The subcellular localization of periwinkle farnesyl diphosphate synthase provides insight into the role of peroxisome in isoprenoid biosynthesis

Farnesyl diphosphate (FPP) synthase (FPS: EC.2.5.1.1, EC.2.5.1.10) catalyzes the formation of FPP from isopentenyl diphosphate and dimethylallyl diphosphate via two successive condensation reactions. A cDNA designated CrFPS, encoding a protein showing high similarities with trans-type short FPS isoforms, was isolated from the Madagascar periwinkle (Catharanthus roseus). This cDNA was shown to functionally complement the lethal FPS deletion mutant in the yeast Saccharomyces cerevisiae. At the subcellular level, while short FPS isoforms are usually described as cytosolic proteins, we showed, using transient transformations of C. roseus cells with yellow fluorescent protein-fused constructs, that CrFPS is targeted to peroxisomes. This finding is discussed in relation to the subcellular distribution of FPS isoforms in plants and animals and opens new perspectives towards the understanding of isoprenoid biosynthesis.     

Engineering linear,branched‐chain triterpene metabolism in monocots

Triterpenes are thirty‐carbon compounds derived from the universal five‐carbon prenyl precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Normally, triterpenes are synthesized via the mevalonate (MVA) pathway operating in the cytoplasm of eukaryotes where DMAPP is condensed with two IPPs to yield farnesyl diphosphate (FPP), catalyzed by FPP synthase (FPS). Squalene synthase (SQS) condenses two molecules of FPP to generate the symmetrical product squalene, the first committed precursor to sterols and most other triterpenes. In the green algae Botryococcus braunii, two FPP molecules can also be condensed in an asymmetric manner yielding the more highly branched triterpene, botryococcene. Botryococcene is an attractive molecule because of its potential as a biofuel and petrochemical feedstock. Because B. braunii, the only native host for botryococcene biosynthesis, is difficult to grow, there have been efforts to move botryococcene biosynthesis into organisms more amenable to large‐scale production. Here, we report the genetic engineering of the model monocot, Brachypodium distachyon, for botryococcene biosynthesis and accumulation. A subcellular targeting strategy was used, directing the enzymes (botryococcene synthase [BS] and FPS) to either the cytosol or the plastid. High titres of botryococcene (>1 mg/g FW in T₀ mature plants) were obtained using the cytosolic‐targeting strategy. Plastid‐targeted BS + FPS lines accumulated botryococcene (albeit in lesser amounts than the cytosolic BS + FPS lines), but they showed a detrimental phenotype dependent on plastid‐targeted FPS, and could not proliferate and survive to set seed under phototrophic conditions. These results highlight intriguing differences in isoprenoid metabolism between dicots and monocots.     

The Arabidopsis thaliana FPP synthase isozymes have overlapping and specific functions in isoprenoid biosynthesis,and complete loss of FPP synthase activity causes early developmental arrest

Farnesyl diphosphate (FPP) synthase (FPS) catalyses the synthesis of FPP, the major substrate used by cytosolic and mitochondrial branches of the isoprenoid pathway. Arabidopsis contains two farnesyl diphosphate synthase genes, FPS1 and FPS2, that encode isozymes FPS1L (mitochondrial), FPS1S and FPS2 (both cytosolic). Here we show that simultaneous knockout of both FPS genes is lethal for Arabidopsis, and embryo development is arrested at the pre‐globular stage, demonstrating that FPP‐derived isoprenoid metabolism is essential. In addition, lack of FPS enzyme activity severely impairs male genetic transmission. In contrast, no major developmental and metabolic defects were observed in fps1 and fps2 single knockout mutants, demonstrating the redundancy of the genes. The levels of sterols and ubiquinone, the major mitochondrial isoprenoid, are only slightly reduced in the single mutants. Although one functional FPS gene is sufficient to support isoprenoid biosynthesis for normal growth and development, the functions of FPS1 and FPS2 during development are not completely redundant. FPS1 activity has a predominant role during most of the plant life cycle, and FPS2 appears to have a major role in seeds and during the early stages of seedling development. Lack of FPS2 activity in seeds, but not of FPS1 activity, is associated with a marked reduction in sitosterol content and positive feedback regulation of 3‐hydroxy‐3‐methylglutaryl CoA reductase activity that renders seeds hypersensitive to the 3‐hydroxy‐3‐methylglutaryl CoA reductase inhibitor mevastatin.     

