enzyme and a Molecular Target of Bisphosphonates

Ling

Miranda

et al. 2007

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115

Farnesyl-diphosphate synthase (FPPS) catalyzes the synthesis of farnesyl diphosphate, an important precursor of sterols, dolichols, ubiquinones, and prenylated proteins. We report the cloning and characterization of two Toxoplasma gondii farnesyl-diphosphate synthase (TgFPPS) homologs. A single genetic locus produces two transcripts, TgFPPS and TgFPPSi, by alternative splicing. Both isoforms were heterologously expressed in Escherichia coli, but only TgFPPS was active. The protein products predicted from the nucleotide sequences have 646 and 605 amino acids and apparent molecular masses of 69.5 and 64.5 kDa, respectively. Several conserved sequence motifs found in other prenyl-diphosphate synthases are present in both TgFPPSs. TgFPPS was also expressed in the baculovirus system and was biochemically characterized. In contrast to the FPPS of other eukaryotic organisms, TgFPPS is bifunctional, catalyzing the formation of both farnesyl diphosphate and geranylgeranyl diphosphate. TgFPPS localizes to the mitochondria, as determined by the co-localisation of the affinity-purified antibodies against the protein with MitoTracker, and in accord with the presence of an N-terminal mitochondria-targeting signal in the protein. This enzyme is an attractive target for drug development, because the order of inhibition of the enzyme by a number of bisphosphonates is the same as that for inhibition of parasite growth. In summary, we report the first bifunctional farnesyl-diphosphate/geranylgeranyldiphosphate synthase identified in eukaryotes, which, together with previous results, establishes this enzyme as a valid target for the chemotherapy of toxoplasmosis.Toxoplasma gondii is a pathogenic protozoan parasite that infects a wide range of vertebrate hosts, including humans. T. gondii has been recognized as a major opportunistic pathogen of fetuses from recently infected mothers and of immunocompromised patients, i.e. those with AIDS. Toxoplasmic encephalitis is associated with high mortality and morbidity and is one of the most common opportunistic infections of the central nervous system in human immunodeficiency virus-infected patients (1). The chemotherapy for toxoplasmosis is not ideal especially for the AIDS patient because there is relapse of the infection if the treatment is halted because of intolerance of the patient to the side effects. Almost all the drugs in use have toxicity associated with their continued use. In addition, most of the recommended treatments do not have an effect on the slow growing bradyzoite forms.Isoprenoids are the most diverse and abundant compounds occurring in nature. Many types of isoprenoids (e.g. steroids, cholesterol, retinoids, carotenoids, ubiquinones, and prenyl groups bound to proteins) are essential components of the cellular machinery of all organisms because of their roles in a variety of biological processes. Despite their structural and functional variety, all isoprenoids derive from a common precursor, isopentenyl diphosphate (IPP), 2 and its isomer, dimethylallyl diphosphate (...

“…1C). Against parasitic organisms (8,9) these agents have been shown in vitro to disrupt cell growth through FPPS inhibition. In people, bisphosphonates are targeted to bone tissue (10) where FPPS inhibition in bone-resorbing osteoclasts is a current therapeutic approach for treating postmenopausal osteoporosis (11,12).…”

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confidence: 99%

Structural Basis for Bisphosphonate-mediated Inhibition of Isoprenoid Biosynthesis

Hosfield¹,

Zhang²,

Dougan³

et al. 2004

252

351

Farnesyl pyrophosphate synthetase (FPPS) synthesizes farnesyl pyrophosphate through successive condensations of isopentyl pyrophosphate with dimethylallyl pyrophosphate and geranyl pyrophosphate. Nitrogen-containing bisphosphonate drugs used to treat osteoclast-mediated bone resorption and tumor-induced hypercalcemia are potent inhibitors of the enzyme. Here we present crystal structures of substrate and bisphosphonate complexes of FPPS. The structures reveal how enzyme conformational changes organize conserved active site residues to exploit metal-induced ionization and substrate positioning for catalysis. The structures further demonstrate how nitrogen-containing bisphosphonates mimic a carbocation intermediate to inhibit the enzyme. Together, these FPPS complexes provide a structural template for the design of novel inhibitors that may prove useful for the treatment of osteoporosis and other clinical indications including cancer.Post-translational modification of C-terminal CAAX sequences by covalent attachment of isoprenyl chains is crucial for intracellular localization and proper function of small GTPases such as Ras, Rac, Rho, and CDC42 (1, 2). The substrates for these modifications are the 15-carbon isoprenoid farnesyl pyrophosphate (FPP) 1 or the 20-carbon isoprenoid geranyl-geranyl pyrophosphate synthesized by enzymes of the mevalonate pathway (3) (Fig. 1A). A key branch point enzyme of the mevalonate pathway is farnesyl pyrophosphate synthetase (FPPS), a ϳ30-kDa Mg 2ϩ -dependent homodimeric enzyme that synthesizes (E,E)-FPP from isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) (4, 5) (Fig. 1B). Interest in understanding FPPS activity stems from the recent discovery that FPPS is the molecular target of nitrogencontaining bisphosphonates (6,7,31,32). Bisphophonates are non-cleavable pyrophosphate (P-O-P) analogues in which the central oxygen is replaced by a carbon (P-C-P) with various side chains (Fig. 1C). Against parasitic organisms (8, 9) these agents have been shown in vitro to disrupt cell growth through FPPS inhibition. In people, bisphosphonates are targeted to bone tissue (10) where FPPS inhibition in bone-resorbing osteoclasts is a current therapeutic approach for treating postmenopausal osteoporosis (11,12). Because of their bone-targeting properties, bisphosphonates have also found use as agents to treat tumor-induced hypercalcemia (13), Paget's disease (14), and osteolytic metastases (15).Although structures of apo-and ligand-bound avian FPPS have been solved (16,17), the active sites are unassembled and do not provide substantial information concerning catalysis. Thus, to resolve the molecular basis of catalysis, and also to understand the structural features governing bisphosphonate recognition, we determined the structures of unliganded Staphylococcus aureus FPPS (FPPS-Sa), as well as two Escherichia coli FPPS (FPPS-Ec) ternary complexes. These ternary complexes include a 2.4-Å "substrate-bound" structure containing IPP and the noncleavable DMAPP analogue dimethyla...