Condensation Of Isopentenyl Diphosphate Research Articles

Discovery, Structure, and Engineering of a cis‐Geranylfarnesyl Diphosphate Synthase

cis‐Prenyltransferases (cis‐PTs) catalyze the sequential head‐to‐tail condensation of isopentenyl diphosphate (IPP) to allylic diphosphates, producing mixed E‐Z prenyl diphosphates of varying lengths; yet, the specific enzymes synthesizing cis‐C25 prenyl diphosphates have not been identified. In this study, we present the discovery and characterization of a cis‐geranylfarnesyl diphosphate synthase (ScGFPPS) from Streptomyces clavuligerus. This enzyme demonstrates high catalytic proficiency in generating six distinct cis‐polyisoprenoids, including three C25 and three C20 variants. We further determine the crystal structure of ScGFPPS. Additionally, we unveil the crystal structure of nerylneryl diphosphate synthase (NNPS), known for synthesizing an all‐cis‐C20 polyisoprenoid. Comparative structural analysis of ScGFPPS and NNPS has identified key differences that influence product specificity. Through site‐directed mutagenesis, we have identified eight single mutations that significantly refine ScGFPPS's selectivity for cis‐polyisoprenoids. Our findings not only expand the functional spectrum of cis‐PTs but also provide a structural comparison strategy in cis‐PTs engineering.

Discovery, Structure, and Engineering of a cis-Geranylfarnesyl Diphosphate Synthase.

cis-Prenyltransferases (cis-PTs) catalyze the sequential head-to-tail condensation of isopentenyl diphosphate (IPP) to allylic diphosphates, producing mixed E-Z prenyl diphosphates of varying lengths; yet, the specific enzymes synthesizing cis-C25 prenyl diphosphates have not been identified. In this study, we present the discovery and characterization of a cis-geranylfarnesyl diphosphate synthase (ScGFPPS) from Streptomyces clavuligerus. This enzyme demonstrates high catalytic proficiency in generating six distinct cis-polyisoprenoids, including three C25 and three C20 variants. We further determine the crystal structure of ScGFPPS. Additionally, we unveil the crystal structure of nerylneryl diphosphate synthase (NNPS), known for synthesizing an all-cis-C20 polyisoprenoid. Comparative structural analysis of ScGFPPS and NNPS has identified key differences that influence product specificity. Through site-directed mutagenesis, we have identified eight single mutations that significantly refine ScGFPPS's selectivity for cis-polyisoprenoids. Our findings not only expand the functional spectrum of cis-PTs but also provide a structural comparison strategy in cis-PTs engineering.

Suppression of phenotype of Escherichia coli mutant defective in farnesyl diphosphate synthase by overexpression of gene for octaprenyl diphosphate synthase.

We investigated suppression of the slow growth of an Escherichia coli ispA null mutant lacking farnesyl diphosphate (FPP) synthase (i.e. IspA) by plasmids carrying prenyl diphosphate synthase genes. The growth rates of ispA mutant-transformants harboring a medium-copy number plasmid that carries ispA or ispB were almost the same as that of the wild-type strain. Although the level of FPP in the transformant with the ispA plasmid was almost the same as that in the wild-type strain, the level in the transformant with the ispB plasmid was as low as that in the ispA mutant. Purified octaprenyl diphosphate synthase (IspB) could condense isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP) to form octaprenyl diphosphate and nonaprenyl diphosphate. It is possible that suppression of the slow growth of the ispA mutant by ispB was due to condensation of IPP not only with FPP but also with DMAPP by octaprenyl diphosphate synthase.