Molecular characterization of farnesyl pyrophosphate synthase from Bacopa monniera by comparative modeling and docking studies

Farnesyl pyrophosphate synthase (FPS; EC 2.5.1.10) is a key enzyme in isoprenoid biosynthetic pathway and provides precursors for the biosynthesis of various pharmaceutically important metabolites. It catalyzes head to tail condensation of two isopentenyl pyrophosphate molecules with dimethylallyl pyrophosphate to form C15 compound farnesyl pyrophosphate. Recent studies have confirmed FPS as a molecular target of bisphosphonates for drug development against bone diseases as well as pathogens. Although large numbers of FPSs from different sources are known, very few protein structures have been reported till date. In the present study, FPS gene from medicinal plant Bacopa monniera (BmFPS) was characterized by comparative modeling and docking. Multiple sequence alignment showed two highly conserved aspartate rich motifs FARM and SARM (DDXXD). The 3-D model of BmFPS was generated based on structurally resolved FPS crystal information of Gallus gallus. The generated models were validated by various bioinformatics tools and the final model contained only α-helices and coils. Further, docking studies of modeled BmFPS with substrates and inhibitors were performed to understand the protein ligand interactions. The two Asp residues from FARM (Asp100 and Asp104) as well as Asp171, Lys197 and Lys262 were found to be important for catalytic activity. Interaction of nitrogen containing bisphosphonates (risedronate, alendronate, zoledronate and pamidronate) with modeled BmFPS showed competitive inhibition; where, apart from Asp (100, 104 and 171), Thr175 played an important role. The results presented here could be useful for designing of mutants for isoprenoid biosynthetic pathway engineering well as more effective drugs against osteoporosis and human pathogens.

Abbreviations

IPP - Isopentenyl Pyrophosphate, DMAPP - Dimethylallyl Pyrophosphate, GPP - Geranyl Pyrophosphate, FPP - FPPFarnesyl Pyrophosphate, DOPE - Discrete Optimized Protein Energy, BmFPS - Bacopa monniera Farnesyl Pyrophosphate Synthase, RMSD - Root Mean square Deviation, OPLS-AA - Optimized Potentials for Liquid Simulations- All Atom, FARM - First Aspartate Rich Motif, SARM - Second Aspartate Rich Motif.     

Molecular analyses of the Chinese herb Leigongteng (Tripterygium wilfordii Hook.f.)

Tripterygium wilfordii Hook.f., known as Leigongteng (Thunder God Vine) in traditional Chinese medicine, has attracted much attention for its applications in relieving autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus, and for treating cancer. Molecular analyses of the ITS and 5S rDNA sequences indicate that T. hypoglaucum and T. doianum are not distinct from T. wilfordii, while T. regelii should be recognized as a separate species. The results also demonstrate potential value of rDNA sequence data in forensic detection of adulterants derived from Celastrusangulatus in commercial samples of Leigongteng.     

Identification and expression analysis of the <Emphasis Type="Italic">Eucommia ulmoides</Emphasis> farnesyl diphosphate synthase gene family to reveal the key gene involved in rubber biosynthesis

Eucommia ulmoides Oliver is rich in trans-polyisoprene rubber (Eu-Rubber), a high-molecular mass polymer of isoprene units with a trans-configuration. Farnesyl diphosphate (FPP) synthase (FPS) is a key enzyme, which involved in the production of important precursors of different terpenoids. In this study, we cloned and characterized five novel FPS genes from E. ulmoides. The full-length synthases were named EuFPS1-5 and their deduced amino acid sequences exhibited high homology to those from other plant isoforms. EuFPS1 and EuFPS4 were observed to be highly expressed in leaves, EuFPS2 and EuFPS3 were present at low levels in leaves and fruit throughout the plant development, and EuFPS5 was highly expressed exclusively in young fruit. Expression of EuFPS5 correlated with the accumulation rate of Eu-Rubber and might be responsible for it. This study is expected to enhance our understanding of the role of EuFPSs in biosynthesis and regulation of useful secondary metabolites in E. ulmoides.     

Molecular cloning and expression of Chimonanthus praecox farnesyl pyrophosphate synthase gene and its possible involvement in the biosynthesis of floral volatile sesquiterpenoids

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.     

