Farnesyl diphosphate synthase. Altering the catalytic site to select for geranyl diphosphate activity

Farnesyl diphosphate synthase (FPPase) catalyzes chain elongation of the C(5) substrate dimethylallyl diphosphate (DMAPP) to the C(15) product farnesyl diphosphate (FPP) by addition of two molecules of isopentenyl diphosphate (IPP). The synthesis of FPP proceeds in two steps, where the C(10) product of the first addition, geranyl diphosphate (GPP), is the substrate for the second addition. The product selectivity of avian FPPase was altered to favor synthesis of GPP by site-directed mutagenesis of residues that form the binding pocket for the hydrocarbon residue of the allylic substrate. Amino acid substitutions that reduced the size of the binding pocket were identified by molecular modeling. FPPase mutants containing seven promising modifications were constructed. Initial screens using DMAPP and GPP as substrates indicated that two of the substitutions, A116W and N144'W, strongly discriminated against binding of GPP to the allylic site. These observations were confirmed by an analysis of the products from reactions with DMAPP in the presence of excess IPP and by comparing the steady-state kinetic constants for the wild-type enzyme and the A116W and N114W mutants.     

Substrate specificities of farnesyl diphosphate synthases from Bacillus stearothermophilus and porcine liver with cyclic substrate homologs

We investigated the substrate specificity of farnesyl diphosphate (FPP) synthase derived from Bacillus stearothermophilus and porcine liver by examining the reactivities of two cyclic substrate homologs, cyclohexylideneethyl diphosphate and cyclohexenylethyl diphosphate.Reaction of geranyl diphosphate with 2-cyclohexenylethyl diphosphate using bacterial or porcine liver FPP synthase produced (S)-geranylcyclohexylideneethyl diphosphate, with relative yields of 13.6% for the bacterial enzyme and 42.2% for the porcine liver enzyme. Reaction of cyclohexylideneethyl diphosphate with isopentenyl diphosphate produced 10-cyclohexyliden-3,7-dimethyldeca-2,6-dien-1-ol as a double condensation product, with relative yields of 23.1% (bacterial enzyme) and 3.0% (porcine liver enzyme). Reaction of cyclohexylideneethyl diphosphate with 2-cyclohexenylethyl diphosphate using bacterial enzyme produced (cyclohexylideneethyl)-cyclohexylideneethyl diphosphate (0.8% yield).     

Characterization of a novel aphid prenyltransferase displaying dual geranyl/farnesyl diphosphate synthase activity

We report on the cDNA cloning and characterization of a novel short-chain isoprenyl diphosphate synthase from the aphid Myzus persicae. Of the three IPPS cDNAs we cloned, two yielded prenyltransferase activity following expression in Escherichia coli; these cDNAs encode identical proteins except for the presence, in one of them, of an N-terminal mitochondrial targeting peptide. Although the aphid enzyme was predicted to be a farnesyl diphosphate synthase by BLASTP analysis, rMpIPPS, when isopentenyl diphosphate and dimethylallyl diphosphate are supplied as substrates, typically generated geranyl diphosphate (C10) as its main product, along with significant quantities of farnesyl diphosphate (C15). Analysis of an MpIPPS homology model pointed to substitutions that could confer GPP/FPP synthase activity to the aphid enzyme.     

Structures of a potent phenylalkyl bisphosphonate inhibitor bound to farnesyl and geranylgeranyl diphosphate synthases

We report the X-ray crystallographic structures of the bisphosphonate N-[methyl(4-phenylbutyl)]-3-aminopropyl-1-hydroxy-1,1-bisphosphonate (BPH-210), a potent analog of pamidronate (Aredia), bound to farnesyl diphosphate synthase (FPPS) from Trypanosoma brucei as well as to geranylgeranyl diphosphate synthase from Saccharomyces cerevisiae. BPH-210 binds to FPPS, together with 3 Mg(2+), with its long, hydrophobic phenylbutyl side-chain being located in the same binding pocket that is occupied by allylic diphosphates and other bisphosphonates. Binding is overwhelmingly entropy driven, as determined by isothermal titration calorimetry. The structure is of interest since it explains the lack of potency of longer chain analogs against FPPS, since these would be expected to have a steric clash with an aromatic ring at the distal end of the binding site. Unlike shorter chain FPPS inhibitors, such as pamidronate, BPH-210 is also found to be a potent inhibitor of human geranylgeranyl diphosphate synthase. In this case, the bisphosphonate binds only to the GGPP product inhibitory site, with only 1 (chain A) or 0 (chain B) Mg(2+), and DeltaS is much smaller and DeltaH is approximately 6 k cal more negative than in the case of FPPS binding. Overall, these results are of general interest since they show that some bisphosphonates can bind to more than one trans-prenyl synthase enzyme which, in some cases, can be expected to enhance their overall activity in vitro and in vivo.     

