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At least 19 recordsLinked to original sources
PubMed1996

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.

Alkyl and Aryl Transferases↗
PubMed2003

Scale-up of Artemisia annua L. hairy root cultures produces complex patterns of terpenoid gene expression.

Hairy roots grow quickly, reach high densities, and can produce significant amounts of secondary metabolites, yet their scale-up to bioreactors remains challenging. Artemisia annua produces a rich array of terpenoids, including the sesquiterpene, artemisinin, and transformed roots of this species provide a good model for studying terpenoid production. These cultures were examined in shake flasks and compared with cultures grown in two types of bioreactors, a mist reactor and a bubble column reactor, which provide very different environments for the growing roots. Mist reactors have been shown previously to result in cultures that produce significantly more artemisinin per gram fresh weight of culture, while bubble column reactors have produced greater biomass. We have compared expression levels of four key terpenoid biosynthetic genes: 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), and farnesyl diphosphate synthase (FPS) in the three culture conditions. In shake flasks we found that although all four genes showed temporal regulation, only FPS expression correlated with artemisinin production. Light also affected the transcription of all four genes. Although expression in reactors was equivalent to or greater than that of roots grown in shake flasks, no correlation was found between expression level within six different zones of each reactor and their respective oxygen levels, light, and root-packing density. Surprisingly, transcriptional regulation of HMGR, DXS, DXR, and FPS was greatly affected by the position of the roots in each reactor. Thus, relying on a single reactor sample to characterize the gene activity in a whole reactor can be misleading, especially if the goal is to examine the difference between reactor types or operating parameters, steps essential in scaling up cultures for production.

Aldose-Ketose Isomerases↗
PubMed2004

Mono and diterpene production in Escherichia coli.

Mono- and diterpenoids are of great industrial and medical value as specialty chemicals and pharmaceuticals. Production of these compounds in microbial hosts, such as Escherichia coli, can be limited by intracellular levels of the polyprenyl diphosphate precursors, geranyl diphosphate (GPP), and geranylgeranyl diphosphate (GGPP). To alleviate this limitation, we constructed synthetic operons that express three key enzymes for biosynthesis of these precursors: (1). DXS,1-deoxy-d-xylulose-5-phosphate synthase; (2). IPPHp, IPP isomerase from Haematococcus pluvialis; and (3). one of two variants of IspA, FPP synthase that produces either GPP or GGPP. The reporter plasmids pAC-LYC and pACYC-IB, which encode enzymes that convert either FPP or GGPP, respectively, to the pigment lycopene, were used to demonstrate that at full induction, the operon encoding the wild-type FPP synthase and mutant GGPP synthase produced similar levels of lycopene. To synthesize di- or monoterpenes in E. coli using the GGPP and GPP encoding operons either a diterpene cyclase [casbene cyclase (Ricinus communis L) and ent-kaurene cyclase (Phaeosphaeria sp. L487)] or a monoterpene cyclase [3-carene cyclase (Picea abies)] was coexpressed with their respective precursor production operon. Analysis of culture extracts or headspace by gas chromatography-mass spectrometry confirmed the in vivo production of the diterpenes casbene, kaur-15-ene, and kaur-16-ene and the monoterpenes alpha-pinene, myrcene, sabinene, 3-carene, alpha-terpinene, limonene, beta-phellandrene, alpha-terpinene, and terpinolene. Construction and functional expression of GGPP and GPP operons provides an in vivo precursor platform host for the future engineering of di- and monoterpene cyclases and the overproduction of terpenes in bacteria.

Alkyl and Aryl Transferases↗
PubMed1994

Isoprenyl diphosphate synthases: protein sequence comparisons, a phylogenetic tree, and predictions of secondary structure.

Isoprenyl diphosphate synthases are ubiquitous enzymes that catalyze the basic chain-elongation reaction in the isoprene biosynthetic pathway. Pairwise sequence comparisons were made for 6 farnesyl diphosphate synthases, 6 geranylgeranyl diphosphate synthases, and a hexaprenyl diphosphate synthase. Five regions with highly conserved residues, two of which contain aspartate-rich DDXX(XX)D motifs found in many prenyltransferases, were identified. A consensus secondary structure for the group, consisting mostly of alpha-helices, was predicted for the multiply aligned sequences from amino acid compositions, computer assignments of local structure, and hydropathy indices. Progressive sequence alignments suggest that the 13 isoprenyl diphosphate synthases evolved from a common ancestor into 3 distinct clusters. The most distant separation is between yeast hexaprenyl diphosphate synthetase and the other enzymes. Except for the chromoplastic geranylgeranyl diphosphate synthase from Capsicum annuum, the remaining farnesyl and geranylgeranyl diphosphate synthases segregate into prokaryotic/archaebacterial and eukaryotic families.

Alkyl and Aryl Transferases↗
PubMed2006

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.

