Cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from Plasmodium falciparum

Abstract Background Isoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites. Methods The isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis–Menten; also, inhibition assays were performed using risedronate. Results The expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68 ± 5 μM, 7.8 ± 1.3 μM and 2.06 ± 0.4 μM. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate. Conclusions The rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria.

Geranyl diphosphate synthase: Cloning, expression, and characterization of this prenyltransferase as a heterodimer

Geranyl diphosphate synthase, which catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to geranyl diphosphate, the key precursor of monoterpene biosynthesis, was purified from isolated oil glands of spearmint. Peptide fragments generated from the pure proteins of 28 and 37 kDa revealed amino acid sequences that matched two cDNA clones obtained by random screening of a peppermint-oil gland cDNA library. The deduced sequences of both proteins showed some similarity to existing prenyltransferases, and both contained a plastid-targeting sequence. Expression of each cDNA individually yielded no detectable prenyltransferase activity; however, coexpression of the two together produced functional geranyl diphosphate synthase. Antibodies raised against each protein were used to demonstrate that both subunits were required to produce catalytically active native and recombinant enzymes, thus confirming that geranyl diphosphate synthase is a heterodimer.

Crystal structure of cis-prenyl chain elongating enzyme, undecaprenyl diphosphate synthase

Undecaprenyl diphosphate synthase (UPS) catalyzes the cis-prenyl chain elongation onto trans, trans-farnesyl diphosphate (FPP) to produce undecaprenyl diphosphate (UPP), which is indispensable for the biosynthesis of bacterial cell walls. We report here the crystal structure of UPS as the only three-dimensional structure among cis-prenyl chain elongating enzymes. The structure is classified into a protein fold family and is completely different from the so-called “isoprenoid synthase fold” that is believed to be a common structure for the enzymes relating to isoprenoid biosynthesis. Conserved amino acid residues among cis-prenyl chain elongating enzymes are located around a large hydrophobic cleft in the UPS structure. A structural P-loop motif, which frequently appears in the various kinds of phosphate binding site, is found at the entrance of this cleft. The catalytic site is determined on the basis of these structural features, from which a possible reaction mechanism is proposed.

Chrysanthemyl diphosphate synthase: Isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium

Chrysanthemyl diphosphate synthase (CPPase) catalyzes the condensation of two molecules of dimethylallyl diphosphate to produce chrysanthemyl diphosphate (CPP), a monoterpene with a non-head-to-tail or irregular c1′-2-3 linkage between isoprenoid units. Irregular monoterpenes are common in Chrysanthemum cinerariaefolium and related members of the Asteraceae family. In C. cinerariaefolium, CPP is an intermediate in the biosynthesis of the pyrethrin ester insecticides. CPPase was purified from immature chrysanthemum flowers, and the N terminus of the protein was sequenced. A C. cinerariaefolium λ cDNA library was screened by using degenerate oligonucleotide probes based on the amino acid sequence to identify a CPPase clone that encoded a 45-kDa preprotein. The first 50 aa of the ORF constitute a putative plastidial targeting sequence. Recombinant CPPase bearing an N-terminal polyhistidine affinity tag in place of the targeting sequence was purified to homogeneity from an overproducing Escherichia coli strain by Ni2+ chromatography. Incubation of recombinant CPPase with dimethylallyl diphosphate produced CPP. The diphosphate ester was hydrolyzed by alkaline phosphatase, and the resulting monoterpene alcohol was analyzed by GC/MS to confirm its structure. The amino acid sequence of CPPase aligns closely with that of the chain elongation prenyltransferase farnesyl diphosphate synthase rather than squalene synthase or phytoene synthase, which catalyze c1′-2-3 cyclopropanation reactions similar to the CPPase reaction.

