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Kyozo Ogura - One of the best experts on this subject based on the ideXlab platform.
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Substrate specificity of thermostable Farnesyl Diphosphate synthase with alkyl group homologs of isopentenyl Diphosphate.
Bioorganic & Medicinal Chemistry Letters, 1998Co-Authors: Masahiko Nagaki, Junji Ishibashi, Hiroki Kannari, Yuji Maki, Kyozo Ogura, Tokuzo Nishino, Tanetoshi KoyamaAbstract:3-Alkyl group homologs of isopentenyl Diphosphate were examined for the reactivity as substrates of the thermostable Farnesyl Diphosphate (FPP) synthase of Bacillus stearothermophilus. Even 3-n-propyl- and 3-n-butyl-but-3-enyl Diphosphates, which are hardly acceptable by animal FPP synthases, are accepted by this bacterial enzyme as substrates to react with dimethylallyl- and geranyl Diphosphates, yielding 7-methyl-3-n-propylocta-2,6-dienyl- and 7,11-dimethyl-3-n-propyldodeca-2,6,10-trienyl Diphosphate, respectively.
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An Artificial Substrate for the Thermostable Farnesyl Diphosphate Synthase from Bacillus stearothermophilus.
Chemistry Letters, 1997Co-Authors: Masahiko Nagaki, Yuji Maki, Tanetoshi Koyama, Tokuzo Nishino, Kanako Shimizu, Kyozo OguraAbstract:Desmethyl homologs of dimethylallyl Diphosphate and of isopentenyl Diphosphate were examined for the reactivity as substrates for the thermostable Farnesyl Diphosphate synthase of Bacillus stearothermophilus. E-But-2-enyl Diphosphate was not accepted as a substrate at all, but but-3-enyl Diphosphate reacted with dimethylallyl- and geranyl Diphosphates to give norgeranyl- and norFarnesyl Diphosphates, respectively, which lack methyl groups at the 3-positions.
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Significance of Phe-220 and Gln-221 in the Catalytic Mechanism of Farnesyl Diphosphate Synthase of Bucillus stearothermophilus
Biochemical and biophysical research communications, 1995Co-Authors: Tanetoshi Koyama, Tokuzo Nishino, M. Tajima, Kyozo OguraAbstract:Abstract 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 Gin 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.
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SUBSTRATE SPECIFICITY OF Farnesyl Diphosphate SYNTHASE FROM BACILLUS STEAROTHERMOPHILUS
Bioorganic & Medicinal Chemistry Letters, 1995Co-Authors: Yuji Maki, Tanetoshi Koyama, Takae Endo, Akiko Masukawa, Hideaki Ono, Kyozo OguraAbstract:Abstract The substrate specificity of Farnesyl Diphosphate synthase from Bacillus stearothermophilus was studied and compared with that of the pig liver counterpart. Allylic Diphosphate analogs having hydrocarbon chains were accepted as good substrates by both enzymes. However, the bacterial enzyme hardly accepted analogs having oxygen atoms in their chains, which are well accepted by the pig liver enzyme, indicating that the former has more stringent specificity than the latter.
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Structural and functional roles of the cysteine residues of Bacillus stearothermophilus Farnesyl Diphosphate synthase.
Biochemistry, 1994Co-Authors: Tanetoshi Koyama, Shusei Obata, Kazuhiro Saito, Ayumi Takeshita-koike, Kyozo OguraAbstract:p-(Chloromercuri)benzoic acid inhibited the catalytic activity of Bacillus stearothermophilus Farnesyl Diphosphate synthase (FPP synthase), which contains only two cysteine residues at positions 73 and 289. In order to explore the role of the cysteine residues, either or both of them were replaced with phenylalanine or serine. Five mutant enzymes, C73F, C73S, C289F, C289S, and C73S-C289S, were overproduced in Escherichia coli and purified to homogeneity. All of them were active as Farnesyl Diphosphate synthase, showing specific activities comparable to that of the wild-type enzyme. These results indicate that neither of the cysteines is essential for catalytic function. The C73F mutant, however, was very sensitive to heat treatment, while C73S was as highly stable as the wild type. The Km value of C289F for isopentenyl Diphosphate is 10 times that of the wild type. The wild-type enzyme was converted into an oxidized form which was separable from the native enzyme on ion-exchange chromatography, and this conversion was accelerated by cupric ions. Similar conversion has previously been reported by several researchers, who found the occurrence of two forms of pig liver FPP synthase and who attributed this phenomenon to the oxidoreduction of sulfhydryl and disulfide groups. However, even the C73S-C289S mutant, which has no cysteine residues, was also converted into an oxidized form as in the case of the wild-type enzyme. These results provide evidence that residues other than cysteine are involved in the conversion of this enzyme into the oxidized form.
