Different reaction mechanisms for cis- and trans-prenyltransferases

Octaprenyl diphosphate synthase (OPPs) and undecaprenyl diphosphate synthases (UPPs) catalyze consecutive condensation reactions of farnesyl diphosphate (FPP) with 5 and 8 isopentenyl diphosphate (IPP) to generate C₄₀ and C₅₅ products with trans- and cis-double bonds, respectively. In this study, we used IPP analogue, 3-bromo-3-butenyl diphosphate (Br-IPP), in conjunction with radiolabeled FPP, to probe the reaction mechanisms of the two prenyltransferases. Using this alternative substrate with electron-withdrawing bromo group at the C3 position to slow down the condensation step, trapping of farnesol in the OPPs reaction from radiolabeled FPP under basic condition was observed, consistent with a sequential mechanism. In contrast, UPPs reaction yielded no farnesyl carbocation intermediate under the same condition with radiolabeled FPP and Br-IPP, indicating a concerted mechanism. Our data demonstrate the different reaction mechanisms for cis- and tran-prenyltransferases although they share the same substrates.     

Mechanism of cis-prenyltransferase reaction probed by substrate analogues

Undecaprenyl pyrophosphate synthase (UPPS) is a cis-type prenyltransferases which catalyzes condensation reactions of farnesyl diphosphate (FPP) with eight isopentenyl pyrophosphate (IPP) units to generate C₅₅ product. In this study, we used two analogues of FPP, 2-fluoro-FPP and [1,1-²H₂]FPP, to probe the reaction mechanism of Escherichia coli UPPS. The reaction rate of 2-fluoro-FPP with IPP under single-turnover condition is similar to that of FPP, consistent with the mechanism without forming a farnesyl carbocation intermediate. Moreover, the deuterium secondary KIE of 0.985 ± 0.022 measured for UPPS reaction using [1,1-²H₂]FPP supports the associative transition state. Unlike the sequential mechanism used by trans-prenyltransferases, our data demonstrate E. coli UPPS utilizes the concerted mechanism.     

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。     

Isolation and characterization of farnesyl diphosphate synthase from the cotton boll weevil,Anthonomus grandis

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.     

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.     

A New Farnesyl Diphosphate Synthase Gene from Taxus media Rehder： Cloning, Characterization and Functional Complementation

Farnesyl dlphosphate synthase （FPS; EC 2.5.1.10） catalyzes the production of 15-carbon farnesyl dlphosphate which Is a branch-point Intermediate for many terpenoids. This reaction Is considered to be a ratelimiting step In terpenold biosynthesis. Here we report for the first time the cloning of a new full-length cDNA encoding farnesyl dlphosphate synthase from a gymnosperm plant species, Taxus media Rehder, designated as TmFPS1. The full-length cDNA of TmFPS1 （GenBank accession number： AY461811） was 1 464 bp with a 1 056-bp open reading frame encoding a 351-amino acid polypeptlde with a calculated molecular weight of 40.3 kDa and a theoretical pl of 5.07. Biolnformatlc analysis revealed that TmFPS1 contained all five conserved domains of prenyltransferases, and showed homology to other FPSs of plant origin. Phylogenetlc analysis showed that farnesyl dlphosphate synthases can be divided Into two groups： one of prokaryotic origin and the other of eukaryotic origin. TmFPS1 was grouped with FPSs of plant origin. Homologybased structural modeling showed that TmFPS1 had the typical spatial structure of FPS, whose most prominent structural feature Is the arrangement of 13 core helices around a large central cavity In which the catalytic reaction takes place. Our blolnformatic analysis strongly suggests that TmFPS1 is a functional gene. Southern blot analysis revealed that TmFPS1 belongs to a small FPSgene family in T. media. Northern blot analysis indicated that TmFPS1 is expressed in all tested tissues, Including the needles, stems and roots of T. media. Subsequently, functional complementatlon with TmFPS1 in a FPS-deflclent mutant yeast demonstrated that TmFPS1 did encode farnesyl dlphosphate synthase, which rescued the yeast mutant. This study will be helpful In future Investigations aiming at understanding the detailed role of FPS In terpenold biosynthesis flux control at the molecular genetic level.     