Organellar and Cytosolic Localization of Four Phosphoribosyl Diphosphate Synthase Isozymes in Spinach

Four cDNAs encoding phosphoribosyl diphosphate (PRPP) synthase were isolated from a spinach (Spinacia oleracea) cDNA library by complementation of an Escherichia coli Δprs mutation. The four gene products produced PRPP in vitro from ATP and ribose-5-phosphate. Two of the enzymes (isozymes 1 and 2) required inorganic phosphate for activity, whereas the others were phosphate independent. PRPP synthase isozymes 2 and 3 contained 76 and 87 amino acid extensions, respectively, at their N-terminal ends in comparison with other PRPP synthases. Isozyme 2 was synthesized in vitro and shown to be imported and processed by pea (Pisum sativum) chloroplasts. Amino acid sequence analysis indicated that isozyme 3 may be transported to mitochondria and that isozyme 4 may be located in the cytosol. The deduced amino acid sequences of isozymes 1 and 2 and isozymes 3 and 4 were 88% and 75% identical, respectively. In contrast, the amino acid identities of PRPP synthase isozyme 1 or 2 with 3 or 4 was modest (22%–25%), but the sequence motifs for binding of PRPP and divalent cation-nucleotide were identified in all four sequences. The results indicate that PRPP synthase isozymes 3 and 4 belong to a new class of PRPP synthases that may be specific to plants.

Sterol regulatory element binding protein binds to a cis element in the promoter of the farnesyl diphosphate synthase gene.

Sterol-regulated transcription of the gene for rat farnesyl diphosphate (FPP) synthase (geranyl-diphosphate:isopentenyl-diphosphate geranyltranstransferase, EC 2.5.1.10) is dependent in part on the binding of the ubiquitous transcription factor NF-Y to a 6-bp element within the proximal promoter. Current studies identify a second element in this promoter that is also required for sterol-regulated transcription in vivo. Mutation of three nucleotides (CAC) within this element blocks the 8-fold induction of FPP synthase promoter-reporter genes that normally occurs when the transfected cells are incubated in medium deprived of sterols. Gel mobility-shift assays demonstrate that the transcriptionally active 68-kDa fragment of the sterol regulatory element (SRE-1)-binding protein assays (SREBP-1) binds to an oligonucleotide containing the wild-type sequence but not to an oligonucleotide in which the CAC has been mutated. DNase 1 protection pattern (footprint) analysis indicates that SREBP-1 binds to nucleotides that include the CAC. Both the in vivo and in vitro assays are affected by mutagenesis of nucleotides adjacent to the CAC. Coexpression of SREBP with a wild-type FPP synthase promoter-reporter gene in CV-1 cells results in very high levels of reporter activity that is sterol-independent. In contrast, the reporter activity remained low when the promoter contained a mutation in the CAC trinucleotide. We conclude that sterol-regulated transcription of FPP synthase is controlled in part by the interaction of SREBP with a binding site that we have termed SRE-3. Identification of this element may prove useful in the identification of other genes that are both regulated by SREBP and involved in lipid biosynthesis.

Regulation of product chain length by isoprenyl diphosphate synthases

An analysis of the x-ray structure of homodimeric avian farnesyl diphosphate synthase (geranyltransferase, EC 2.5.1.10) coupled with information about conserved amino acids obtained from a sequence alignment of 35 isoprenyl diphosphate synthases that synthesize farnesyl (C15), geranylgeranyl (C20), and higher chain length isoprenoid diphosphates suggested that the side chains of residues corresponding to F112 and F113 in the avian enzyme were important for determining the ultimate length of the hydrocarbon chains. This hypothesis was supported by site-directed mutagenesis to transform wild-type avian farnesyl diphosphate synthase (FPS) into synthases capable of producing geranylgeranyl diphosphate (F112A), geranylfarnesyl (C25) diphosphate (F113S), and longer chain prenyl diphosphates (F112A/F113S). An x-ray analysis of the structure of the F112A/F113S mutant in the apo state and with allylic substrates bound produced the strongest evidence that these mutations caused the observed change in product specificity by directly altering the size of the binding pocket for the growing isoprenoid chain in the active site of the enzyme. The proposed binding pocket in the apo mutant structure was increased in depth by 5.8 Å as compared with that for the wild-type enzyme. Allylic diphosphates were observed in the holo structures, bound through magnesium ions to the aspartates of the first of two conserved aspartate-rich sequences (D117–D121), with the hydrocarbon tails of all the ligands growing down the hydrophobic pocket toward the mutation site. A model was constructed to show how the growth of a long chain prenyl product may proceed by creation of a hydrophobic passageway from the FPS active site to the outside surface of the enzyme.