Alendronate↗
PubMed2006

Characterization and tissue-specific expression of two lepidopteran farnesyl diphosphate synthase homologs: implications for the biosynthesis of ethyl-substituted juvenile hormones.

The sesquiterpenoid juvenile hormone (JH) regulates insect development and reproduction. Most insects produce only one chemical form of JH, but the Lepidoptera produce four derivatives featuring ethyl branches. The biogenesis of these JHs requires the synthesis of ethyl-substituted farnesyl diphosphate (FPP) by FPP synthase (FPPS). To determine if there exist more than one lepidopteran FPPS, and whether one FPPS homolog is better adapted for binding the bulkier ethyl-branched substrates/products, we cloned three lepidopteran FPPS cDNAs, two from Choristoneura fumiferana and one from Pseudaletia unipuncta. Amino acid sequence comparisons among these and other eukaryotic FPPSs led to the recognition of two lepidopteran FPPS types. Type-I FPPSs display unique active site substitutions, including several in and near the first aspartate-rich motif, whereas type-II proteins have a more "conventional" catalytic cavity. In a yeast assay, a Drosophila FPPS clone provided full complementation of an FPPS mutation, but lepidopteran FPPS clones of either type yielded only partial complementation, suggesting unusual catalytic features and/or requirements of these enzymes. Although a structural analysis of lepidopteran FPPS active sites suggested that type-I enzymes are better suited than type-II for generating ethyl-substituted products, a quantitative real-time PCR assessment of their relative abundance in insect tissues indicated that type-I expression is ubiquitous whereas that of type-II is essentially confined to the JH-producing glands, where its transcripts are approximately 20 times more abundant than those of type-I. These results suggest that type-II FPPS plays a leading role in lepidopteran JH biosynthesis in spite of its apparently more conventional catalytic cavity.

Amino Acid Sequence↗
PubMed1995

Farnesyl pyrophosphate synthase from white lupin: molecular cloning, expression, and purification of the expressed protein.

Plants produce a variety of sesquiterpenoid compounds with diverse biological functions, whose synthesis is initiated by farnesyl pyrophosphate synthase [EC 2.5.1.1, EC 2.5.1.10]. The lack of availability of molecular tools to analyze this enzyme has, thus far, prevented the study of its expression and regulation in plants. A DNA fragment corresponding to a portion of the farnesyl pyrophosphate synthase gene was amplified by the polymerase chain reaction using was amplified by the polymerase chain reaction using degenerate primers designed from two highly conserved domains (FLV(A/L)DD(I/M)MD and FQIQDDYLD) found in eukaryotic farnesyl pyrophosphate synthase sequences. A clone, pS19, of a 438-bp PCR fragment was used to screen a white lupin root cDNA library. Two full-length cDNA clones (pFPS1 and pFPS2) were isolated and sequenced, and one of them (pFPS2) was expressed in a bacterial system and the enzyme protein encoded by the clone was purified. The 1345-bp insert of pFPS2 contains a 1026-bp open reading frame, encoding a 342-amino-acid peptide with a calculated molecular mass of 39,310 Da. The deduced amino acid sequence of lupin farnesyl pyrophosphate synthase pFPS2 shares 90 and 79% identity with those from Lupinus albus (pFPS1) and Arabidopsis thaliana, respectively, 51% with the yeast enzyme, and 44% identity with those from rat and human. The overexpressed protein, which was purified to near homogeneity, displayed both dimethylallyl transferase and geranyl transferase activities. Polyclonal antibodies raised against the purified protein immunorecognized a ca 39-kDa protein in lupin root extracts.

Alkyl and Aryl Transferases↗
PubMed1996

Cloning, characterization, and heterologous expression of cDNAs for farnesyl diphosphate synthase from the guayule rubber plant reveals that this prenyltransferase occurs in rubber particles.

Two farnesyl diphosphate synthase (FPS) cDNA's from a guayule stembark library were isolated and characterized. Both encode M(r) 39,000 proteins containing 432 amino acids that differ slightly in their deduced molecular weights and isoelectric points. They both contain the DDXXD motifs that are characteristic of prenyltransferases, and both isoforms show high homology to other plant FPS sequences but less overall homology to FPS sequences from nonplant sources. The two isoforms differ by 5% in their amino acid sequence. When expressed in Escherichia coli, each guayule isoform exhibits high specific activity that produces farnesyl diphosphate as the major isoprenoid and small amounts of geranyl diphosphate. Biochemical and immunological evidence also indicates that FPS is associated with guayule rubber particles. Antibodies to chicken FPS cross-react with both guayule isoforms expressed in E. coli and recognize a low abundance M(r) 39,000 protein in rubber particles purified from guayule stembark. Guayule FPS sequences show high homology to peptide fragments of the prenyltransferase associated with rubber particles from Hevea brasiliensis, suggesting that this enzyme may be important for rubber biosynthesis in both species.

