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1.
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Farnesyl pyrophosphate (FPP) synthase catalyzes the biosynthesis of FPP, which is the precursors of sesquiterpenoids such as floral scent volatiles, from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). cDNA encoding wintersweet (Chimonanthus praecox L.) FPP synthase was isolated by the RT-PCR and RACE methods. The deduced amino acid sequence showed a high identity to plant FPP synthases. Expression of the gene in Escherichia coli yielded FPPS activity that catalyzed the synthesis of FPP as a main product. Tissue-specific and developmental analyses of the mRNA levels of CpFPPS and volatile sesquiterpenoids levels in C. praecox flowers revealed that the FPPS may play a regulatory role in floral volatile sesquiterpenoids of wintersweet.  相似文献   

3.
Farnesyl diphosphate synthase (FPPS) catalyzes the consecutive condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate to form farnesyl diphosphate (FPP). In insects, FPP is used for the synthesis of ubiquinones, dolicols, protein prenyl groups, and juvenile hormone. A full‐length cDNA of FPPS was cloned from the cotton boll weevil, Anthonomus grandis (AgFPPS). AgFPPS cDNA consists of 1,835 nucleotides and encodes a protein of 438 amino acids. The deduced amino acid sequence has high similarity to previously isolated insect FPPSs and other known FPPSs. Recombinant AgFPPS expressed in E. coli converted labeled isopentenyl diphosphate in the presence of dimethylallyl diphosphate to FPP. Southern blot analysis indicated the presence of a single copy gene. Using molecular modeling, the three‐dimensional structure of coleopteran FPPS was determined and compared to the X‐ray crystal structure of avian FPPS. The α‐helical fold is conserved in AgFPPS and the size of the active site cavity is consistent with the enzyme being a FPPS. © 2009 Wiley Periodicals, Inc.  相似文献   

4.
Farnesyl diphosphate synthase (FPPS) is a key enzyme in isoprenoid biosynthesis, it catalyzes the head-to-tail condensation of dimethylallyl diphosphate (DMAPP) with two molecules of isopentenyl diphosphate (IPP) to generate farnesyl diphosphate (FPP), a precursor of juvenile hormone (JH). In this study, we functionally characterized an Aedes aegypti FPPS (AaFPPS) expressed in the corpora allata. AaFPPS is the only FPPS gene present in the genome of the yellow fever mosquito, it encodes a 49.6 kDa protein exhibiting all the characteristic conserved sequence domains on prenyltransferases. AaFPPS displays its activity in the presence of metal cofactors; and the product condensation is dependent of the divalent cation. Mg2+ ions lead to the production of FPP, while the presence of Co2+ ions lead to geranyl diphosphate (GPP) production. In the presence of Mg2+ the AaFPPS affinity for allylic substrates is GPP > DMAPP > IPP. These results suggest that AaFPPS displays “catalytic promiscuity”, changing the type and ratio of products released (GPP or FPP) depending on allylic substrate concentrations and the presence of different metal cofactors. This metal ion-dependent regulatory mechanism allows a single enzyme to selectively control the metabolites it produces, thus potentially altering the flow of carbon into separate metabolic pathways.  相似文献   

5.
A farnesyl diphosphate synthase gene (FPPS2), which contains 11 introns and 12 exons, was isolated from the apple cultivar “White Winter Pearmain”. When it was compared to our previously reported FPPS1, its each intron size was different, its each exon size was the same as that of FPPS1 gene, 30 nucleotide differences were found in its coding sequence. Based on these nucleotide differences, specific primers were designed to perform expression analysis; the results showed that it expressed in both fruit and leaf, its expression level was obviously lower than that of FPPS1 gene in fruit which was stored at 4 °C for 5 weeks. This is the first report concerning two FPPS genes and their expression comparison in apples.  相似文献   

