Purification and characterization of recombinant human farnesyl diphosphate synthase expressed in <i>Escherichia coli</i>

Ding, Victor D.-H.; Sheares, B T; Bergstrom, James D.; Ponpipom, Mitree M.; Perez, Lawrence; Poulter, C. Dale

doi:10.1042/bj2750061

Cited by 24 publications

(19 citation statements)

References 32 publications

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“…As shown in Table I and Fig. 4A, the protein we have expressed has optimum activity in the presence of 0.5-10 mM MgCl 2 and in the pH range 7.4 -8.5, basically as found with the human enzyme (41). In addition, our results show that TbFPPS is potently inhibited by a number of bisphosphonates (Table II).…”

Section: Discussionsupporting

confidence: 56%

Farnesyl Pyrophosphate Synthase Is an Essential Enzyme in Trypanosoma brucei

Montalvetti

Fernández

Sanders

et al. 2003

Journal of Biological Chemistry

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We report the cloning and sequencing of a gene encoding the farnesyl pyrophosphate synthase (FPPS) of Trypanosoma brucei. The protein (TbFPPS) is an attractive target for drug development because the growth of T. brucei has been shown to be inhibited by analogs of its substrates, the nitrogen containing bisphosphonates currently in use in bone resorption therapy. The protein predicted from the nucleotide sequence of the gene has 367 amino acids and a molecular mass of 42 kDa. Several sequence motifs found in other FPPSs are present in TbFPPS, including an 11-mer peptide insertion present also in the Trypanosoma cruzi FPPS. Heterologous expression of TbFPPS in Escherichia coli produced a functional enzyme that was inhibited by several nitrogencontaining bisphosphonates, such as pamidronate and risedronate. Risedronate was active in vivo against T. brucei infection in mice (giving a 60% survival rate), but pamidronate was not effective. The essential nature of TbFPPS was studied using RNA interference (RNAi) to inhibit the expression of the gene. Expression of TbF-PPS double-stranded RNA in procyclic trypomastigotes caused specific degradation of mRNA. After 4 days of RNAi, the parasite growth rate declined and the cells subsequently died. Similar results were obtained with bloodstream form trypomastigotes, except that the RNAi system in this case was leaky and mRNA levels and parasites recovered with time. Molecular modeling and structure-activity investigations of enzyme and in vitro growth inhibition data resulted in similar pharmacophores, further validating TbFPPS as the target for bisphosphonates. These results establish that FPPS is essential for parasite viability and validate this enzyme as a target for drug development.The Trypanosoma brucei group of parasites causes African trypanosomiasis (sleeping sickness) in humans, and nagana in animals and is responsible for heavy socioeconomic losses in most countries of sub-Saharan Africa (1). Therapy against African sleeping sickness is unsatisfactory because of the toxicity of currently available drugs, together with the development of drug resistance (2).A number of bisphosphonates have recently been shown to have significant activity against the proliferation of T. brucei and other parasites in vitro (3, 4) and also have curative effects in in vivo models of visceral (5) and cutaneous (6) leishmaniasis. Bisphosphonates are pyrophosphate analogs in which the oxygen bridge between the two phosphorus atoms has been replaced by a carbon substituted with various side chains. Several bisphosphonates are potent inhibitors of bone resorption and are in clinical use for the treatment and prevention of osteoporosis, Paget's disease, hypercalcemia caused by malignancy, and tumor metastases in bone (7-10). Many bisphosphonates, such as pamidronate, alendronate, and risedronate, are known to inhibit farnesyl pyrophosphate synthase (FPPS) 1 (11-16), and in so doing, they inhibit the formation of farnesyl pyrophosphate, a compound used in protein prenylation and in the syn...

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Section: Discussionsupporting

confidence: 56%

Farnesyl Pyrophosphate Synthase Is an Essential Enzyme in Trypanosoma brucei

Montalvetti

Fernández

Sanders

et al. 2003

Journal of Biological Chemistry

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“…As shown in Table I and Fig. 5A, the protein we have expressed has optimum activity in the presence of 1-5 mM MgCl 2 and in the pH range 8 -9, basically as found with the human enzyme (40). In addition, our results show that TcFPPS is potently inhibited by a number of bisphosphonates (Table II).…”

Section: Isolation Of the Tcfppssupporting

confidence: 56%

Bisphosphonates Are Potent Inhibitors of Trypanosoma cruzi Farnesyl Pyrophosphate Synthase

Montalvetti¹,

Bailey²,

Martin³

et al. 2001

Journal of Biological Chemistry

148

163

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We report the cloning and sequencing of a gene encoding the farnesyl pyrophosphate synthase of Trypanosoma cruzi. The protein (T. cruzi farnesyl pyrophosphate synthase, TcFPPS) is an attractive target for drug development, since the growth of T. cruzi is inhibited by carbocation transition state/reactive intermediate analogs of its substrates, the nitrogen-containing bisphosphonates currently in use in bone resorption therapy. The protein predicted from the nucleotide sequence of the gene has 362 amino acids and a molecular mass of 41.2 kDa. Several sequence motifs found in other FPPSs are present in TcFPPS. Heterologous expression of TcF-PPS in Escherichia coli produced a functional enzyme that was inhibited by the nitrogen-containing bisphosphonates alendronate, pamidronate, homorisedronate, and risedronate but was less sensitive to the non-nitrogen-containing bisphosphonate etidronate, which, unlike the nitrogen-containing bisphosphonates, does not affect parasite growth. The protein contains a unique 11-mer insertion located near the active site, together with other sequence differences that may facilitate the development of novel anti-Chagasic agents.