Alkyl and Aryl Transferases↗
PubMed2000

Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase.

Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other N-containing bisphosphonates inhibit the isoprenoid biosynthesis pathway and interfere with protein prenylation, as a result of reduced geranylgeranyl diphosphate levels. This study identified farnesyl disphosphate synthase as the mevalonate pathway enzyme inhibited by bisphosphonates. HPLC analysis of products from a liver cytosolic extract narrowed the potential targets for alendronate inhibition (IC(50) = 1700 nM) to isopentenyl diphosphate isomerase and farnesyl diphosphate synthase. Recombinant human farnesyl diphosphate synthase was inhibited by alendronate with an IC(50) of 460 nM (following 15 min preincubation). Alendronate did not inhibit isopentenyl diphosphate isomerase or GGPP synthase, partially purified from liver cytosol. Recombinant farnesyl diphosphate synthase was also inhibited by pamidronate (IC(50) = 500 nM) and risedronate (IC(50) = 3.9 nM), negligibly by etidronate (IC50 = 80 microM), and not at all by clodronate. In osteoclasts, alendronate inhibited the incorporation of [(3)H]mevalonolactone into proteins of 18-25 kDa and into nonsaponifiable lipids, including sterols. These findings (i) identify farnesyl diphosphate synthase as the selective target of alendronate in the mevalonate pathway, (ii) show that this enzyme is inhibited by other N-containing bisphosphonates, such as risendronate, but not by clodronate, supporting a different mechanism of action for different bisphosphonates, and (iii) document in purified osteoclasts alendronate inhibition of prenylation and sterol biosynthesis.

Alendronate↗
PubMed1995

The molecular mechanism of the induction of the low density lipoprotein receptor by chenodeoxycholic acid in cultured human cells.

In a cultured human hepatoblastoma cell line, Hep G2, chenodeoxycholic acid (CDCA) induced LDL receptor mRNA levels approximately 4 fold and mRNA levels for HMG-CoA reductase and HMG-CoA synthase two fold. In contrast, the mRNA levels for mevalonate kinase, farnesyl pyrophosphate synthase and squalene synthase were not changed significantly. The pattern of the induction of the sterol-sensitive genes was similar to the induction by N-acetyl-leucyl-leucyl-norleucinal (ALLN), an SREBP degradation inhibitor, suggesting that CDCA may increase mature SREBPs. CDCA could inhibit the 25-hydroxycholesterol mediated inactivation of SREBP without affecting mRNA levels of SREBPs. These results suggest that CDCA can affect sterol metabolism by a novel mechanism involving the inhibition of the oxysterol-mediated inactivation of SREBP.

Alkyl and Aryl Transferases↗
PubMed1995

Significance of Phe-220 and Gln-221 in the catalytic mechanism of farnesyl diphosphate synthase of Bacillus stearothermophilus.

Farnesyl diphosphate synthase [EC 2.5.1.10] from Bacillus stearothermophilus was specifically altered at two amino acid residues by using site-directed mutagenesis. The highly conserved Phe and Gln residues at the sequential amino acid positions 220-221 in an upstream part of the putative substrate binding site were replaced with Ala and Glu, respectively. These mutageneses (F220A and Q221E) resulted in 10(-5) and 10(-3) decreases in catalytic activity of farnesyl diphosphate synthesis, respectively. Michaelis constants of the Q221E mutant for the allylic substrates (dimethylallyl- and geranyl diphosphates) increased approximately 25- and 2-folds, respectively, compared to wild type, whereas those for the homoallylic substrate (isopentenyl diphosphate) were not altered much. These results suggest that the Phe-Gln motif is involved not only in the binding of allylic substrates but also in the catalysis by farnesyl diphosphate synthase.

Alkyl and Aryl Transferases↗
PubMed1999

Nitrogen-containing bisphosphonates as carbocation transition state analogs for isoprenoid biosynthesis.

Nitrogen-containing bisphosphonates are potent bone antiresorptive agents as well as having herbicidal and antiparasitic activity, and are thought to act by inhibiting enzymes of the mevalonate pathway. Using molecular modeling and ab initio quantum chemical calculations, we show that bisphosphonates can act as aza-isoprenoid transition state analogs, thereby inhibiting isoprenoid biosynthesis. The two phosphonate groups of the 1,1-bisphosphonates readily dock into the diphosphate-Mg(2+) binding site in farnesyl diphosphate synthase, while the charged ammonium (or pyridinium or imidazolium) groups act as carbocation transition state analogs, whose binding is stabilized by a cluster of oxygen atoms in the active site cleft, and an overall negative electrostatic potential in this region. Enhanced activity is shown to correlate with increasing van der Waals stabilization due to N-alkylation, or the presence of a charged, planar (sp(2)-hybridized) aromatic residue in the carbocation binding site. These results are of general interest since they suggest a rational approach to bisphosphonate drug design.