Identifying the Metal That Activates the Prenyltransferase That Catalyzes Formation of Geranyl Diphosphate in the Diatom Pseudo‐nitzschia multiseries

Domoic acid (DA) is a neurotoxin synthesized by marine diatoms of the genus Pseudo‐nitzschia which causes amnesiac shellfish poisoning in humans and death of marine wildlife. Geranyl diphosphate (GPP), a key intermediate in domoic acid biosynthesis, is formed from the “head‐to‐tail” condensation of isopentenyl diphosphate and dimethylallyl diphosphate in a reaction catalyzed by a member of the prenyltransferase family of enzymes. These prenyltransferases can generate different chain‐length products, including geranyl diphosphate (C10), farnesyl diphosphate (FPP, C15), and geranylgeranyl diphosphate (GGPP, C20), all of which are precursors to terpenoid natural products. Prenyltransferases require a divalent metal cation for activity. Previous work has shown that a single prenyltransferase can generate multiple products (GPP, FPP, and GGPP), and that the product distribution will vary depending on the species of divalent cation used to support the reaction (Mg2+, Mn2+, or Co2+). Here, we measured the levels of Mg2+, Mn2+, and Co2+ in Pseudo‐nitzschia cells to determine which ion is most likely to support GPP formation. Three samples of Pseudo‐nitzschia multiseries cultured in sea water were harvested by centrifugation. Samples were digested with HNO3 before analysis using atomic absorption spectroscopy. The average concentration of Mg2+ present, per cell and per μm3, is over 100‐fold higher than the concentrations of Mn2+ and Co2+. This is consistent with results from studies in other systems, suggesting that Mg2+ is the main cofactor that activates the prenyltransferase that generates GPP for domoic acid biosynthesis in Pseudo‐nitzschia.Support or Funding InformationThis project is supported by the SURE (Summer Undergraduate Research Experience) Award Program from the College of Natural Science and Mathematics, California State University Sacramento.

CDNA cloning and expression analysis of farnesyl pyrophosphate synthase from Ornithogalum saundersiae.

Farnesyl pyrophosphate synthase (FPPS, EC 2.5.1.10) catalyzes the consecutive head-to-tail condensations of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP) to form farnesyl pyrophosphate (FPP), a key precursor of sesquiterpenoids, triterpenoids, sterols, and farnesylated proteins. Here we report the molecular cloning and functional identification of a new full-length cDNA encoding FPPS from Ornithogalum saundersiae, a potential medicinal plant that produces a promising antitumour sterol glycoside, OSW-1. An 1327 bp long unigene with an open reading frame of 1044 bp was retrieved from the transcriptome sequencing of O. saundersiae. The full-length FPPS cDNA, designated OsaFPPS, was isolated from O. saundersiae with gene-specific primers. The resultant OsaFPPS encodes a 347-amino acids protein with a calculated molecular mass of 40,085.6 Da, and a theoretical isoelectric point of 5.01. Phylogenetic tree analysis indicated that OsaFPPS belongs to the plant FPPS super-family. Expression of soluble OsaFPPS in E. coli was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Functional analysis of the purified OsaFPPS protein was carried out using IPP and DMAPP as substrates, and the product was unambiguously determined by gas chromatography-mass spectrometry (GC-MS) analyses.

Farnesyl diphosphate synthase assay.

Farnesyl diphosphate synthase (FPS) catalyzes the sequential head-to-tail condensation of isopentenyl diphosphate (IPP, C5) with dimethylallyl diphosphate (DMAPP, C5) and geranyl diphosphate (GPP, C10) to produce farnesyl diphosphate (FPP, C15). This short-chain prenyl diphosphate constitutes a key branch-point of the isoprenoid biosynthetic pathway from which a variety of bioactive isoprenoids that are vital for normal plant growth and survival are produced. Here we describe a protocol to obtain highly purified preparations of recombinant FPS and a radiochemical assay method for measuring FPS activity in purified enzyme preparations and plant tissue extracts.