Substrate Specificities of Wild and Mutated Farnesyl Diphosphate Synthases from Bacillus Stearothermophilus with Artificial Substrates

To determine the substrate specificities of wild and mutated types of farnesyl diphosphate (FPP) synthases from Bacillus stearothermophilus, we examined the reactivities of 8-hydroxygeranyl diphosphate (HOGPP) and 8-methoxygeranyl diphosphate (CH₃OGPP) as allylic substrate homologs.

The wild-type FPP synthase reaction of HOGPP (and CH₃OGPP) with isopentenyl diphosphate (IPP) gave hydroxyfarnesyl- (and methoxyfarnesyl-) diphosphates that stopped at the first stage of condensation.

On the other hand, with mutated type FPP synthase (Y81S), the former gave hydroxygeranylgeranyl diphosphate as the main double-condensation product together with hydroxyfarnesyl diphosphate as a single-condensation product and a small amount of hydroxygeranylfarnesyl diphosphate as a triple-condensation product. Moreover, the latter gave a double-condensation product, methoxygeranylgeranyl diphosphate, as the main product and only a trace of methoxyfarnesyl diphosphate was obtained.     

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.     

Overexpression of Farnesyl Diphosphate Synthase in Arabidopsis Mitochondria Triggers Light-dependent Lesion Formation and Alters Cytokinin Homeostasis

To investigate the role of mitochondrial farnesyl diphosphate synthase (FPS) in plant isoprenoid biosynthesis we characterized transgenic Arabidopsis thaliana plants overexpressing FPS1L isoform. This overexpressed protein was properly targeted to mitochondria yielding a mature and active form of the enzyme of 40 kDa. Leaves from transgenic plants grown under continuous light exhibited symptoms of chlorosis and cell death correlating to H₂O₂ accumulation, and leaves detached from the same plants displayed accelerated senescence. Overexpression of FPS in mitochondria also led to altered leaf cytokinin profile, with a reduction in the contents of physiologically active trans-zeatin- and isopentenyladenine-type cytokinins and their corresponding riboside monophosphates as well as enhanced levels of cis-zeatin 7-glucoside and storage cytokinin O-glucosides. Overexpression of 3-hydroxy-3-methylglutaryl coenzyme A reductase did not prevent chlorosis in plants overexpressing FPS1L, but did rescue accelerated senescence of detached leaves and restored wild-type levels of cytokinins. We propose that the overexpression of FPS1L leads to an enhanced uptake and metabolism of mevalonic acid-derived isopentenyl diphosphate and/or dimethylallyl diphosphate by mitochondria, thereby altering cytokinin homeostasis and causing a mitochondrial dysfunction that renders plants more sensitive to the oxidative stress induced by continuous light.     

枸骨IcFPS1基因的克隆、表达及生物信息学分析

法尼基焦磷酸合酶(farnesyl diphosphate synthase,FPS)是三萜皂苷生物合途径的一个关键酶,为研究FPS基因在枸骨中的功能,该研究采用PCR技术将一个FPS基因的cDNA序列从枸骨叶中分离出来,并命名为IcFPS1。结果表明:根据测序结果分析发现扩增获得的IcFPS1基因cDNA长度为1 591 bp,包含一个完整的开放阅读框,大小为1 029 bp。通过序列分析发现枸骨IcFPS1基因编码342个氨基酸,分子量和等电点分别为39.58 kDa和5.18。通过理化性质预测分析发现IcFPS1蛋白不含信号肽,不含有跨膜区域,该IcFPS1蛋白为亲水性蛋白质。通过序列多重比对发现IcFPS1蛋白质与其他植物的FPS蛋白质高度同源,有共同的保守区域和氨基酸序列,其中与西洋参FPS序列的相似性高达89%。通过系统进化树分析发现枸骨FPS蛋白与同属于被子植物的五加科植物FPS蛋白亲缘关系较近,说明FPS基因在进化过程中相对比较保守。根据蛋白调控网络预测分析结果发现该蛋白可能与IPP1、IPP2、GGPS3、GGPS6和ERA1相互作用,参与类异戊二烯的合成代谢过程。通过实时荧光定量PCR分析发现IcFPS1基因在枸骨各个组织部位中均有表达,其中在枸骨根中表达量最高,在茎和雌花中表达量最低。     

Activities of Soluble and Microsomal Farnesyl Diphosphatases in Datura stramonium

