Cloning and nucleotide sequence of the ispA gene responsible for farnesyl diphosphate synthase activity in Escherichia coli

The molecular cloning and the determination of the nucleotide sequence of the ispA gene responsible for farnesyl diphosphate (FPP) synthase [EC 2.5.1.1] activity in Escherichia coli are described. E. coli ispA strains have temperature-sensitive FPP synthase, and the defective gene is located at about min 10 on the chromosome. The wild-type ispA gene was subcloned from a lambda phage clone containing the chromosomal fragment around min 10, picked up from the aligned genomic library of Kohara et al. [Kohara, Y., Akiyama, K., & Isono, K. (1987) Cell 50, 495-508]. The cloned gene was identified as the ispA gene by the recovery and amplification of FPP synthase activity in an ispA strain. A 1,452-nucleotide sequence of the cloned fragment was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected molecular weights of 8,951 and 32,158, respectively. A part of the deduced amino acid sequence of ORF-2 showed similarity to the sequences of eucaryotic FPP synthases and of crtE product of a photosynthetic bacterium. The plasmid carrying ORF-2 downstream of the lac promoter complemented the defect of FPP synthase activity of the ispA mutant, showing that the product encoded by ORF-2 is the ispA product. The maxicell analysis indicated that a protein of molecular weight 36,000, approximately consistent with the molecular weight of the deduced ORF-2-encoded protein, is the gene product.     

Directed evolution of Escherichia coli farnesyl diphosphate synthase (IspA) reveals novel structural determinants of chain length specificity

Directed evolution of farnesyl diphosphate (FPP, C15) synthase (IspA) of Escherichia coli was carried out by error-prone PCR with a color complementation screen utilizing C40 carotenoid pathway enzymes. This allowed IspA mutants with enhanced production of the C40 carotenoid precursor geranylgeranyl diphosphate (GGPP, C20) to be readily identified. Analysis of these mutants was carried out in order to better understand the mechanisms of product chain length specificity in this enzyme. The 12 evolved clones having enhanced C20 GGPP production have characteristic mutations in the conserved regions of prenyl diphosphate synthases (designated regions I through VII). Some of these mutations (I76T, Y79S, Y79H, C75Y, H83Y, and H83Q) are found near or before the conserved first aspartate rich motif (FARM), which is involved in the mechanism for chain elongation reaction of all prenyl synthases. Molecular modeling suggested a mechanism for chain length determination for these mutations including substitutions at the 1st and 9th amino acids upstream of the FARM that have not been reported previously. In addition, a mutation on a helix adjacent to the FARM within the substrate-binding pocket (D115G) suggests a novel mechanism for chain length determination. One mutant IspA clone carries a mutation of C155G at the 2nd amino acid upstream of conserved region IV (GQxxDL), which was recently found to be an important region controlling the chain elongation of a Type III GGPP synthase. One IspA clone carries mutations (T234A and T249I) near the conserved second aspartate rich motif (SARM). As a verification of the in vivo activity of the mutant clones (represented as C40 carotenoid formation), we confirmed the product distribution of wild-type and mutant IspA using an in vitro assay.     

Isolation and characterization of an Escherichia coli mutant having temperature-sensitive farnesyl diphosphate synthase.

The screening of a collection of highly mutagenized strains of Escherichia coli for defects in isoprenoid synthesis led to the isolation of a mutant that had temperature-sensitive farnesyl diphosphate synthase. The defective gene, named ispA, was mapped at about min 10 on the E. coli chromosome, and the gene order was shown to be tsx-ispA-lon. The mutant ispA gene was transferred to the E. coli strain with a defined genetic background by P1 transduction for investigation of its function. The in vitro activity of farnesyl diphosphate synthase of the mutant was 21% of that of the wild-type strain at 30 degrees C and 5% of that at 40 degrees C. At 42 degrees C the ubiquinone level was lower (66% of normal) in the mutant than in the wild-type strain, whereas at 30 degrees C the level in the mutant was almost equal to that in the wild-type strain. The polyprenyl phosphate level was slightly higher in the mutant than in the wild-type strain at 30 degrees C and almost the same in both strains at 42 degrees C. The mutant had no obvious phenotype regarding its growth properties.     