Tanetoshi Koyama - One of the best experts on this subject based on the ideXlab platform.
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Substrate specificities of Farnesyl Diphosphate synthases of Bacillus stearothermophilus and porcine liver with allylic substrate homologs having vinyl or ethynyl group
Journal of Molecular Catalysis B-enzymatic, 2009Co-Authors: Masahiko Nagaki, Yuji Maki, Tohru Musashi, Yuji Hirano, Hidenori Tanaka, Junji Ichita, Tanetoshi KoyamaAbstract:Abstract To investigate substrate specificities of Farnesyl Diphosphate synthases from porcine liver and Bacillus stearothermophilus , we have examined the reactivities of vinyldimethylallyl and ethynyldimethylallyl Diphosphates as allylic substrate homologs. The reaction of vinyldimethylallyl Diphosphate with isopentenyl Diphosphate by Farnesyl Diphosphate synthase of porcine liver gave vinylgeranyl and vinylFarnesyl Diphosphates, which shows that the reaction stopped at the single or double condensation of isopentenyl Diphosphate, respectively. However, the similar reaction by the use of wild-type Farnesyl Diphosphate synthase of B. stearothermophilus gave vinylFarnesyl Diphosphate, exclusively. On the other hand, the reaction of Z -ethynyldimethylallyl Diphosphates with isopentenyl Diphosphate by the use of wild-type of Farnesyl Diphosphate synthase of B. stearothermophilus gave Z -ethynylFarnesyl Diphosphate, as the sole product. Moreover, a mutated Farnesyl Diphosphate synthase (Y81D) reaction of Z -ethynyldimethylallyl Diphosphates with isopentenyl Diphosphate gave three kinds of products: ethynylgeranyl, ethynylFarnesyl, and ethynylgeranylgeranyl Diphosphates. Using wild-type of Farnesyl Diphosphate synthase of B. stearothermophilus , the reaction of E -ethynyldimethylallyl Diphosphate with isopentenyl Diphosphate gave only E -ethynylFarnesyl Diphosphate as double condensation product.
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Substrate specificities of wild and mutated Farnesyl Diphosphate synthases: Reactivity of allylic substrate homologs having hydrophilic groups at ω-position
Journal of Molecular Catalysis B: Enzymatic, 2009Co-Authors: Masahiko Nagaki, Yuji Maki, Minori Nakada, Tohru Musashi, Jun Kawakami, Takae Endo, Tanetoshi KoyamaAbstract:Abstract To investigate substrate specificities of wild and mutated types of Farnesyl Diphosphate synthases from Bacillus stearothermophilus , we have examined the reactivities of methoxymethoxydimethylallyl- and propoxygeranyl Diphosphates as allylic substrate homologs. The wild type Farnesyl Diphosphate synthase reaction of methoxymethoxydimethylallyl and propoxygeranyl Diphosphates with isopentenyl Diphosphate gave methoxymethoxygeranyl and propoxyFarnesyl Diphosphates which stopped at the first stage of the condensation. Using a mutated Farnesyl Diphosphate synthase (Y81D FPS), the reaction of methoxymethoxydimethylallyl Diphosphate with isopentenyl Diphosphate gave only methoxymethoxyFarnesyl Diphosphate as single condensation product. Moreover, both of mutated Farnesyl Diphosphate synthase reaction with propoxygeranyl Diphosphate of isopentenyl Diphosphate gave propoxyFarnesyl- and propoxygeranylgeranyl Diphosphate.
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Product chain-length determination mechanism of Z,E-Farnesyl Diphosphate synthase.