Product chain-length determination mechanism of Z,E-farnesyl diphosphate synthase

cis-Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic prenyl diphosphates, producing Z,E-mixed prenyl diphosphate. The Mycobacterium tuberculosis Z,E-farnesyl diphosphate synthase Rv1086 catalyzes the condensation of one molecule of IPP with geranyl diphosphate to yield Z,E-farnesyl diphosphate and is classified as a short-chain cis-prenyltransferase. To elucidate the chain-length determination mechanism of the short-chain cis-prenyltransferase, we introduced some substitutive mutations at the characteristic amino acid residues of Rv1086. Among the mutants constructed, L84A showed a dramatic change of catalytic function to synthesize longer prenyl chain products than that of wild type, indicating that Leu84 of Rv1086 plays an important role in product chain-length determination. Mutagenesis at the corresponding residue of a medium-chain cis-prenyltransferase, Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase also resulted in the production of different prenyl chain length from the intrinsic product, suggesting that this position also plays an important role in product chain-length determination for medium-chain cis-prenyltransferases.     

Geranyl diphosphate synthase from Abies grandis: cDNA isolation,functional expression,and characterization

Geranyl diphosphate synthase catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to generate geranyl diphosphate, the essential precursor of monoterpene biosynthesis. Using geranylgeranyl diphosphate synthase from Taxus canadensis as a hybridization probe, four full length cDNA clones, sharing high sequence identity to each other (>69%) and to the Taxus geranylgeranyl diphosphate synthase (>66%), were isolated from a grand fir (Abies grandis) cDNA library. When expressed in Escherichia coli, three of the recombinant enzymes produced geranyl diphosphate and one produced geranylgeranyl diphosphate as the dominant product when supplied with isopentenyl diphosphate and dimethylallyl diphosphate as cosubstrates. One enzyme (AgGPPS2) was confirmed as a specific geranyl diphosphate synthase, in that it accepted only dimethylallyl diphosphate as the allylic cosubstrate and it produced exclusively geranyl diphosphate as product, with a k(cat) of 1.8s(-1). Gel filtration experiments performed on the recombinant geranyl diphosphate synthases, in which the plastidial targeting sequences had been deleted, revealed that these enzymes are homodimers similar to other short-chain prenyltransferases but different from the heterotetrameric geranyl diphosphate synthase of mint.     

Quantitative analysis of isoprenoid diphosphate intermediates in recombinant and wild-type Escherichia coli strains

In biotechnology, the heterologous biosynthesis of isoprenoid compounds in Escherichia coli is a field of great interest and growth. In order to achieve higher isoprenoid yields in heterologous E. coli strains, it is necessary to quantify the pathway intermediates and adjust gene expression. In this study, we developed a precise and sensitive nonradioactive method for the simultaneous quantification of the isoprenoid precursors farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) in recombinant and wild-type E. coli cells. The method is based on the dephosphorylation of FPP and GGPP into the respective alcohols and involves their in situ extraction followed by separation and detection using gas chromatography–mass spectrometry. The integration of a geranylgeranyl diphosphate synthase gene into the E. coli chromosome leads to the accumulation of GGPP, generating quantities as high as those achieved with a multicopy expression vector. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. T. Vallon and S. Ghanegaonkar contributed equally to this work.     

Inhibition of monoterpene cyclases by inert analogues of geranyl diphosphate and linalyl diphosphate

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.     

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.     

Farnesyl diphosphate synthase activity affects ergosterol level and proliferation of yeast Saccharomyces cerevisae

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.     

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 (CH(3)OGPP) as allylic substrate homologs. The wild-type FPP synthase reaction of HOGPP (and CH(3)OGPP) 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.     