Farnesyl Phosphatase,a Corpora allata Enzyme Involved in Juvenile Hormone Biosynthesis in Aedes aegypti

Background

The juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. The late steps of JH III biosynthesis in the mosquito Aedes aegypti involve the hydrolysis of farnesyl pyrophosphate (FPP) to farnesol (FOL), which is then successively oxidized to farnesal and farnesoic acid, methylated to form methyl farnesoate and finally transformed to JH III by a P450 epoxidase. The only recognized FPP phosphatase (FPPase) expressed in the corpora allata (CA) of an insect was recently described in Drosophila melanogaster (DmFPPase). In the present study we sought to molecularly and biochemically characterize the FPP phosphatase responsible for the transformation of FPP into FOL in the CA of A. aegypti.

Methods

A search for orthologs of the DmFPPase in Aedes aegypti led to the identification of 3 putative FPPase paralogs expressed in the CA of the mosquito (AaFPPases-1, -2, and -3). The activities of recombinant AaFPPases were tested against general phosphatase substrates and isoprenoid pyrophosphates. Using a newly developed assay utilizing fluorescent tags, we analyzed AaFPPase activities in CA of sugar and blood-fed females. Double-stranded RNA (dsRNA) was used to evaluate the effect of reduction of AaFPPase mRNAs on JH biosynthesis.

Conclusions

AaFPPase-1 and AaFPPase-2 are members of the NagD family of the Class IIA C2 cap-containing haloalkanoic acid dehalogenase (HAD) super family and efficiently hydrolyzed FPP into FOL. AaFPPase activities were different in CA of sugar and blood-fed females. Injection of dsRNAs resulted in a significant reduction of AaFPPase-1 and AaFPPase-2 mRNAs, but only reduction of AaFPPase-1 caused a significant decrease of JH biosynthesis. These results suggest that AaFPPase-1 is predominantly involved in the catalysis of FPP into FOL in the CA of A. aegypti.     

Spatial and temporal patterns of GUS expression directed by 5′ regions of the Arabidopsis thaliana farnesyl diphosphate synthase genes FPS1 and FPS2

Farnesyl diphosphate synthase (FPS), the enzyme that catalyses the synthesis of farnesyl diphosphate (FPP) from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), is considered a regulatory enzyme of plant isoprenoid biosynthesis. The promoter regions of the FPS1 and FPS2 genes controlling the expression of isoforms FPS1S and FPS2, respectively, were fused to the -glucuronidase (GUS) reporter gene and introduced into Arabidopsis thaliana plants. The FPS1S:GUS gene is widely expressed in all plant tissues throughout development, thus supporting a role for FPS1S in the synthesis of isoprenoids serving basic plant cell functions. In contrast, the FPS2:GUS gene shows a pattern of expression restricted to specific organs at particular stages of development. The highest levels of GUS activity are detected in flowers, especially in pollen grains, from the early stages of flower development. After pollination, much lower levels of GUS activity are detected in the rest of floral organs, with the exception of the ovary valves, which remain unstained throughout flower development. GUS activity is also detected in developing and mature seeds. In roots, GUS expression is primarily detected at sites of lateral root initiation and in junctions between primary and secondary roots. No GUS activity is detected in root apical meristems. GUS expression is also observed in junctions between primary and secondary stems. Overall, the pattern of expression of FPS2:GUS suggests a role for FPS2 in the synthesis of particular isoprenoids with specialized functions. Functional FPS2 gene promoter deletion analysis in transfected protoplasts and transgenic A. thaliana plants indicate that all the cis-acting elements required to establish the full pattern of expression of the FPS2 gene are contained in a short region extending from positions –111 to +65. The potential regulatory role of specific sequences within this region is discussed.     

In silico study of binding motifs in squalene synthase enzyme of secondary metabolic pathway solanaceae family

Solanaceae is an important family with several plants of medicinal importance. These medicinal plants have distinctive pathways for secondary metabolite biosynthesis. In most of the plants, two important compounds, dimethylallyl diphosphate and isopentenyl diphosphate, synthesize isoprenoid or terpenoids. Squalene synthase (SQS) is a key enzyme of the biosynthesis of isoprenoid (farnesyl pyrophosphate (FPP) → squalene). Withania somnifera (ashwagandha), an important medicinal plant of family solanaceae produces withanolides. Withanolides are secondary metabolites synthesized through isoprenoid pathway. In this study, 13 SQS protein sequences from the plants of solanacae family and Arabidopsis thaliana were analyzed. The conserved domains in corresponding sequences were searched. The multiple sequence alignment of conserved domains revealed the important motifs and identified the residue substitution in each motif. Our result further indicated that residue substitution in motifs might not lead to functional variation, although it may affect the binding affinity of Mg⁺⁺, FPP and NAD(P)H. In addition, the homology modelling of SQS enzyme of W. somnifera was done for the prediction of three-dimensional structure. Molecular docking study of considered substrates with WsSQS was performed and the docked structure were analyzed further. The docked structures showed binding affinity for motif 2 of WsSQS. Our analysis revealed that 29 residues of motif 2 might be important for catalytic/functional activity of SQS enzyme of W. somnifera. This study may provide an understanding of metabolic pathways responsible for the production of secondary metabolites. The motifs may play a key role in regulating the pathway towards enhanced production of metabolites.     