Farnesyl diphosphatase (FDPase; EC 3.1.7.1) produces farnesol from farnesyl diphosphate (FDP) in a reaction that does not require Mg²⁺. This report shows that FDPase is constitutively expressed at a high level in the soluble and the microsomal fractions of Datura stramonium. Soluble and microsomal FDPase have a similar pH optimum (5.0 – 6.0) and a similar substrate specificity. Geranyl diphosphate (GDP) and geranylgeranyl diphosphate (GGDP) compete for FDP, but isopentenyl diphosphate (IDP), ATP, and para-nitrophenyl phosphate do not. Soluble FDPase activity was highest in fruit and flower followed by root, and leaf. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inhibition of geranylgeranyl diphosphate synthesis in in vitro systems

The incorporation of [¹⁴C]mevalonate and [¹⁴C]isopentenyl diphosphate into geranylgeranyl diphosphate was investigated in in vitro systems from Cucurbita pepo (pumpkin) endosperm and from Avena sativa etioplasts. Mevalonate incorporation was effectively inhibited in the pumpkin system by geranylgeranyl diphosphate and geranylgeranyl monophosphate but less effectively by phytyl diphosphate or inorganic diphosphate. Membrane lipids, geranyllinalool, or lecithin enhanced mevalonate incorporation in the Cucurbita system. Incorporation of isopentenyl diphosphate was also enhanced by lecithin and inhibited by geranylgeranyl diphosphate in the Cucurbita system. No lipid enhancement was found in the Avena system; inhibition by GGPP required a much higher GGPP concentration than in the Cucurbita system.     

Farnesyl Diphosphate Synthase. A Paradigm for Understanding Structure and Function Relationships in E-polyprenyl Diphosphate Synthases

The chain elongation reaction catalyzed by polyprenyl diphosphate synthases is the fundamental building reaction in the isoprenoid pathway. During chain elongation, the hydrocarbon moiety in an allylic isoprenoid diphosphate is added to the carbon–carbon double bond of isopentenyl diphosphate (IPP). The chain elongation enzymes can be divided into two genetically different families depending on whether the stereochemistry of the newly formed double bond during each cycle of chain elongation is E or Z. Farnesyl diphosphate (FPP) synthase, a member of the E-double bond family, is the best studied of the chain elongation enzymes and serves as a paradigm for understanding the reactions catalyzed by E-polyprenyl diphosphate synthases. The mechanism for chain elongation is a stereoselective electrophilic alkylation of the carbon–carbon double bond in IPP by the allylic substrate. X-ray structures of avian and E. coli FPP synthases have provided important insights about the mechanism for chain elongation and a structural basis for understanding the stereochemistry of the reaction.This review is dedicated to Professor Rodney Croteau on the occasion of his 60th birthday.     

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.     

Cloning of an Arabidopsis thaliana cDNA coding for farnesyl diphosphate synthase by functional complementation in yeast

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.     

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.     

Molecular cloning and functional analysis of an organ-specific expressing gene coding for farnesyl diphosphate synthase from <Emphasis Type="Italic">Michelia chapensis</Emphasis> Dandy

Farnesyl diphosphate synthase (FPS; EC 2.5.1.1/EC 2.5.1.10) catalyzes the synthesis of farnesyl diphosphate, a key intermediate in the biosynthesis of sesquiterpenes. This present study described the cloning and characterization of a cDNA encoding FPS from leaves of Michelia chapensis Dandy (designated as McFPS, GenBank accession number: GQ214406) for the first time. McFPS was 1,432 bp and contained an open reading frame (ORF) of 1,056 bp, encoding a protein of 351 amino acids with a calculated molecular mass of 40.52 kDa. Bioinformatic analysis revealed that the deduced McFPS had high homology with FPSs from other plant species. Phylogenetic tree analysis indicated that McFPS belonged to the plant FPS group and had the closest relationship with FPS from Chimonanthus praecox. Southern blot analysis revealed that there were at most two copies of McFPS gene existed in M. chapensis genome. The organ expression pattern analysis showed that McFPS expressed strongly only in leaves, and there were no expression in stems and roots, implying that McFPS was an organ-specific expressing gene. Functional complementation of McFPS in a FPS-deficient yeast strain demonstrated that cloned cDNA encoded a farnesyl diphosphate synthase.