Substrate Specificities of <i>E</i>-and <i>Z</i>-Farnesyl Diphosphate Synthases with Artificial Substrates

Graduate School of Science and Technology, Hirosaki University, 3 Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan *Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Kojirakawa-machi, Yamagata 990-8560, Japan **Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Aoba-ku, Sendai, Miyagi 980-8577, Japan Fax: 81-172-39-3947, e-mail: nagaki@cc.hirosaki-u.ac.jp Here, to assess substrate specificities of E-and Z-farnesyl diphosphate synthases from Bacillus stearothermophilus and Thermobifida fusca, we examined the reactivities of isopentenyl diphosphate homologs having an alkyl group at the 3-position. E- and Z-FPP synthase reactions of geranyl diphosphate (GPP) with 3-ethylisopentenyl diphosphate produced E- and Z-3-ethylfarnesyl diphosphates (yield: 70.8%, and 11.3%, respectively). The E-FPP synthase reaction of GPP with 3-propylisopentenyl diphosphate produced E-3-propylfarnesyl diphosphate (yield: 40.1%); however, when reacting GPP with 3-propylisopentenyl diphosphate by Z-FPP synthase reaction, only trace product was acquired. Further, neither E- nor Z-FPP synthase reaction of GPP with 3-butylisopentanyl diphosphate produced any product. Key words: E- and Z-farnesyl diphosphate synthases, substrate specificity, homologs of isopentenyl diphosphate, Bacillus stearothermophilus, Thermobifida fusca 1. INTRODUCTION Prenyltransferases can be roughly divided into Z- and E-prenyl chain elongating enzymes[1-5]. E-farnesyl diphosphate (FPP) synthase, a short prenyl chain elongating enzyme, has long been the subject of intense research. The E-FPP synthase has been solved to quite detailed portions, such as a chain length determination mechanism [6-8]. However, after Crick et al. successfully cloned Z-FPP synthase from Mycobacterium tubercule in 2000, a great number of studies began focusing on Z-prenyltransferase[9-11]. Subsequent to Crick et al.’s findings, Koyama et al. recently successfully cloned Z-FPP synthase from several other bacterial species, including Thermobifida fusca and Corynebacterium glutamicum [12-14]. C. Sallaud et al. identified the genes encoding Z-FPP synthase in the wild tomato, Solanum habrochaites, and reported that the recombinant enzymatic reaction between dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) produced all-Z-FPP [15]. E-FPP synthase [EC 2.5.1.10] catalyzes the head-to-tail condensation of IPP with DMAPP (or geranyl diphosphate [GPP]) as an allylic substrate, producing FPP as the final product (Scheme 1). Several previous studies have investigated the substrate specificities of FPP synthase with both allylic and homoallylic substrate homologs [16-20]. Scheme 1. FPP Synthase Reaction of DMAPP withIPP Our team also recently reported on the reactivities of several allylic substrate homologs which have a hydrophilic group at the ω-position, using B. stearothermophilus and porcine liver FPP synthases [21-23]. Previously, to examine the substrate specificity of FPP synthase derived from B. stearothermophilus, we reported on the reaction of GPP with IPP homologs which have an alkyl group at the 3-position[24]. Here, we compared E- and Z-FPP synthases by examining several new, similar reactions. We then assessed the substrate specificities of the synthases by examining the reactivity of artificial substrate homologs.

Cloning and functional analysis of cis-prenyltransferase from Thermobifida fusca

cis-Prenyltransferase catalyzes the synthesis of Z,E-mixed prenyl diphosphates by a condensation of isopentenyl diphosphate to an allylic diphosphate. A novel gene encoding a cis-prenyltransferase is cloned from Thermobifida fusca. It showed a unique substrate specificity accepting dimethylallyl diphosphate as a shortest allylic substrate, and synthesizes polyprenyl products up to C(70).

The Farnesyl-diphosphate/Geranylgeranyl-diphosphate Synthase of Toxoplasma gondii Is a Bifunctional Enzyme and a Molecular Target of Bisphosphonates

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.

Short-chain prenyl diphosphate synthase that condenses isopentenyl diphosphate with dimethylallyl diphosphate in ispA null Escherichia coli strain lacking farnesyl diphosphate synthase

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.