法呢基焦磷酸(FPP)的生物合成及其分子调控

法呢基焦磷酸合酶作为异戊二烯途径中的重要调节酶,是许多萜类物质的合成前体。FPS的cDNA克隆在许多生物体中也已得到了分离并进行了表达特性研究。从FPP的生物合成途径入手,对FPP生物学特性、FPS酶基因调控的相关信息进行了综述,同时对FPS在基因工程方面的应用进行了展望。     

Cyclooxygenase-2 and Prostaglandin E2 Signaling through Prostaglandin Receptor EP-2 Favor the Development of Myocarditis during Acute Trypanosoma cruzi Infection

Inflammation plays an important role in the pathophysiology of Chagas disease, caused by Trypanosoma cruzi. Prostanoids are regulators of homeostasis and inflammation and are produced mainly by myeloid cells, being cyclooxygenases, COX-1 and COX-2, the key enzymes in their biosynthesis from arachidonic acid (AA). Here, we have investigated the expression of enzymes involved in AA metabolism during T. cruzi infection. Our results show an increase in the expression of several of these enzymes in acute T. cruzi infected heart. Interestingly, COX-2 was expressed by CD68⁺ myeloid heart-infiltrating cells. In addition, infiltrating myeloid CD11b⁺Ly6G^- cells purified from infected heart tissue express COX-2 and produce prostaglandin E₂ (PGE₂) ex vivo. T. cruzi infections in COX-2 or PGE₂-dependent prostaglandin receptor EP-2 deficient mice indicate that both, COX-2 and EP-2 signaling contribute significantly to the heart leukocyte infiltration and to the release of chemokines and inflammatory cytokines in the heart of T. cruzi infected mice. In conclusion, COX-2 plays a detrimental role in acute Chagas disease myocarditis and points to COX-2 as a potential target for immune intervention.     

Cloning, expression and characterization of squalene synthase from Inonotus obliquus

The chaga mushroom Inonotus obliquus has been widely used as a folk medicine in Russia, Poland and most of the Baltic countries. The total triterpene saponins of I. obliquus have significant pharmacological activity. Though the triterpene component has been well characterized in terms of its pharmaceutical activity, there is little information on the genes responsible for the biosynthesis of these compounds in I. obliquus. Squalene synthase represents a potential branching point and the first committed step to diverge the carbon flux from the main isoprenoid pathway towards sterol biosynthesis. In this study, we cloned and characterized squalene synthase from I. obliquus. A 1476-bp full-length cDNA consisting of the entire coding region of squalene synthase (GenBank accession number is KC182754) was cloned by RT-PCR. The DNA sequence showed as much as 76 % similarity with the sequence of Fomitiporia mediterranea squalene synthase, and phylogenetic analysis indicated that it is most closely related to F. mediterranea squalene synthase at both DNA and protein levels. I. obliquus squalene synthase was actively expressed in the yeast Pichia pastoris as a secreted form and purified by gel filtration using Superdex G-75 column. The purified recombinant squalene synthase was able to convert farnesyl diphosphate (FPP) to squalene in an NADPH-dependent reaction. The result of this study could serve as an important step toward the manipulation of triterpenoids biosynthesis in I. obliquus at the level of squalene through engineering better SQS for reintroduction into the mushroom.     

Three-step pathway engineering results in more incidence rate and higher emission of nerolidol and improved attraction of Diadegma semiclausum

The concentration and ratio of terpenoids in the headspace volatile blend of plants have a fundamental role in the communication of plants and insects. The sesquiterpene (E)-nerolidol is one of the important volatiles with effect on beneficial carnivores for biologic pest management in the field. To optimize de novo biosynthesis and reliable and uniform emission of (E)-nerolidol, we engineered different steps of the (E)-nerolidol biosynthesis pathway in Arabidopsis thaliana.Introduction of a mitochondrial nerolidol synthase gene mediates de novo emission of (E)-nerolidol and linalool. Co-expression of the mitochondrial FPS1 and cytosolic HMGR1 increased the number of emitting transgenic plants (incidence rate) and the emission rate of both volatiles. No association between the emission rate of transgenic volatiles and their growth inhibitory effect could be established. (E)-Nerolidol was to a large extent metabolized to non-volatile conjugates.     