Biochemical and biophysical research communications, 2008Co-Authors: Motoyoshi Noike, Sebabrata Mahapatra, Dean C. Crick, Takanori Ambo, Sayaka Kikuchi, Toshihide Suzuki, Satoshi Yamashita, Seiji Takahashi, Hirofumi Kurokawa, Tanetoshi KoyamaAbstract:cis-Prenyltransferases catalyze the consecutive condensation of isopentenyl Diphosphate (IPP) with allylic prenyl Diphosphates, producing Z,E-mixed prenyl Diphosphate. The Mycobacterium tuberculosis Z,E-Farnesyl Diphosphate synthase Rv1086 catalyzes the condensation of one molecule of IPP with geranyl Diphosphate to yield Z,E-Farnesyl Diphosphate and is classified as a short-chain cis-prenyltransferase. To elucidate the chain-length determination mechanism of the short-chain cis-prenyltransferase, we introduced some substitutive mutations at the characteristic amino acid residues of Rv1086. Among the mutants constructed, L84A showed a dramatic change of catalytic function to synthesize longer prenyl chain products than that of wild type, indicating that Leu84 of Rv1086 plays an important role in product chain-length determination. Mutagenesis at the corresponding residue of a medium-chain cis-prenyltransferase, Micrococcus luteus B-P 26 undecaprenyl Diphosphate synthase also resulted in the production of different prenyl chain length from the intrinsic product, suggesting that this position also plays an important role in product chain-length determination for medium-chain cis-prenyltransferases.
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Substrate specificity of thermostable Farnesyl Diphosphate synthase with alkyl group homologs of isopentenyl Diphosphate.
Bioorganic & Medicinal Chemistry Letters, 1998Co-Authors: Masahiko Nagaki, Junji Ishibashi, Hiroki Kannari, Yuji Maki, Kyozo Ogura, Tokuzo Nishino, Tanetoshi KoyamaAbstract:3-Alkyl group homologs of isopentenyl Diphosphate were examined for the reactivity as substrates of the thermostable Farnesyl Diphosphate (FPP) synthase of Bacillus stearothermophilus. Even 3-n-propyl- and 3-n-butyl-but-3-enyl Diphosphates, which are hardly acceptable by animal FPP synthases, are accepted by this bacterial enzyme as substrates to react with dimethylallyl- and geranyl Diphosphates, yielding 7-methyl-3-n-propylocta-2,6-dienyl- and 7,11-dimethyl-3-n-propyldodeca-2,6,10-trienyl Diphosphate, respectively.
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An Artificial Substrate for the Thermostable Farnesyl Diphosphate Synthase from Bacillus stearothermophilus.
Chemistry Letters, 1997Co-Authors: Masahiko Nagaki, Yuji Maki, Tanetoshi Koyama, Tokuzo Nishino, Kanako Shimizu, Kyozo OguraAbstract:Desmethyl homologs of dimethylallyl Diphosphate and of isopentenyl Diphosphate were examined for the reactivity as substrates for the thermostable Farnesyl Diphosphate synthase of Bacillus stearothermophilus. E-But-2-enyl Diphosphate was not accepted as a substrate at all, but but-3-enyl Diphosphate reacted with dimethylallyl- and geranyl Diphosphates to give norgeranyl- and norFarnesyl Diphosphates, respectively, which lack methyl groups at the 3-positions.
C. Dale Poulter - One of the best experts on this subject based on the ideXlab platform.
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Structure–Function Studies of Artemisia tridentata Farnesyl Diphosphate Synthase and Chrysanthemyl Diphosphate Synthase by Site-Directed Mutagenesis and Morphogenesis
Journal of the American Chemical Society, 2017Co-Authors: J. Scott Lee, Gurusankar Ramamoorthy, Jianjung Pan, C. Dale PoulterAbstract:The amino acid sequences of Farnesyl Diphosphate synthase (FPPase) and chrysanthemyl Diphosphate synthase (CPPase) from Artemisia tridentata ssp. Spiciformis, minus their chloroplast targeting regions, are 71% identical and 90% similar. FPPase efficiently and selectively synthesizes the “regular” sesquiterpenoid Farnesyl Diphosphate (FPP) by coupling isopentenyl Diphosphate (IPP) to dimethylallyl Diphosphate (DMAPP) and then to geranyl Diphosphate (GPP). In contrast, CPPase is an inefficient promiscuous enzyme, which synthesizes the “irregular” monoterpenes chrysanthemyl Diphosphate (CPP), lavandulyl Diphosphate (LPP), and trace quantities of maconelliyl Diphosphate (MPP) from two molecules of DMAPP, and couples IPP to DMAPP to give GPP. A. tridentata FPPase and CPPase belong to the chain elongation protein family (PF00348), a subgroup of the terpenoid synthase superfamily (CL0613) whose members have a characteristic α terpene synthase α-helical fold. The active sites of A. tridentata FPPase and CPPase ar...
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Synthesis and enzymatic studies of bisubstrate analogues for Farnesyl Diphosphate synthase.