Localization of farnesyl diphosphate synthase in chloroplasts.

The subcellular localization of plant farnesyl diphosphate synthase (FPPS) was examined. Immunocytochemical staining using anti-FPPS1 antibody followed by electron microscopy showed that FPPS1 was localized to chloroplasts of rice mesophyll cells. Subcellular fractions from wheat leaves were examined by immunoblot analysis. FPPS was detected in the chloroplast fraction in wheat, and was protected from proteolysis following trypsin treatment of chloroplasts. FPPS was also detected in the chloroplast fraction of a dicot plant, tobacco.     

The ispB gene encoding octaprenyl diphosphate synthase is essential for growth of Escherichia coli.

The Escherichia coli ispB gene encoding octaprenyl diphosphate synthase is responsible for the synthesis of the side chain of isoprenoid quinones. We tried to construct an E. coli ispB-disrupted mutant but could not isolate the chromosomal ispB disrupted mutant unless the ispB gene or its homolog was supplied on a plasmid. The chromosomal ispB disruptants that harbored plasmids carrying the ispB homologs from Haemophilus influenzae and Synechocystis sp. strain PCC6803 produced mainly ubiquinone 7 and ubiquinone 9, respectively. Our results indicate that the function of the ispB gene is essential for normal growth and that this function can be substituted for by homologs of the ispB gene from other organisms that produce distinct forms of ubiquinone.     

Temperature-dependent modulation of farnesyl diphosphate/geranylgeranyl diphosphate synthase from hyperthermophilic archaea

Enzyme characteristics of trans-prenyl diphosphate synthase (Tk-IdsA) from Thermococcus kodakaraensis, which catalyzes the consecutive trans-condensation of isopentenyl diphosphate (C(5)) units with allylic diphosphate, were examined. Product analysis revealed that Tk-IdsA is a bifunctional enzyme, farnesyl diphosphate (FPP, C(15))/geranylgeranyl diphosphate (GGPP, C(20)) synthase, and mainly yields both C(15) and C(20). The FPP/GGPP product ratio increases with the rise of the reaction temperature. The kinetic parameters obtained at 70 and 90 degrees C demonstrated that the rise of the temperature elevates the k(0) value for the C(10) allylic substrate to more than those for the C(5) and C(15) allylic substrates. These data suggest that Tk-IdsA contributes to adjust the membrane composition to the cell growth temperature by modulating its substrate and product specificities. Mutation study indicated that the aromatic side chain of Tyr-81 acts as a steric hindrance to terminate the chain elongation and defines the final product length.     

卷叶贝母法尼基焦磷酸合酶基因的克隆及表达分析

为了探究卷叶贝母(Fritillaria cirrhosa)法尼基焦磷酸合酶基因(FcFPPS)是否参与甾类生物碱合成、萜类合成等代谢过程,该研究基于转录组测序结果,通过PCR技术克隆卷叶贝母FPPS基因(FcFPPS)开放阅读框(Open Reading Frame,ORF)序列,运用生物信息学方法对该基因进行分析,预测其编码蛋白的结构与功能,并通过qRT-PCR检测FcFPPS基因在野生鳞茎和再生鳞茎(通过激素组合刺激获得的组织培养物)中的表达情况,以及利用煎煮法测定野生鳞茎和再生鳞茎的总生物碱含量。结果表明:获得了1 059bp的FcFPPS ORF片段,编码352个氨基酸,并与NCBI上公布的麝香百合、虎眼万年青、春兰等植物FPPS蛋白的相似性在85%以上;对FcFPPS蛋白的二级、三级结构预测发现FcFPPS蛋白主要由α螺旋构成;qRT-PCR与总生物碱含量测定结果显示FcFPPS基因的表达水平与总生物碱含量的变化趋势一致,都是再生鳞茎高于野生鳞茎。FcFPPS蛋白质特征区及同源性等生物信息学分析结合qRT-PCR的测定结果证明FcFPPS可能是一个有生物学功能的蛋白质,这为后续利用基因工程手段提高卷叶贝母中生物碱含量奠定了理论基础。     