The First Step of Gibberellin Biosynthesis in Pumpkin Is Catalyzed by at Least Two Copalyl Diphosphate Synthases Encoded by Differentially Regulated Genes

The first step in gibberellin biosynthesis is catalyzed by copalyl diphosphate synthase (CPS) and ent-kaurene synthase. We have cloned from pumpkin (Cucurbita maxima L.) two cDNAs, CmCPS1 and CmCPS2, that each encode a CPS. Both recombinant fusion CmCPS proteins were active in vitro. CPS are translocated into plastids and processed by cleavage of transit peptides. For CmCPS1 and CmCPS2, the putative transit peptides cannot exceed the first 99 and 107 amino acids, respectively, because longer N-terminal deletions abolished activity. Levels of both CmCPS transcripts were strictly regulated in an organ-specific and developmental manner. Both transcripts were almost undetectable in leaves and were abundant in petioles. CmCPS1 transcript levels were high in young cotyledons and low in roots. In contrast, CmCPS2 transcripts were undetectable in cotyledons but present at significant levels in roots. In hypocotyls, apices, and petioles, CmCPS1 transcript levels decreased with age much more rapidly than those of CmCPS2. We speculate that CmCPS1 expression is correlated with the early stages of organ development, whereas CmCPS2 expression is correlated with subsequent growth. In contrast, C. maxima ent-kaurene synthase transcripts were detected in every organ at almost constant levels. Thus, ent-kaurene biosynthesis may be regulated through control of CPS expression.

Bisphosphonate Inhibition of Prostate Cancer Cell Invasion, Migration and Cytoskeletal Organization

Metastatic bone lesions are commonly associated with prostate cancer affecting approximately 60-80% of the patients. The progression of prostate cancer into an advanced stage is a complex process and its molecular mechanisms are poorly understood. So far, no curative treatment is available for advanced stages of prostate cancer. Bisphosphonates (BPs) are synthetic pyrophosphate analogues, which are used as therapeutics for various metabolic bone diseases because of their ability to inhibit osteoclastic bone resorption. Nitrogen-containing bisphosphonates block the function of osteoclasts by disturbing the vesicular traffic and the mevalonate pathway -related enzymes, for example farnesyl diphosphate synthase, which is involved in post-translational isoprenylation of small GTPases. In addition, the anti-proliferative, anti-invasive and pro-apoptotic effects of nitrogen-containing bisphosphonates on various cancer cell lines have been reported. The aim of this thesis work was to clarify the effects of bisphosphonates on prostate cancer cells, focusing on the mechanisms of adhesion, invasion and migration. Furthermore, the role of the mevalonate pathway and prenylation reactions in invasion and regulation of the cytoskeleton of prostate cancer cells were examined. Finally, the effects of alendronate on cytoskeleton- and actin-related proteins in prostate cancer cells were studied in vitro and in vivo. The results showed that the nitrogen-containing bisphosphonate alendronate inhibited the adhesion of prostate cancer cells to various extracellular matrix proteins and migration and invasion in vitro. Inhibition of invasion and migration was reversed by mevalonate pathway intermediates. The blockage of the prenylation transferases GGTase I and FTase inhibited the invasion, migration and actin organization of prostate cancer cells. The marked decrease of cofilin was observed by the prenylation inhibitors used. Inhibition of GGTase I also disrupted the regulation of focal adhesion kinase and paxillin. In addition, alendronate disrupted the cytoskeletal organization and decreased the level of cofilin in vitro and in vivo. The decrease of the cofilin level by alendronate could be one of the key mechanisms behind the observed inhibition of migration and invasion. Based on the effects of nitrogen-containing bisphosphonates on tumor cell invasion and cytoskeletal organization, they can be suggested to be developed as therapeutics for inhibiting prostate cancer metastasis.