The Journal of organic chemistry, 2015Co-Authors: Gurusankar Ramamoorthy, Mark L. Pugh, Boxue Tian, Richard M. Phan, Lawrence Blas Perez, Matthew P. Jacobson, C. Dale PoulterAbstract:Farnesyl Diphosphate synthase catalyzes the sequential chain elongation reactions between isopentenyl Diphosphate (IPP) and dimethylallyl Diphosphate (DMAPP) to form geranyl Diphosphate (GPP) and between IPP and GPP to give Farnesyl Diphosphate (FPP). Bisubstrate analogues containing the allylic and homoallylic substrates were synthesized by joining fragments for IPP and the allylic Diphosphates with a C–C bond between the methyl group at C3 in IPP and the Z-methyl group at C3 in DMAPP (3-OPP) and GPP (4-OPP), respectively. These constructs placed substantial limits on the conformational space available to the analogues relative to the two substrates. The key features of the synthesis of bisubstrate analogues 3-OPP and 4-OPP are a regioselective C-alkylation of the dianion of 3-methyl-3-buten-1-ol (5), a Z-selective cuprate addition of alkyl groups to an α,β-alkynyl ester intermediate, and differential activation of allylic and homoallylic alcohols in the analogues, followed by a simultaneous displacement...
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Synthesis and evaluation of chlorinated substrate analogues for Farnesyl Diphosphate synthase.
The Journal of organic chemistry, 2011Co-Authors: Nicole A. Heaps, C. Dale PoulterAbstract:Substrate analogues for isopentenyl Diphosphate (IPP) and dimethylallyl Diphosphate (DMAPP), where the C3 methyl groups were replaced by chlorine, were synthesized and evaluated as substrates for avian Farnesyl Diphosphate synthase (FPPase). The IPP analogue (3-ClIPP) was a cosubstrate when incubated with dimethylallyl Diphosphate (DMAPP) or geranyl Diphosphate (GPP) to give the corresponding chlorinated analogues of geranyl Diphosphate (3-ClGPP) and Farnesyl Diphosphate (3-ClFPP), respectively. No products were detected in incubations of 3-ClIPP with 3-ClDMAPP. Incubation of IPP with 3-ClDMAPP gave 11-ClFPP as the sole product. Values of K(M)(3-ClIPP) (with DMAPP) and K(M)(3-ClDMAPP) (with IPP) were similar to those for IPP and DMAPP; however, values of k(cat) for both analogues were substantially lower. These results are consistent with a dissociative electrophilic alkylation mechanism where the rate-limiting step changes from heterolytic cleavage of the carbon-oxygen bond in the allylic substrate to alkylation of the double bond of the homoallylic substrate.
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Farnesyl Diphosphate Analogues with ω-Bioorthogonal Azide and Alkyne Functional Groups for Protein Farnesyl Transferase-Catalyzed Ligation Reactions
The Journal of organic chemistry, 2007Co-Authors: Guillermo R. Labadie, Rajesh Viswanathan, C. Dale PoulterAbstract:Eleven Farnesyl Diphosphate analogues, which contained ω-azide or alkyne substituents suitable for bioorthogonal Staudinger and Huisgen [3 + 2] cycloaddition coupling reactions, were synthesized. The analogues were evaluated as substrates for the alkylation of peptide cosubstrates by yeast protein Farnesyl transferase. Five of the Diphosphates were good alternative substrates for Farnesyl Diphosphate (FPP). Steady-state kinetic constants were measured for the active compounds, and the products were characterized by HPLC and LC-MS. Two of the analogues gave steady-state kinetic parameters (kcat and Km) very similar to those of the natural substrate.
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Enzymes encoded by the Farnesyl Diphosphate synthase gene family in the Big Sagebrush Artemisia tridentata ssp. spiciformis.