Farnesyl Diphosphate Synthase: The Art of Compromise between Substrate Selectivity and Stereoselectivity

Farnesyl diphosphate (FPP) synthase catalyzes the consecutive head-to-tail condensations of isopentenyl diphosphate (IPP, C5) with dimethylallyl diphosphate (DMAPP, C5) and geranyl diphosphate (GPP, C10) to give (E,E)-FPP (C15). The enzyme belongs to a genetically distinct family of chain elongation enzymes that install E-double bonds during each addition of a five-carbon isoprene unit. Analysis of the C10 and C15 products from incubations with avian FPP synthase reveals that small amounts of neryl diphosphate (Z-C10) and (Z,E)-FPP are formed along with the E-isomers during the C5 --> C10 and C10 --> C15 reactions. Similar results were obtained for FPP synthase from Escherichia coli, Artemisia tridentata (sage brush), Pyrococcus furiosus, and Methanobacter thermautotrophicus and for GPP and FPP synthesized in vivo by E. coli FPP synthase. When (R)-[2-2H]IPP was a substrate for chain elongation, no deuterium was found in the chain elongation products. In contrast, the deuterium in (S)-[2-2H]IPP was incorporated into all of the products. Thus, the pro-R hydrogen at C2 of IPP is lost when the E- and Z-double bond isomers are formed. The synthesis of Z-double bond isomers by FPP synthase during chain elongation is unexpected for a highly evolved enzyme and probably reflects a compromise between optimizing double bond stereoselectivity and the need to exclude DMAPP from the IPP binding site.

Disruption of the Structural Gene for Farnesyl Diphosphate Synthase in Escherichia coli

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.

The fellowship of natural abundance 2H-isotopomers of monoterpenes

Site-specific natural abundance hydrogen isotope ratios have been measured by deuterium-NMR in a wide variety of monoterpenes from numerous kinds of plants grown in different environments. Once the NMR signals have been assigned to the whole sets of isotopomers in the different molecules and schemes of connections to the parent isotopomers in the geranyl diphosphate (GPP) precursor have been defined, a very consistent set of isotopic profiles is evidenced. The results, which are incompatible with the mevalonate pathway, can be satisfactorily interpreted by considering the deoxyxylulose pathway (DOXP), which is now recognized as the usual route for monoterpene biosynthesis in plants. Strong deuterium depletion at ex-site 2 of GPP, accompanied by high isotope ratio values at site ex-6, are consistent with synthesis of GPP from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) molecules independently produced by the DOXP pathway. However, for a given molecular species, significant differences are observed as a function of the plant source, in particular at site ex-6 of GPP. Thus, monoterpenes from plants with a C3 metabolism are mostly characterized by relatively high values of ( D/ H) 6, whereas C4 plants tend to show much lower values. This behavior may be attributed to more or less significant contributions of GPP resulting from the condensation of IPP with DMAPP produced by isomerization. The isotopic profile therefore enables the role of physiological and environmental factors on the relative importance of the “independent” and “isomerized” model to be estimated. More generally, isotope ratios at individual sites in geraniol can be traced back to the corresponding sites in GPP, then to sites of the IPP and DMAPP building blocks, then to the pyruvate and glyceraldehyde 3-phosphate DOXP active molecules, and finally to the carbohydrate photosynthetic precursor. Furthermore, the methylenic hydrogen atoms, which are enantiotopic in geraniol, become diastereotopic in chiral, and more specially in cyclic, monoterpenes. This provides an isotopic verification for the complete stereochemical chain of affiliation, and a way of estimating enantiomeric purity and whether intermolecular exchanges have taken place.