Alkyl and Aryl Transferases↗
PubMed1999

Mechanism of aminobisphosphonate action: characterization of alendronate inhibition of the isoprenoid pathway.

Alendronate (ALN), an aminobisphosphonate compound used for the treatment of osteoporosis and other disorders of bone resorption, has been suggested to act by inhibition of the formation of GGPP. In the present study we used an S(10) homogenate fraction of rat liver to show that ALN causes a dose-dependent inhibition of [(3)H]MVA incorporation into sterols and a concomitant increase in incorporation of radiolabel into IPP and DMAPP. We further show that ALN is a potent inhibitor of cytosolic trans-prenyltransferase (FPP synthase). The inhibition is competitive with respect to allylic pyrophosphate substrates, but not IPP, suggesting that ALN acts as an allylic pyrophosphate analog and binds to the free enzyme. The K(i) is in the 0.5 microM range.

Alendronate↗
PubMed2000

The regulation of activity of main mevalonic acid pathway enzymes: farnesyl diphosphate synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, and squalene synthase in yeast Saccharomyces cerevisiae.

The co-regulation of the main mevalonic acid pathway enzymes was investigated in the yeast Saccharomyces cerevisiae. It was found that a 6-fold increase in FPPS activity compared with that of the wild-type strain FL100 did not cause significant changes in HMG-CoA reductase activity, while the amounts of synthesized dolichols and ergosterol increased by 80 and 32%, respectively. The disruption of the SQS gene in the strain grown in the presence of ergosterol repressed the activities of both FPP synthase and HMG-CoA reductase to a comparable degree, whereas in the same strain starved for ergosterol the activity of FPPS was 10-fold higher and HMG-CoA reductase activity was practically unchanged. We show that FPPS is the enzyme that regulates the flow rate of synthesized mevalonic acid pathway products independent of HMG-CoA reductase and SQS.

Alkyl and Aryl Transferases↗
PubMed2002

Identification of a bisphosphonate that inhibits isopentenyl diphosphate isomerase and farnesyl diphosphate synthase.

We and others have recently shown that the major molecular target of nitrogen-containing bisphosphonate drugs is farnesyl diphosphate synthase, an enzyme in the mevalonate pathway. In an in vitro screen, we discovered a bisphosphonate, NE21650, that potently inhibited farnesyl diphosphate synthase but, unlike other N-BPs investigated, was also a weak inhibitor of isopentenyl diphosphate isomerase. NE21650 was a more potent inhibitor of protein prenylation in osteoclasts and macrophages, and a more potent inhibitor of bone resorption in vitro, than alendronate, despite very similar IC(50) values for inhibition of farnesyl diphosphate synthase. Our observations show that minor changes to the structure of bisphosphonates allow inhibition of more than one enzyme in the mevalonate pathway and suggest that loss of protein prenylation due to inhibition of more than one enzyme in the mevalonate pathway may lead to an increase in antiresorptive potency compared to bisphosphonates that only inhibit farnesyl diphosphate synthase.

Alendronate↗
PubMed1993

Crystallization and preliminary X-ray diffraction studies of Bacillus stearothermophilus farnesyl diphosphate synthase expressed in Escherichia coli.

Thermostable farnesyl diphosphate synthase (EC 2.5.1.10) from Bacillus stearothermophilus, which was overexpressed in Escherichia coli, has been crystallized by the vapor-diffusion procedure. Tetragonal crystals were obtained using ammonium sulfate as a precipitant. The crystals diffracted X-rays to about 3 A resolution. The diffraction pattern indicated that the space group is I4(1)22 with unit-cell dimensions of a = b = 114 A and c = 247 A. It is thought that the asymmetric unit comprises two or three molecules of farnesyl diphosphate synthase.

Alkyl and Aryl Transferases↗
PubMed2000

Activation of promoters for cellular lipogenic genes by hepatitis B virus large surface protein.

Hepatitis B virus large surface protein has the unusual property of accumulating in a particulate form within a preGolgi compartment, leading to marked proliferation of intracellular membranes. We show here that large surface protein activates the promoters for two lipogenic genes that code for farnesyl diphosphate synthase and fatty acid synthase. This activation is transduced, in part, by the transcription factor NF-Y. Although NF-Y is also necessary for the transcriptional induction of chaperone proteins residing in the endoplasmic reticulum by unfolded proteins, other inducers of chaperone synthesis do not activate the promoters for farnesyl diphosphate synthase and fatty acid synthase. Our results suggest the presence of a novel signaling pathway from the endoplasmic reticulum to the nucleus that causes the intracellular membrane proliferation seen in the hepatocytes of persons with accumulated large surface protein particles.

Alkyl and Aryl Transferases↗