Molecular cloning and expression of Hedychium coronarium farnesyl pyrophosphate synthase gene and its possible involvement in the biosynthesis of floral and wounding/herbivory induced leaf volatile sesquiterpenoids

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.     

Stereochemistry of 2-aminopimelic acid and related amino acids in three species of asplenium

We previously reported two free D-amino acids, D-2-aminopimelic acid (D-APA) and trans-3,4-dehydro-D-2-aminopimelic acid (D-Δ-APA), from Asplenium unilaterale. In the present work we isolated 4-hydroxy-2-aminopimelic acid (OH-APA) from the same plant and determined it to be the α-L-form. We also investigated the configurations of these amino acids isolated from A. prolongatum and A. wilfordii which are morphologically distinct from A. unilaterale. In A. prolongatum, APA was the D- and OH-APA was the L-isomer. In contrast, APA from A. wilfordii was partially racemized and the degree of racemization was significantly different in plant material collected in July and November, L:D = 3:2 and 3:7, respectively. In A. wilfordii OH-APA was almost pure L- and Δ-APA was mostly the D-isomer.     

Aphid-repellent pheromone E-β-farnesene is generated in transgenic Arabidopsis thaliana over-expressing farnesyl diphosphate synthase2

Background and Aims Plant-synthesized sesquiterpenes play a pivotal role in chemotactic interactions with insects. Biosynthesis of functionally diverse sesquiterpenes is dependent on the availability of a pool of the precursor farnesyldiphosphate (FDP). In Arabidopsis thaliana, FPS2, encoding cytosolic farnesyldiphosphate synthase, is implicated in the synthesis of cytosolic FDP, but it is not known whether enhanced levels of FDP have a commensurate effect on sesquiterpene-mediated defence responses. This study examined transgenic arabidopsis plants generated to over-express FPS2 in order to determine if any effects could be observed in the response of aphids, Myzus persicae.Methods Transgenic arabidopsis plants were generated to over-express FPS2 to produce FPS2 in either the cytosol or the chloroplasts. Morphochemical analyses of the transgenic plants were carried out to detremine growth responses of roots and shoots, and for GC-MS profiling of sesquiterpenes. Aphid response to hyrdo-distillate extracts and head-space volatiles from transgenic plants was assessed using a bioassay.Key Results Either over-expression of FPS2 in the cytosol or targetting of its translated product to chlorplasts resulted in stimulatory growth responses of transgenic arabidopsis at early and late developmental stages. GC-MS analysis of hydro-distillate extracts from aerial parts of the plants revealed biosynthesis of several novel sesquiterpenes, including E-β-farnesene, an alarm pheromone of aphids. Both entrapped volatiles and hydro-distillate extracts of the transgenic leaves triggered agitation in aphids, which was related to both time and dose of exposure.Conclusions Over-expression of FPS2 in the cytosol and targeting of its translated product to chloroplasts in arabidopsis led to synthesis of several novel sesquiterpenes, including E-β-farnesene, and induced alarm responses in M. persicae. The results suggest a potential for engineering aphid-resistant strains of arabidopsis.     

Structures,mechanisms and inhibitors of undecaprenyl diphosphate synthase: A cis-prenyltransferase for bacterial peptidoglycan biosynthesis

Isoprenoids are an intensive group of compounds made from isopentenyl diphosphate (IPP), catalyzed by prenyltransferases such as farnesyl diphosphate (FPP) cyclases, squalene synthase, protein farnesyltransferases and geranylgeranyltransferases, aromatic prenyltransferases as well as a group of prenyltransferases (cis- and trans-types) catalyzing consecutive condensation reactions of FPP with specific numbers of IPP to generate linear products with designate chain lengths. These prenyltransferases play significant biological functions and some of them are drug targets. In this review, structures, mechanisms, and inhibitors of a cis-prenyltransferase, undecaprenyl diphosphate synthase (UPPS) that mediates bacterial peptidoglycan biosynthesis, are summarized for comparison with the most related trans-prenyltransferases and other prenyltransferases.