Regulation of carotenoid biosynthesis in plants: evidence for a key role of hydroxymethylbutenyl diphosphate reductase in controlling the supply of plastidial isoprenoid precursors

Carotenoids are isoprenoid pigments that function as photoprotectors, precursors of the hormone abscisic acid (ABA), colorants and nutraceuticals. A major problem for the metabolic engineering of high carotenoid levels in plants is the limited supply of their isoprenoid precursor geranylgeranyl diphosphate (GGPP), formed by condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) units usually synthesized by the methylerythritol phosphate (MEP) pathway in plastids. Our earlier work with three of the seven MEP pathway enzymes suggested that the first reaction of the pathway catalyzed by deoxyxylulose 5-phosphate synthase (DXS) is limiting for carotenoid biosynthesis during tomato (Lycopersicon esculentum) fruit ripening. Here we investigate the contribution of the enzyme hydroxymethylbutenyl diphosphate reductase (HDR), which simultaneously synthesizes IPP and DMAPP in the last step of the pathway. A strong upregulation of HDR gene expression was observed in correlation with carotenoid production during both tomato fruit ripening and Arabidopsis thaliana seedling deetiolation. Constitutive overexpression of the tomato cDNA encoding HDR in Arabidopsis did not increase carotenoid levels in etioplasts. By contrast, light-grown transgenic plants showed higher carotenoid levels and an enhanced seed dormancy phenotype suggestive of increased ABA levels. The analysis of double transgenic Arabidopsis plants overproducing both the enzyme taxadiene synthase (which catalyzes the production of the non-native isoprenoid taxadiene from GGPP) and either HDR or DXS showed a twofold stronger effect of HDR in increasing taxadiene levels. Together, the data support a major role for HDR in controlling the production of MEP-derived precursors for plastid isoprenoid biosynthesis.

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 μM) with both dimethylallyl diphosphate and geranyl diphosphate ( K m=0.8±0.4 μM). The enzyme requires the presence of detergent, glycerol, and non-specific protein–protein interactions for stability and maximum catalytic activity.

Partial Purification and Characterization of the Short-Chain Prenyltransferases, Geranyl Diphosphate Synthase and Farnesyl Diphosphate Synthase, from Abies grandis (Grand Fir)

In the conifer Abies grandis (grand fir), a secreted oleoresin rich in mono-, sesqui-, and diterpenes serves as a constitutive and induced defense against insects and pathogenic fungi. Geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) synthase, two enzymes which form the principal precursors of the oleoresin mono- and sesquiterpenes, were isolated from the stems of 2-year-old grand fir saplings. These enzymes were partially purified by sequential chromatography on DEAE–Sepharose, Mono-Q, and phenyl–Sepharose to remove competing phosphohydrolase and isopentenyl diphosphate (IPP) isomerase activities. GPP and FPP synthase formed GPP and E,E-FPP, respectively, as the sole products of the enzymatic condensation of IPP and dimethylallyl diphosphate (DMAPP). The properties of both enzymes are broadly similar to those of other prenyltransferases. The apparent native molecular masses are 54 ± 3 kDa for GPP synthase and 110 ± 6 kDa for FPP synthase, as determined by gel permeation chromatography. Michaelis-Menten constants for GPP synthase are 14.3 and 16.7 μM for IPP and DMAPP, respectively. For IPP synthase, Km values of 15.3 and 9.0 μM were determined for IPP and DMAPP, respectively, and the Km value for GPP was 1.8 μM. Both enzymes require a divalent metal ion for catalytic activity. GPP synthase prefers Mg2+ as cofactor, whereas in the case of FPP synthase, Mg2+ can be substituted almost equivalently by Mn2+, Zn2+, or Co2+. The products of both prenyltransferase reactions, including inorganic diphosphate, GPP, and FPP, are inhibitory. No significant induction of either enzyme activity was observed in stem tissue over a 20-day time course after mechanical wounding.