Molecular characterization of ginseng farnesyl diphosphate synthase gene and its up-regulation by methyl jasmonate

We isolated a gene encoding for farnesyl diphosphate synthase (FPS) from Panax ginseng, a species that produces a large quantity of triterpene saponins such as ginsenosides. The deduced amino acid sequence of PgFPS was 77, 84 and 95 % identical to those of Arabidopsis, Hevea, and Centella. Southern blot analysis indicated that P. ginseng contained more than two genes encoding for FPS. When the cDNA of PgFPS was expressed in Escherichia coli, the recombinant enzyme, purified with a His-tag column, was found to possess FPS activity. When cultures of ginseng hairy root were treated with 0.1 mM methyl jasmonate (MJ), PgFPS mRNA was detected within 12 h of the treatment, and achieved maximum after 24 h. Also FPS activity in the hairy root cultures after 12 h of MJ treatment was higher than that of the control.     

Disruption of the Escherichia coli cls gene responsible for cardiolipin synthesis

The cls gene of Escherichia coli is responsible for the synthesis of a major membrane phospholipid, cardiolipin, and has been proposed to encode cardiolipin synthase. This gene cloned on a pBR322 derivative was disrupted by either insertion of or replacement with a kanamycin-resistant gene followed by exchange with the homologous chromosomal region. The proper genomic disruptions were confirmed by Southern blot hybridization and a transductional linkage analysis. Both types of disruptants had essentially the same properties; cardiolipin synthase activity was not detectable, but the strains grew well, although their growth rates and final culture densities were lower than those of the corresponding wild-type strains and strains with the classical cls-1 mutation. A disruptant harboring a plasmid that carried the intact cls gene grew normally. The results indicate that the cls gene and probably the cardiolipin synthase are dispensable for E. coli but may confer growth or survival advantages. Low but definite levels of cardiolipin were synthesized by all the disruptants. Cardiolipin content of the cls mutants depended on the dosage of the pss gene, and attempts to transfer a null allele of the cls gene into a pss-1 mutant were unsuccessful. We point out the possibilities of minor cardiolipin formation by phosphatidylserine synthase and of the essential nature of cardiolipin for the survival of E. coli cells.     

Molecular characterization and expression analysis of a gene encoding for farnesyl diphosphate synthase from Euphorbia pekinensis Rupr

The root of Euphorbia pekinensis as a traditional herbal medicine has been recorded in Chinese pharmacopoeias for the treatment of oedema, gonorrhea, migraine and wart cures. In this work, we reported on the cDNA cloning and characterization of a novel farnesyl diphosphate synthase (FPS) from E. pekinensis. The full-length cDNA named EpFPS (Genbank Accession Number FJ755465) contained 1431 bp with an open reading frame of 1029 bp encoding a polypeptie of 342 amino acids. The deduced amino acid sequence of the EpFPS named EpFPS exhibited a high homology with other plant FPSs, and contained five conserved domains. Phylogenetic analysis showed that EpFPS belonged to the plant FPS group. Southern blot analysis revealed that there exists a small FPS gene family in E. pekinensis. Expression pattern analysis revealed that EpFPS expressed strongly in root, weak in leaf and stem. In callus, expression of EpFPS gene and biosynthesis of triterpenoids were strongly induced by Methyl jasmonate and slightly induced by Salicylic acid. Functional complementation of EpFPS in an ergosterol auxotrophic yeast strain indicated that the cloned cDNA encoded a functional farnesyl diphosphate synthase.     

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.     