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“…The kinetics of FPP synthase has been reported previously (8,19) specifically for the condensation of IPP and DMAPP in a 2:1 molar ratio. However, a complete kinetic analysis of FPP formation through the discrete GPP intermediate has not been done.…”

Section: Resultsmentioning

confidence: 99%

Preparation, Characterization, and Optimization of an In Vitro C₃₀Carotenoid Pathway

Jeong

Mijts

et al. 2005

Appl Environ Microbiol

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The ispA gene encoding farnesyl pyrophosphate (FPP) synthase from Escherichia coli and the crtM gene encoding 4,4-diapophytoene (DAP) synthase from Staphylococcus aureus were overexpressed and purified for use in vitro. Steady-state kinetics for FPP synthase and DAP synthase, individually and in sequence, were determined under optimized reaction conditions. For the two-step reaction, the DAP product was unstable in aqueous buffer; however, in situ extraction using an aqueous-organic two-phase system resulted in a 100% conversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate into DAP. This aqueous-organic two-phase system is the first demonstration of an in vitro carotenoid synthesis pathway performed with in situ extraction, which enables quantitative conversions. This approach, if extended to a wide range of isoprenoidbased pathways, could lead to the synthesis of novel carotenoids and their derivatives.Carotenoids are naturally occurring pigments found in a wide variety of plants and microorganisms (22,23). Recent studies indicate that these isoprenoid-based natural products possess biologically active properties (21, 24), thereby making them of interest to the medicinal chemist. Most carotenoids belong to the C 30 and C 40 classes, being distinguished by the number of iterative steps of isopentenyl pyrophosphate (IPP) condensation to add C 5 isoprene units. The initial synthetic step is the condensation of IPP with ␥,␥-dimethylallyl pyrophosphate (DMAPP) catalyzed by farnesyl pyrophosphate (FPP) synthase (ispA) to give geranyl pyrophosphate (GPP) and, sequentially, FPP (9). This C 15 compound serves as a central node for the synthesis of sterols (29), farnesylated proteins (4), hemes (20), sesquiterpenes (6), and dolichols (5), as well as the crucial precursor for carotenoids.C 30 carotenoids are present in the nonphotosynthetic bacteria, such as Streptococcus faecium, Staphylococcus aureus, and Methylobacterium rhodinum, and in the photosynthetic Heliobacterium species (16,25,27,28). Only the genes encoding 4,4Ј-diapophytoene (DAP) synthase (crtM) and 4,4Ј-diapophytoene desaturase (crtN) from S. aureus have been cloned and functionally expressed in Escherichia coli, resulting in the yellow 4,4Ј-diaponeurosporene from FPP (30) (Fig. 1). The first committed step in C 30 carotenoid biosynthesis is the condensation of two molecules of FPP catalyzed by DAP synthase to form the colorless carotenoid 4,4Ј-diapophytoene.Until now, only a few reports have discussed the formation of DAP through a reconstituted C 30 carotenoid metabolic pathway in vitro (13, 17), and none have provided a full kinetic analysis or reaction optimization of the in vitro pathway. Nevertheless, such a study would be valuable in order to design novel carotenoids and carotenoid-based hybrid natural products. Therefore, in the current work, we set out to optimize the C 30 carotenoid pathway reconstructed in vitro for the synthesis of DAP from IPP and DMAPP. Furthermore, we elucidated the kinetic behavior of both the FPP synthase a...

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Purification and characterization of recombinant human farnesyl diphosphate synthase expressed in Escherichia coli

Cited by 24 publications

References 32 publications

Farnesyl Pyrophosphate Synthase Is an Essential Enzyme in Trypanosoma brucei

Farnesyl Pyrophosphate Synthase Is an Essential Enzyme in Trypanosoma brucei

Bisphosphonates Are Potent Inhibitors of Trypanosoma cruzi Farnesyl Pyrophosphate Synthase

Preparation, Characterization, and Optimization of an In Vitro C₃₀Carotenoid Pathway

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Purification and characterization of recombinant human farnesyl diphosphate synthase expressed in Escherichia coli

Cited by 24 publications

References 32 publications

Farnesyl Pyrophosphate Synthase Is an Essential Enzyme in Trypanosoma brucei

Farnesyl Pyrophosphate Synthase Is an Essential Enzyme in Trypanosoma brucei

Bisphosphonates Are Potent Inhibitors of Trypanosoma cruzi Farnesyl Pyrophosphate Synthase

Preparation, Characterization, and Optimization of an In Vitro C30Carotenoid Pathway

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Preparation, Characterization, and Optimization of an In Vitro C₃₀Carotenoid Pathway