Journal of Biological Chemistry, 2003Co-Authors: Andréa Hemmerlin, Susan B. Rivera, Hans K. Erickson, C. Dale PoulterAbstract:Abstract Farnesyl Diphosphate synthase catalyzes the sequential head-to-tail condensation of two molecules of isopentenyl Diphosphate with dimethylallyl Diphosphate. In plants the presence of Farnesyl Diphosphate synthase isozymes offers the possibility of differential regulation. Three full-length cDNAs encoding putative isoprenoid synthases, FDS-1, FDS-2, and FDS-5, with greater than 89% similarity were isolated from a Big Sagebrush Artemisia tridentata cDNA library using a three-step polymerase chain reaction protocol. One of the open reading frames, FDS-5, encoded a protein with an N-terminal amino acid extension that was identified as a plastidial targeting peptide. Recombinant histidine-tagged versions of three proteins were purified, and their enzymatic properties were characterized. FDS-1 and FDS-2 synthesized Farnesyl Diphosphate as the final chain elongation product, but their kinetic behavior varied. FDS-1 prefers geranyl Diphosphate over dimethylallyl Diphosphate as an allylic substrate and is active at acidic pH values compared with FDS-2. In contrast, FDS-5 synthesized two irregular monoterpenoids, chrysanthemyl Diphosphate and lavandulyl Diphosphate, when incubated with dimethylallyl Diphosphate and an additional product, the regular monoterpene geranyl Diphosphate, when incubated with isopentenyl Diphosphate and dimethylallyl Diphosphate. Specific cellular functions are proposed for each of the three enzymes, and a scenario for evolution of isoprenyl synthases in plants is presented.
Eric Oldfield - One of the best experts on this subject based on the ideXlab platform.
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Farnesyl Diphosphate synthase inhibitors with unique ligand binding geometries
ACS Medicinal Chemistry Letters, 2015Co-Authors: Yi Liang Liu, Rong Cao, Yang Wang, Eric OldfieldAbstract:Farnesyl Diphosphate synthase (FPPS) is an important drug target for bone resorption, cancer, and some infectious diseases. Here, we report five new structures including two having unique bound ligand geometries. The diamidine inhibitor 7 binds to human FPPS close to the homoallylic (S2) and allosteric (S3) sites and extends into a new site, here called S4. With the bisphosphonate inhibitor 8, two molecules bind to Trypanosoma brucei FPPS, one molecule in the allylic site (S1) and the other close to S2, the first observation of two bisphosphonate molecules bound to FPPS. We also report the structures of apo-FPPS from T. brucei, together with two more bisphosphonate-bound structures (2,9), for purposes of comparison. The diamidine structure is of particular interest because 7 could represent a new lead for lipophilic FPPS inhibitors, while 8 has low micromolar activity against T. brucei, the causative agent of human African trypanosomiasis.
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Farnesyl Diphosphate Synthase Inhibitors from In Silico Screening
Chemical biology & drug design, 2013Co-Authors: Steffen Lindert, Eric Oldfield, Wei Zhu, Yi Liang Liu, Ran Pang, J. Andrew MccammonAbstract:The relaxed complex scheme is an in silico drug screening method that accounts for receptor flexibility using molecular dynamics simulations. Here, we used this approach combined with similarity searches and experimental inhibition assays to identify several low micromolar, non-bisphosphonate inhibitors, bisamidines, of Farnesyl Diphosphate synthase (FPPS), an enzyme targeted by some anticancer and antimicrobial agents and for the treatment of bone resorption diseases. This novel class of Farnesyl Diphosphate synthase inhibitors have more drug-like properties than existing bisphosphonate inhibitors, making them interesting pharmaceutical leads.
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the Farnesyl Diphosphate geranylgeranyl Diphosphate synthase of toxoplasma gondii is a bifunctional enzyme and a molecular target of bisphosphonates
Journal of Biological Chemistry, 2007Co-Authors: Yan Ling, Eric Oldfield, Kildare Miranda, Silvia N. J. MorenoAbstract: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/geranylgeranyl-Diphosphate synthase identified in eukaryotes, which, together with previous results, establishes this enzyme as a valid target for the chemotherapy of toxoplasmosis.
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The Farnesyl-Diphosphate/Geranylgeranyl-Diphosphate Synthase of Toxoplasma gondii Is a Bifunctional Enzyme and a Molecular Target of Bisphosphonates
The Journal of biological chemistry, 2007Co-Authors: Yan Ling, Eric Oldfield, Kildare Miranda, Silvia N. J. MorenoAbstract: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/geranylgeranyl-Diphosphate synthase identified in eukaryotes, which, together with previous results, establishes this enzyme as a valid target for the chemotherapy of toxoplasmosis.
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Structure and mechanism of the Farnesyl Diphosphate synthase from Trypanosoma cruzi: Implications for drug design
Proteins, 2006Co-Authors: Sandra B. Gabelli, Eric Oldfield, Andrea Montalvetti, Roberto Docampo, Jason S. Mclellan, L. Mario AmzelAbstract: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.
Tokuzo Nishino - One of the best experts on this subject based on the ideXlab platform.