The cyclization of farnesyl diphosphate and nerolidyl diphosphate by a purified recombinant delta-cadinene synthase

The first step in the conversion of the isoprenoid intermediate, farnesyl diphosphate (FDP), to sesquiterpene phytoalexins in cotton (Gossypium barbadense) plants is catalyzed by delta-cadinene (CDN) synthase. CDN is the precursor of desoxyhemigossypol and hemigossypol defense sesquiterpenes. In this paper we have studied the mechanism for the cyclization of FDP and the putative intermediate, nerolidyl diphosphate, to CDN. A purified recombinant CDN synthase (CDN1-C1) expressed in Escherichia coli from CDN1-C1 cDNA isolated from Gossypium arboreum cyclizes (1RS)-[1-2H](E, E)-FDP to >98% [5-2H]and [11-2H]CDN. Enzyme reaction mixtures cyclize (3RS)-[4,4,13,13,13-2H5]-nerolidyl diphosphate to 62.1% [8,8,15,15,15-2H5]-CDN, 15.8% [6,6,15,15,15-2H5]-alpha-bisabolol, 8.1% [6,6,15,15,15-2H5]-(beta)-bisabolene, 9.8% [4,4,13,13-2H4]-(E)-beta-farnesene, and 4.2% unknowns. Competitive studies show that (3R)-nerolidyl diphosphate is the active enantiomer of (3RS)-nerolidyl diphosphate that cyclized to CDN. The kcat/Km values demonstrate that the synthase uses (E,E)-FDP as effectively as (3R)-nerolidyl diphosphate in the formation of CDN. Cyclization studies with (3R)-nerolidyl diphosphate show that the formation of CDN, (E)-beta-farnesene, and beta-bisabolene are enzyme dependent, but the formation of alpha-bisabolol in the reaction mixtures was a Mg2+-dependent solvolysis of nerolidyl diphosphate. Enzyme mechanisms are proposed for the formation of CDN from (E,E)-FDP and for the formation of CDN, (E)-beta-farnesene, and beta-bisabolene from (3RS)-nerolidyl diphosphate. The primary structures of cotton CDN synthase and tobacco epi-aristolochene synthase show 48% identity, suggesting similar three-dimensional structures. We used the SWISS-MODEL to test this. The two enzymes have the same overall structure consisting of two alpha-helical domains and epi-aristolochene synthase is a good model for the structure of CDN synthase. Several amino acids in the primary structures of both synthases superimpose. The amino acids having catalytic roles in epi-aristochene synthase are substituted in the CDN synthase and may be related to differences in catalytic properties.     

Expression of the nopaline synthase gene in Escherichia coli

The Agrobacterium tumor-inducing (Ti) plasmid pTiT37 encodes nopaline synthase (NOS) gene (nos) with eukaryotic promoter elements that is expressed in transformed plant cells but not in the bacterial host. We have fused the nos gene to the Escherichia coli trp promoter, and observed synthesis of NOS in E. coli. The nopaline produced by this enzyme is excreted into the culture medium. NOS is enzymatically active at 30 degrees C but not 37 degrees C, as based on nopaline production. NOS protein is produced at both temperatures, based on production in minicells.     

Enzymes encoded by the farnesyl diphosphate synthase gene family in the Big Sagebrush Artemisia tridentata ssp. spiciformis

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.     

Partial purification of a farnesyl diphosphate synthase from whole-body Manduca sexta

Farnesyl diphosphate synthase (FPP synthase) is a ubiquitous enzyme that is required for the biosynthesis of sesquiterpenes, dolichols ubiquinones, and prenylated proteins in insects. We report on the partial purification and characterization of an FPP synthase, obtained from whole-body preparations of the lepidopteran insect, Manduca sexta. The larval enzyme was separated from isopentenyl diphosphate (IPP) isomerase, phosphatase, and GGPP synthase by preparative isoelectric focusing, and was further purified by DEAE Sepharose, hydroxyapatite, and size exclusion chromatography. Whole-body M. sexta FPP synthase has a native molecular weight of 60.5+/-3.5 kDa and consists of two subunits of 28.5+/-0.5 kDa. As seen with other prenyltransferases, the enzyme has an absolute requirement for divalent cation and both Mn(2+) and Mg(2+) stimulated activity, although the former was inhibitory at higher concentrations. Insect FPP synthase catalyzes the condensation of IPP (K(m)=2.9+/-1.2 microM) with both dimethylallyl diphosphate and geranyl diphosphate (K(m)=0.8+/-0.4 microM). The enzyme requires the presence of detergent, glycerol, and non-specific protein-protein interactions for stability and maximum catalytic activity.