6.
A cDNA encoding farnesyl diphosphate synthase, an enzyme that synthesizes C15 isoprenoid diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate, was cloned from an Arabidopsis thaliana cDNA library by complementation of a mutant of Saccharomyces cerevisiae deficient in this enzyme. The A. thaliana cDNA was also able to complement the lethal phenotype of the erg20 deletion yeast mutant. As deduced from the full-length 1.22 kb cDNA nucleotide sequence, the polypeptide contains 343 amino acids and has a relative molecular mass of 39689. The predicted amino acid sequence presents about 50% identity with the yeast, rat and human FPP synthases. Southern blot analyses indicate that A. thaliana probably contains a single gene for farnesyl diphosphate synthase.  相似文献   

7.
Farnesyl diphosphate synthase (FPPS) catalyzes the condensation of the non-allylic diphosphate, isopentenyl diphosphate (IPP; C5), with the allylic diphosphate primer dimethylallyl diphosphate (DMAPP; C5) to generate the C15 prenyl chain (FPP) used for protein prenylation as well as sterol and terpene biosynthesis. Here, we designed and prepared a series of pyridinium bisphosphonate (PyrBP) compounds, with the aim of selectively inhibiting FPPS of the lepidopteran insect order. FPPSs of Drosophila melanogaster and the spruce budworm, Choristoneura fumiferana, were inhibited by several PyrBPs, and as hypothesized, larger bisphosphonates were more selective for the lepidopteran protein and completely inactive towards dipteran and vertebrate FPPSs. Cell growth of a D. melanogaster cell line was adversely affected by exposure to PyrPBs that were strongly inhibitory to insect FPPS, although their effect was less pronounced than that observed upon exposure to the electron transport disrupter, chlorfenapyr. To assess the impact of PyrBPs on lepidopteran insect growth and development, we performed feeding and topical studies, using the tobacco hornworm, Manduca sexta, as our insect model. The free acid form of a PyrBP and a known bisphosphonate inhibitor of vertebrate FPPS, alendronate, had little to no effect on larval M. sexta; however, the topical application of more lipophilic ester PyrBPs caused decreased growth, incomplete larval molting, cuticle darkening at the site of application, and for those insects that survived, the formation of larval–pupal hybrids. To gain a better understanding of the structural differences that produce selective lepidopteran FPPS inhibition, homology models of C. fumiferana and D. melanogaster FPPS (CfFPPS2, and DmFPPS) were prepared. Docking of substrates and PyrBPs demonstrates that differences at the −3 and −4 positions relative to the first aspartate rich motif (FARM) are important factors in the ability of the lepidopteran enzyme to produce homologous isoprenoid structure and to be selectively inhibited by larger PyrBPs.  相似文献   

8.
A clone encoding farnesyl diphosphate synthase (FPPS) was obtained by PCR from a cDNA library made from young leaves of Artemisia annua. A cDNA clone encoding the tobacco epi-aristolochene synthase (eAS) was kindly supplied by J. Chappell (University of Kentucky, Lexington, KY, USA). Two fusions were constructed, i.e. FPPS/eAS and eAS/FPPS. The stop codon of the N-terminal enzyme was removed and replaced by a short peptide (Gly-Ser-Gly) to introduce a linker between the two ORFs. These two fusions and the two single cDNA clones were separately introduced into a bacterial expression vector (pET32). Escherichia coli was transformed with the expression vectors and enzymatically active soluble proteins were obtained after induction with isopropyl thio-beta-d-thiogalactoside. The recombinant enzymes were purified using immobilized metal affinity chromatography on Co2+ columns. The fusion enzymes produced epi-aristolochene from isopentenyl diphosphate through a coupled reaction. The Km values of FPPS and eAS for isopentenyl diphosphate and farnesyl diphosphate, respectively, were essentially the same for the single and fused enzymes. The bifunctional enzymes showed a more efficient conversion of isopentenyl diphosphate to epi-aristolochene than the corresponding amount of single enzymes.  相似文献   