Biosynthesis of Isoprenoids in Escherichia coli: Stereochemistry of the Reaction Catalyzed by Farnesyl Diphosphate Synthase

[formula: see text] Farnesyl diphosphate (FPP) synthase from Escherichia coli catalyzes the condensation of isopentenyl diphosphate (IPP) and geranyl diphosphate (GPP) with selective removal of the pro-R hydrogen at C2 of IPP, the same stereochemistry observed for the pig liver, yeast, and avian enzymes.

Arabidopsis thaliana Contains Two Differentially Expressed Farnesyl-Diphosphate Synthase Genes

The enzyme farnesyl-diphosphate synthase (FPS; EC 2.5.1.1/EC 2.5.1.10) catalyzes the synthesis of farnesyl diphosphate (FPP) from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This reaction is considered to be a rate-limiting step in isoprenoid biosynthesis. Southern blot analysis indicates that Arabidopsis thaliana contains at least 2 genes (FPS1 and FPS2) encoding FPS. The FPS1 and FPS2 genes have been cloned and characterized. The two genes have a very similar organization with regard to intron positions and exon sizes and share a high level of sequence similarity, not only in the coding region but also in the intronic sequences. Northern blot analysis showed that FPS1 and FPS2 have a different pattern of expression. FPS1 mRNA accumulates preferentially in roots and inflorescences, whereas FPS2 mRNA is predominantly expressed in inflorescences. The cDNA corresponding to the FPS1 gene was isolated by functional complementation of a mutant yeast strain defective in FPS activity (Delourme, D., Lacroute, F., and Karst, F. (1994) Plant Mol. Biol. 26, 1867-1873). By using a reverse transcription-polymerase chain reaction strategy we have cloned the cDNA corresponding to the FPS2 gene. Analysis of the FPS2 cDNA sequence revealed an open reading frame encoding a protein of 342 amino acid residues with a predicted molecular mass of 39,825 Da. FPS1 and FPS2 isoforms share an overall amino acid identity of 90.6%. Arabidopsis FPS2 was able to rescue the lethal phenotype of an ERG20-disrupted yeast strain. We demonstrate that FPS2 catalyzes the two successive condensations of IPP with both DMAPP and geranyl diphosphate leading to FPP. The significance of the occurrence of different FPS isoforms in plants is discussed in the context of the complex organization of the plant isoprenoid pathway.

Effect of site-directed mutagenesis of conserved aspartate and arginine residues upon farnesyl diphosphate synthase activity.

All polyprenyl synthases catalyze the condensation of the allylic substrate, isopentenyl diphosphate, with a specific homoallylic diphosphate substrate. Polyprenyl synthases from Homo sapiens, Ratus rattus, Escherichia coli, Saccharomyces cerevisiae, Neurospora crassa, and Erwinia herbicola contain two conserved "aspartate-rich domains" (Ashby, M.N., and Edwards, P.A. (1992) J. Biol. Chem. 267, 4128-4136). In order to determine the importance of these domains in catalysis, the conserved aspartates or arginines in domains I and II of rat farnesyl diphosphate synthase were individually mutated to glutamate or lysine, respectively. The putative "active site" arginine (Brems, D.N., Breunger, E., and Rilling, H. C. (1981) Biochemistry 20, 3711-3718) was mutated to lysine. Each mutant enzyme was overexpressed in E. coli and purified to apparent homogeneity. Detailed kinetic analyses of the wild type and mutant enzymes indicated that mutagenesis of Asp104, Asp107, Arg112, Arg113, and Asp243 resulted in a decreased Vmax of approximately 1000-fold compared to wild type. However, no significant change in the Km values for either the isopentenyl diphosphate or geranyl diphosphate substrate were observed. The results strongly suggest that these amino acids, and to a lesser extent Asp244, are involved in either the condensation of isopentenyl diphosphate and geranyl diphosphate to form farnesyl diphosphate and/or the release of the farnesyl diphosphate product from farnesyl diphosphate synthase. The conservation of these amino acid residues in different enzymes from several species suggests that these domains play a similar role in other polyprenyl synthases.