Genetic dissection of vitamin E biosynthesis in tomato

Vegetables are critical for human health as they are a source of multiple vitamins including vitamin E (VTE). In plants, the synthesis of VTE compounds, tocopherol and tocotrienol, derives from precursors of the shikimate and methylerythritol phosphate pathways. Quantitative trait loci (QTL) for alpha-tocopherol content in ripe fruit have previously been determined in an Solanum pennellii tomato introgression line population. In this work, variations of tocopherol isoforms (alpha, beta, gamma, and delta) in ripe fruits of these lines were studied. In parallel all tomato genes structurally associated with VTE biosynthesis were identified and mapped. Previously identified VTE QTL on chromosomes 6 and 9 were confirmed whilst novel ones were identified on chromosomes 7 and 8. Integrated analysis at the metabolic, genetic and genomic levels allowed us to propose 16 candidate loci putatively affecting tocopherol content in tomato. A comparative analysis revealed polymorphisms at nucleotide and amino acid levels between Solanum lycopersicum and S. pennellii candidate alleles. Moreover, evolutionary analyses showed the presence of codons evolving under both neutral and positive selection, which may explain the phenotypic differences between species. These data represent an important step in understanding the genetic determinants of VTE natural variation in tomato fruit and as such in the ability to improve the content of this important nutriceutical.

Sugarcane Phosphoribosyl Pyrophosphate Synthetase: Molecular Characterization of a Phosphate-independent PRS

Phosphoribosyl pyrophosphate synthetase (PRS-EC:2.7.6.1) is an important enzyme present in several metabolic pathways, thus forming a complex family of isoenzymes. However, plant PRS enzymes have not been extensively investigated. In this study, a sugarcane prs gene has been characterized from the Sugar Cane Expressed Sequence Tag Genome Project. This gene contains a 984-bp open reading frame encoding a 328-amino acid protein. The predicted amino acid sequence has 77% and 78% amino acid sequence identity to Arabidopsis thaliana and Spinacia oleracea PRS4, respectively. The assignment of sugarcane PRS as a phosphate-independent PRS isoenzyme (Class II PRS) is verified following enzyme assay and phylogenetic reconstruction of PRS homologues. To gain further insight into the structural framework of the phosphate independence of sugarcane PRS, a molecular model is described. This model reveals the formation of two conserved domains elucidating the structural features involved in sugarcane PRS phosphate independence. The recombinant PRS retains secondary structure elements and a quaternary arrangement consistent with known PRS homologues, based on circular dichroism measurements.

Proteomic analysis of papaya fruit ripening using 2DE-DIGE

Papayas have a very short green life as a result of their rapid pulp softening as well as their susceptibility to physical injury and mold growth. The ripening-related changes take place very quickly, and there is a continued interest in the reduction of postharvest losses. Proteins have a central role in biological processes, and differential proteomics enables the discrimination of proteins affected during papaya ripening. A comparative analysis of the proteomes of climacteric and pre-climacteric papayas was performed using 2DE-DIGE. Third seven proteins corresponding to spots with significant differences in abundance during ripening were submitted to MS analysis, and 27 proteins were identified and classified into six main categories related to the metabolic changes occurring during ripening. Proteins from the cell wall (alpha-galactosidase and invertase), ethylene biosynthesis (methionine synthase), climacteric respiratory burst, stress response, synthesis of carotenoid precursors (hydroxymethylbutenyl 4-diphosphate synthase, GcpE), and chromoplast differentiation (fibrillin) were identified. There was some correspondence between the identified proteins and the data from previous transcript profiling of papaya fruit, but new, accumulated proteins were identified, which reinforces the importance of differential proteomics as a tool to investigate ripening and provides potentially useful information for maintaining fruit quality and minimizing postharvest losses. (C) 2011 Elsevier B.V. All rights reserved.

Characterization of Juvenile Hormone Biosynthetic Enzymes in the Mosquito, Aedes aegypti

The juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. They are synthesized and secreted by a pair of small endocrine glands, the corpora allata (CA), which are intimately connected to the brain. The enzymes involved in the biosynthesis of JH are attractive targets for the control of mosquito populations. This dissertation is a comprehensive functional study of five Aedes aegypti CA enzymes, HMG-CoA synthase (AaHMGS), mevalonate kinase (AaMK), phosphomevalonate kinase (AaPMK), farnesyl diphosphate synthase (AaFPPS) and farnesyl pyrophosphate phosphatase (AaFPPase). The enzyme AaHMGS catalyzes the condensation of acetoacetyl-CoA and acetyl-CoA to produce HMG-CoA. The enzyme does not require any co-factor, although its activity is enhanced by addition of Mg2+. The enzyme AaMK is a class I mevalonate kinase that catalyzes the ATP-dependent phosphorylation of mevalonic acid to form mevalonate 5-phosphate. Activity of AaMK is inhibited by isoprenoids. The enzyme AaPMK catalyzes the cation-dependent reversible reaction of phosphomevalonate and ATP to form diphosphate mevalonate and ADP. The enzyme AaFPPS catalyzes the condensation of isopentenyl diphosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to form geranyl diphosphate (GPP) and farnesyl pyrophosphate (FPP). The enzyme AaFPPS shows an unusual product regulation mechanism, with chain length final product of 10 or 15 C depending on the metal cofactor present. The enzymes AaFPPase-1 and AaFPPase-2 efficiently hydrolyze FPP into farnesol, although RNAi experiments demonstrate that only AaFPPase-1 is involved in the catalysis of FPP into FOL in the CA of A. aegypti. This dissertation also explored the inhibition of the activity of some of the JH biosynthesis enzymes as tools for insect control. We described the effect of N-acetyl-S-geranylgeranyl-L-cysteine as a potent inhibitor of AaFPPase 1 and AaFPPase-2. In addition, inhibitors of AaMK and AaHMGS were also investigated using purified recombinant proteins. The present study provides an important contribution to the characterization of recombinant proteins, the analysis of enzyme kinetics and inhibition constants, as well as the understanding of the importance of these five enzymes in the control of JH biosynthesis rates.

Electron transfer in acetohydroxy acid synthase as a side reaction of catalysis. implications for the reactivity and partitioning of the carbanion/enamine form of (alpha-hydroxyethyl)thiamin diphosphate in a "nonredox" flavoenzyme

Acetohydroxy acid synthases (AHAS) are thiamin diphosphate- (ThDP-) and FAD-dependent enzymes that catalyze the first common step of branched-chain amino acid biosynthesis in plants, bacteria, and fungi. Although the flavin cofactor is not chemically involved in the physiological reaction of AHAS, it has been shown to be essential for the structural integrity and activity of the enzyme. Here, we report that the enzyme-bound FAD in AHAS is reduced in the course of catalysis in a side reaction. The reduction of the enzyme-bound flavin during turnover of different substrates under aerobic and anaerobic conditions was characterized by stopped-flow kinetics using the intrinsic FAD absorbance. Reduction of enzyme-bound FAD proceeds with a net rate constant of k' = 0.2 s(-1) in the presence of oxygen and approximately 1 s(-1) under anaerobic conditions. No transient flavin radicals are detectable during the reduction process while time-resolved absorbance spectra are recorded. Reconstitution of the binary enzyme-FAD complex with the chemically synthesized intermediate 2-(hydroxyethyl)-ThDP also results in a reduction of the flavin. These data provide evidence for the first time that the key catalytic intermediate 2-(hydroxyethyl)ThDP in the carbanionic/enamine form is not only subject to covalent addition of 2-keto acids and an oxygenase side reaction but also transfers electrons to the adjacent FAD in an intramolecular redox reaction yielding 2-acetyl-ThDP and reduced FAD. The detection of the electron transfer supports the idea of a common ancestor of acetohydroxy acid synthase and pyruvate oxidase, a homologous ThDP- and FAD-dependent enzyme that, in contrast to AHASs, catalyzes a reaction that relies on intercofactor electron transfer.

Crystal structure of yeast acetohydroxyacid synthase: A target for herbicidal inhibitors

Acetohydroxyacid synthase (AHAS; EC 4.1.3.18) catalyzes the first step in branched-chain amino acid biosynthesis. The enzyme requires thiamin diphosphate and FAD for activity, but the latter is unexpected, because the reaction involves no oxidation or reduction. Due to its presence in plants, AHAS is a target for sulfonylurea and imidazolinone herbicides. Here, the crystal structure to 2.6 A resolution of the catalytic subunit of yeast AHAS is reported. The active site is located at the dimer interface and is near the proposed herbicide-binding site. The conformation of FAD and its position in the active site are defined. The structure of AHAS provides a starting point for the rational design of new herbicides. (C) 2002 Elsevier Science Ltd.