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10.
Eucommia ulmoides Oliver is one of a few woody plants capable of producing abundant quantities of trans-polyisoprene rubber in their leaves, barks, and seed coats. One cDNA library each was constructed from its outer stem tissue and inner stem tissue. They comprised a total of 27,752 expressed sequence tags (ESTs) representing 10,520 unigenes made up of 4,302 contigs and 6,218 singletons. Homologues of genes coding for rubber particle membrane proteins that participate in the synthesis of high-molecular poly-isoprene in latex were isolated, as well as those encoding known major latex proteins (MLPs). MLPs extensively shared ESTs, indicating their abundant expression during trans-polyisoprene rubber biosynthesis. The six mevalonate pathway genes which are implicated in the synthesis of isopentenyl diphosphate (IPP), a starting material of poly-isoprene biosynthesis, were isolated, and their role in IPP biosynthesis was confirmed by functional complementation of suitable yeast mutants. Genes encoding five full-length trans-isoprenyl diphosphate synthases were also isolated, and two among those synthesized farnesyl diphosphate from IPP and dimethylallyl diphosphate, an assumed intermediate of rubber biosynthesis. This study should provide a valuable resource for further studies of rubber synthesis in E. ulmoides.  相似文献   

11.
The yeast farnesyl diphosphate synthase (FPPS) gene was engineered so as to construct allelic forms giving various activities of the enzyme. One of the substitutions was F96W in the chain length determination region. The other, K197, conserved within a consensus sequence found in the majority of FPP and GGPP synthases, was substituted by R, E and V. An intricate correlation has been found between the FPPS activity, the amount of ergosterol synthesized and cell growth of a mutant strain defective in FPPS. About 40% of wt FPPS activity was sufficient to support normal growth of the mutant. With further decline of FPPS activity (20 down to 3%) the amount of ergosterol remained unchanged at approximately 0.16% (vs dry weight), whereas growth yield decreased and lag times increased. We postulate that, in addition to ergosterol initiating and maintaining growth of yeast cells, FPP and/or its derivatives participate in these processes.  相似文献   

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13.
The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which then cyclizes to the various monoterpene skeletons. X-ray crystal structures of these enzymes complexed with suitable analogues of the substrate and intermediate could provide a clearer view of this universal, but cryptic, step of monoterpenoid cyclase catalysis. Toward this end, the functionally inert analogues 2-fluorogeranyl diphosphate, (±)-2-fluorolinalyl diphosphate, and (3R)- and (3S)-homolinalyl diphosphates (2,6-dimethyl-2-vinyl-5-heptenyl diphosphates) were prepared, and compared to the previously described substrate analogue 3-azageranyl diphosphate (3-aza-2,3-dihydrogeranyl diphosphate) as inhibitors and potential crystallization aids with two representative monoterpenoid cyclases, (-)-limonene synthase and (+)-bornyl diphosphate synthase. Although these enantioselective synthases readily distinguished between (3R)- and (3S)-homolinalyl diphosphates, both of which were more effective inhibitors than was 3-azageranyl diphosphate, the fluorinated analogues proved to be the most potent competitive inhibitors and have recently yielded informative liganded structures with limonene synthase.  相似文献   

14.
The farnesyl diphosphate synthase (FPPS) has previously been characterized in trypanosomes as an essential enzyme for their survival and as the target for bisphosphonates, drugs that are effective both in vitro and in vivo against these parasites. Enzymes from the isoprenoid pathway have been assigned to different compartments in eukaryotes, including trypanosomatids. We here report that FPPS localizes to the cytoplasm of both Trypanosoma cruzi and T. brucei, and is not present in other organelles such as the mitochondria and glycosomes.  相似文献   

15.
Farnesyl pyrophosphate synthase (FPPS EC 2.5.1.10) catalyzes the production of farnesyl pyrophosphate (FPP), which is a key precursor for many sesquiterpenoids such as floral scent and defense volatiles against herbivore attack. Here we report a new full-length cDNA encoding farnesyl diphosphate synthase from Hedychium coronarium. The open reading frame for full-length HcFPPS encodes a protein of 356 amino acids, which is 1068 nucleotides long with calculated molecular mass of 40.7 kDa. Phylogenetic tree analysis indicates that HcFPPS belongs to the plant FPPS super-family and has strong relationship with FPPS from Musa acuminata. Expression of the HcFPPS gene in Escherichia coli yielded FPPS activity. Tissue-specific and developmental analyses of the HcFPPS mRNA and corresponding volatile sesquiterpenoid levels in H. coronarium flowers revealed that the HcFPPS might play a regulatory role in floral volatile sesquiterpenoid biosynthesis. The emission of the FPP-derived volatile terpenoid correlates with strong expression of HcFPPS induced by mechanical wounding and Udaspes folus-damage in leaves, which suggests that HcFPPS may have an important ecological function in H. coronarium vegetative organ.  相似文献   