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Short-chain prenyl Diphosphate synthase that condenses isopentenyl Diphosphate with dimethylallyl Diphosphate in ispA null Escherichia coli strain lacking Farnesyl Diphosphate synthase.
Journal of bioscience and bioengineering, 2007Co-Authors: Ken Saito, Shingo Fujisaki, Tokuzo NishinoAbstract:A short-chain prenyl Diphosphate synthase in an Escherichia coli mutant that lacked the gene coding for Farnesyl Diphosphate synthase, ispA, was separated from other prenyl Diphosphate synthases by DEAE-Toyopearl column chromatography. The purified enzyme catalyzed the condensation of isopentenyl Diphosphate with dimethylallyl Diphosphate to form Farnesyl Diphosphate and geranylgeranyl Diphosphate.
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Disruption of the structural gene for Farnesyl Diphosphate synthase in Escherichia coli.
Journal of biochemistry, 2005Co-Authors: Shingo Fujisaki, Tokuzo Nishino, Isao Takahashi, Hiroshi Hara, Kensuke Horiuchi, Yukinobu NishimuraAbstract:The chromosomal ispA gene encoding Farnesyl Diphosphate synthase of Escherichia coli was disrupted by inserting a neo gene cassette. The null ispA mutants were viable. The growth yield of the mutants was 70% to 80% of that of the wild-type strain under aerobic conditions, and was almost the same as the wild-type under anaerobic conditions. The levels of ubiquinone-8 and menaquinone-8 were both significantly lower (less than 13% and 18% of normal, respectively) in the mutants than in the wild-type. The undecaprenyl phosphate level in the mutants was modestly lower (40% to 70% of normal) than in the wild-type strain. Thus the synthesis of all-E-octaprenyl Diphosphate, the precursor of ubiquinone-8 and menaquinone-8, was decreased more severely than that of Z,E-mixed undecaprenyl Diphosphate, the precursor of undecaprenyl monophosphates, under the conditions where the synthesis of Farnesyl Diphosphate was decreased. The condensation of isopentenyl Diphosphate with dimethylallyl Diphosphate was detected in the cell-free extracts of the mutants, although it was 5% of that in the wild-type strain. A low level of Farnesyl Diphosphate seems to be synthesized in the mutants by other prenyltransferases such as octaprenyl Diphosphate synthase or undecaprenyl Diphosphate synthase.
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Substrate specificity of thermostable Farnesyl Diphosphate synthase with alkyl group homologs of isopentenyl Diphosphate.
Bioorganic & Medicinal Chemistry Letters, 1998Co-Authors: Masahiko Nagaki, Junji Ishibashi, Hiroki Kannari, Yuji Maki, Kyozo Ogura, Tokuzo Nishino, Tanetoshi KoyamaAbstract:3-Alkyl group homologs of isopentenyl Diphosphate were examined for the reactivity as substrates of the thermostable Farnesyl Diphosphate (FPP) synthase of Bacillus stearothermophilus. Even 3-n-propyl- and 3-n-butyl-but-3-enyl Diphosphates, which are hardly acceptable by animal FPP synthases, are accepted by this bacterial enzyme as substrates to react with dimethylallyl- and geranyl Diphosphates, yielding 7-methyl-3-n-propylocta-2,6-dienyl- and 7,11-dimethyl-3-n-propyldodeca-2,6,10-trienyl Diphosphate, respectively.
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An Artificial Substrate for the Thermostable Farnesyl Diphosphate Synthase from Bacillus stearothermophilus.
Chemistry Letters, 1997Co-Authors: Masahiko Nagaki, Yuji Maki, Tanetoshi Koyama, Tokuzo Nishino, Kanako Shimizu, Kyozo OguraAbstract:Desmethyl homologs of dimethylallyl Diphosphate and of isopentenyl Diphosphate were examined for the reactivity as substrates for the thermostable Farnesyl Diphosphate synthase of Bacillus stearothermophilus. E-But-2-enyl Diphosphate was not accepted as a substrate at all, but but-3-enyl Diphosphate reacted with dimethylallyl- and geranyl Diphosphates to give norgeranyl- and norFarnesyl Diphosphates, respectively, which lack methyl groups at the 3-positions.
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Significance of Phe-220 and Gln-221 in the Catalytic Mechanism of Farnesyl Diphosphate Synthase of Bucillus stearothermophilus
Biochemical and biophysical research communications, 1995Co-Authors: Tanetoshi Koyama, Tokuzo Nishino, M. Tajima, Kyozo OguraAbstract:Abstract 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 Gin 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.