A corpora allata farnesyl diphosphate synthase in mosquitoes displaying a metal ion dependent substrate specificity

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.     

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

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

Human geranylgeranyl diphosphate synthase. cDNA cloning and expression

Geranylgeranyl diphosphate (GGPP) synthase (GGPPSase) catalyzes the synthesis of GGPP, which is an important molecule responsible for the C20-prenylated protein biosynthesis and for the regulation of a nuclear hormone receptor (LXR.RXR). The human GGPPSase cDNA encodes a protein of 300 amino acids which shows 16% sequence identity with the known human farnesyl diphosphate (FPP) synthase (FPPSase). The GGPPSase expressed in Escherichia coli catalyzes the GGPP formation (240 nmol/min/mg) from FPP and isopentenyl diphosphate. The human GGPPSase behaves as an oligomeric molecule with 280 kDa on a gel filtration column and cross-reacts with an antibody directed against bovine brain GGPPSase, which differs immunochemically from bovine brain FPPSase. Northern blot analysis indicates the presence of two forms of the mRNA.     

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.     

Quantitative determination of farnesyl and geranylgeranyl diphosphate levels in mammalian tissue

Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are branch point intermediates of isoprenoid biosynthesis. Inhibitors of isoprenoid biosynthesis, such as the statins and bisphosphonates, are widely used therapeutic agents. However, little is known about the degree to which they alter levels of upstream and downstream isoprenoids, including FPP and GGPP. Therefore, we developed a method to isolate and quantify FPP and GGPP from mammalian tissues. Tissues from mice were collected, snap frozen in liquid nitrogen, and stored at −80 °C. FPP and GGPP were isolated by a combined homogenization and extraction procedure and were purified with a C18 solid phase extraction column. Farnesyl protein transferase (FTase) or geranylgeranyl protein transferase I (GGTase I) were used to conjugate FPP and GGPP with fluorescent dansylated peptides. FPP and GGPP were quantified by high-performance liquid chromatography (HPLC). The respective concentrations of FPP and GGPP are as follows: 0.355 ± 0.030 and 0.827 ± 0.082 units of nmol/g wet tissues in brain, 0.320 ± 0.019 and 0.293 ± 0.035 units of nmol/g wet tissues in kidney, 0.326 ± 0.064 and 0.213 ± 0.029 units of nmol/g wet tissues in liver, and 0.364 ± 0.015 and 0.349 ± 0.023 units of nmol/g wet tissues in heart (means ± SEM). This method allows for determination of FPP and GGPP concentrations in any tissue type and is sensitive enough to detect changes following treatment with inhibitors of isoprenoid biosynthesis.     

Crystal structure of heterodimeric hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26 reveals that the small subunit is directly involved in the product chain length regulation

Hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26 (Ml-HexPPs) is a heterooligomeric type trans-prenyltransferase catalyzing consecutive head-to-tail condensations of three molecules of isopentenyl diphosphates (C(5)) on a farnesyl diphosphate (FPP; C(15)) to form an (all-E) hexaprenyl diphosphate (HexPP; C(30)). Ml-HexPPs is known to function as a heterodimer of two different subunits, small and large subunits called HexA and HexB, respectively. Compared with homooligomeric trans-prenyltransferases, the molecular mechanism of heterooligomeric trans-prenyltransferases is not yet clearly understood, particularly with respect to the role of the small subunits lacking the catalytic motifs conserved in most known trans-prenyltransferases. We have determined the crystal structure of Ml-HexPPs both in the substrate-free form and in complex with 7,11-dimethyl-2,6,10-dodecatrien-1-yl diphosphate ammonium salt (3-DesMe-FPP), an analog of FPP. The structure of HexB is composed of mostly antiparallel α-helices joined by connecting loops. Two aspartate-rich motifs (designated the first and second aspartate-rich motifs) and the other characteristic motifs in HexB are located around the diphosphate part of 3-DesMe-FPP. Despite the very low amino acid sequence identity and the distinct polypeptide chain lengths between HexA and HexB, the structure of HexA is quite similar to that of HexB. The aliphatic tail of 3-DesMe-FPP is accommodated in a large hydrophobic cleft starting from HexB and penetrating to the inside of HexA. These structural features suggest that HexB catalyzes the condensation reactions and that HexA is directly involved in the product chain length control in cooperation with HexB.