16.
Farnesyl diphosphate synthase (FPPS) has been identified as an androgen-response gene in the rat ventral prostate using a highly sensitive PCR-based cDNA subtraction technique. FPPS is an essential enzyme that catalyzes the synthesis of farnesyl diphosphate (FPP), which is required for cholesterol biosynthesis as well as protein prenylation. We have characterized the expression of FPPS in the rat prostate in response to androgen manipulation. Northern blot analysis showed that castration induced a 10-fold down-regulation of FPPS mRNA within 24 h in the ventral prostate and androgen replacement up-regulated FPPS mRNA rapidly in the regressed ventral prostate of a castrated rat. The expression of FPPS was also regulated by androgen in the lateral and dorsal prostate, indicating that FPPS is important to androgen action in all three lobes of the prostate. Western blot analysis showed that FPPS protein level was also regulated by androgen in the prostate. Northern blot analysis of tissue specificity indicated that FPPS was most abundantly expressed in the ventral prostate of a mature rat and was responsive to androgen manipulation in the prostate and seminal vesicles, but not in other tissues. In situ hybridization study showed that FPPS mRNA was localized to the prostatic epithelium. Interestingly, the expression of FPPS was elevated in Dunning rat prostate tumor cell lines. The above findings suggest that FPPS has the potential to play an important role in androgen action and prostate cancer progression.  相似文献   

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18.
《Gene》1997,189(1):79-82
A cDNA clone, blpl14, corresponding to the large subunit of ADP-glucose pyrophosphorylase (AGPase), has been isolated from a cDNA library prepared from leaves of barley (Hordeum vulgare L.). An open reading frame encodes a protein of 503 aa, with a calculated molecular weight of 54 815. The derived aa sequence contains a putative transit peptide sequence, required for targeting to plastids, and has a highly conserved positioning of critical Lys residues that are believed to be involved in effector binding. The derived aa sequence shows 97% identity with the corresponding protein from wheat, but only 36% identity with AGPase from E. coli. The blpl14 gene is expressed predominantly in leaves and to a lesser degree in seed endosperm, but not roots, of barley.  相似文献   

19.
《Gene》1996,172(2):211-215
We have sequenced a cDNA and a gene, AtRPC14, from Arabidopsis thaliana (At) (ecotype Columbia) that encode a protein related to the yeast RNA polymerases (Pol) I and III subunits, yAC19. Polyclonal antibodies raised against the recombinant At polypeptide (AtC14) bind to the Pol I and/or III subunits of about 13–15 kDa, but do not bind to any Pol II subunit in Pol purified from cauliflower, wheat or At. The amino acid (aa) sequence derived from the AtRPC14 cDNA and genomic clones consists of 122 aa, as compared to the 142 aa in the yeast yAC19 subunit and 143 aa in a putative Caenorhabditis elegans CeAC16 subunit. AtC14, yAC19 and CeAC16 contain a conserved sequence of about 85 aa which is related to two motifs in the α subunit of Escherichia coli (Ec) Pol. AtC14 lacks a highly charged N terminus of about 50 aa found in both yAC19 and CeAC16, but has a highly charged C terminus of about 30 aa not found in yAC19 and CeAC16.  相似文献   

20.
cDNAs specifying four active phosphoribosyl diphosphate synthase isozymes were isolated from an Arabidopsis thaliana cDNA library. In contrast to other phosphoribosyl diphosphate synthases the activity of two of the A. thaliana isozymes are independent of Pi. Amino acid sequence comparison and phylogenetic analysis indicate that these two isozymes belong to a novel class of phosphoribosyl diphosphate synthases.  相似文献   

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