Mechanism of the prenyl-transfer reaction. Studies with (E)- and (Z)-3-trifluoromethyl-2-buten-1-yl pyrophosphate

The prenyl-transfer reaction catalyzed by porcine farnesyl pyrophosphate synthetase has been studied using (E)- and (Z)-3-trifluoromethyl-2-buten-1-yl pyrophosphates as substrates and inhibitors. The rate of condensation between isopentenyl pyrophosphate (IPP) and the allylic fluoro analogues is drastically depressed relative to the normal catalytic rate observed with dimethylallyl pyrophosphate (DMAPP) or geranyl pyrophosphate (GPP). A similar depression is found in the rates of solvolysis for methanesulfonate derivatives of the fluoro analogues in aqueous actone under typical SN1 reaction conditions. Prolonged incubation of [14C] IPP and (E)- or (Z)-CF3-DMAPP with the enzyme, followed by treatment with alkaline phosphatase, gave a product that comigrated with geranylgeraniol on a polystyrene column. Both fluoro analogues showed mixed linear inhibition patterns with DMAPP or GPP as the variable substrate. We interpret these results in terms of an ionization-condensation-elimination mechanism for the prenyl-transfer reaction.     

Rubber elongation by farnesyl pyrophosphate synthases involves a novel switch in enzyme stereospecificity

A prenyltransferase purified from the commercial rubber tree, Hevea brasiliensis, that elongates existing cis-polyisoprene rubber molecules also catalyzes the formation of all trans-farnesyl pyrophosphate (t,t-FPP) from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). In assays of the latter activity trans-geranyl pyrophosphate is the only other product identified. In contrast to this limited addition of IPP to DMAPP, we measured 7000 additions of isoprene per rubber molecule in a previous titration of active allylic ends of rubber molecules by purified prenyltransferase (Light, D. R., and Dennis, M. S. (1989) J. Biol. Chem. 264, 18589-18597). In order to confirm that purified prenyltransferase extensively elongates rubber molecules, doubly labeled [1-14C]isopentenyl [U-32P]pyrophosphate ([14C,32P]IPP) was synthesized. Using this reagent we show that both prenyltransferase purified from H. brasiliensis and prenyltransferase purified from avian liver (FPP synthase) add greater than 15 isoprene units to existing rubber molecules, consistent with the previous titration data. For confirmation that the prenyltransferase purified from H. brasiliensis adds isoprene units to rubber to make cis-polyisoprene, chirally tritiated [14C]IPP ([14C,2S-3H]IPP) was synthesized. Retention of the tritium label in FPP synthesized from [14C,2S-3H]IPP and DMAPP, geranyl pyrophosphate, or neryl pyrophosphate by prenyltransferase from H. brasiliensis or avian liver confirms trans addition to these substrates. In contrast, when [14C,2S-3H]IPP is incubated with serum-free rubber particles and prenyltransferase purified from H. brasiliensis, avian liver, or yeast, no tritium is incorporated into the rubber particles indicating cis addition. Thus, rubber particles have the ability to alter the stereoselective removal of the 2R-prochiral proton in favor of the removal of the 2S-prochiral proton. This apparent inversion of carbon 2 of IPP during the proton abstraction step by rubber particles represents a novel example of a switch in enzyme stereospecificity. In addition to being enzymatically similar to other prenyltransferases, rubber transferase also appears to be related immunologically to FPP synthases, since polyclonal antibodies to the H. brasiliensis prenyltransferase cross-react with the purified yeast prenyltransferase. In order to investigate potential primers of greater molecular weight than that of FPP, cis-undecaprenyl pyrophosphate (C55PP) was synthesized. C55PP stimulates the incorporation of [14C]IPP into rubber particles suggesting that it may prime new rubber molecules. However, in contrast to DMAPP, C55PP is not incorporated into any detectable products when incubated with prenyltransferase and [14C]IPP in the absence of rubber particles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Farnesyl pyrophosphate synthase is the molecular target of nitrogen-containing bisphosphonates.

Bisphosphonates (Bps), inhibitors of osteoclastic bone resorption, are used in the treatment of skeletal disorders. Recent evidence indicated that farnesyl pyrophosphate (FPP) synthase and/or isopentenyl pyrophosphate (IPP) isomerase is the intracellular target(s) of bisphosphonate action. To examine which enzyme is specifically affected, we determined the effect of different Bps on incorporation of [(14)C]mevalonate (MVA), [(14)C]IPP, and [(14)C]dimethylallyl pyrophosphate (DMAPP) into polyisoprenyl pyrophosphates in a homogenate of bovine brain. HPLC analysis revealed that the three intermediates were incorporated into FPP and geranylgeranyl pyrophosphate (GGPP). In contrast to clodronate, the nitrogen-containing Bps (NBps), alendronate, risedronate, olpadronate, and ibandronate, completely blocked FPP and GGPP formation and induced in incubations with [(14)C]MVA a 3- to 5-fold increase in incorporation of label into IPP and/or DMAPP. Using a method that could distinguish DMAPP from IPP on basis of their difference in stability in acid, we found that none of the NBps affected the conversion of [(14)C]IPP into DMAPP, catalyzed by IPP isomerase, excluding this enzyme as target of NBp action. On the basis of these and our previous findings, we conclude that none of the enzymes up- or downstream of FPP synthase are affected by NBps, and FPP synthase is, therefore, the exclusive molecular target of NBp action.     

Structure, mechanism and function of prenyltransferases.

In this review, we summarize recent progress in studying three main classes of prenyltransferases: (a) isoprenyl pyrophosphate synthases (IPPSs), which catalyze chain elongation of allylic pyrophosphate substrates via consecutive condensation reactions with isopentenyl pyrophosphate (IPP) to generate linear polymers with defined chain lengths; (b) protein prenyltransferases, which catalyze the transfer of an isoprenyl pyrophosphate (e.g. farnesyl pyrophosphate) to a protein or a peptide; (c) prenyltransferases, which catalyze the cyclization of isoprenyl pyrophosphates. The prenyltransferase products are widely distributed in nature and serve a variety of important biological functions. The catalytic mechanism deduced from the 3D structure and other biochemical studies of these prenyltransferases as well as how the protein functions are related to their reaction mechanism and structure are discussed. In the IPPS reaction, we focus on the mechanism that controls product chain length and the reaction kinetics of IPP condensation in the cis-type and trans-type enzymes. For protein prenyltransferases, the structures of Ras farnesyltransferase and Rab geranylgeranyltransferase are used to elucidate the reaction mechanism of this group of enzymes. For the enzymes involved in cyclic terpene biosynthesis, the structures and mechanisms of squalene cyclase, 5-epi-aristolochene synthase, pentalenene synthase, and trichodiene synthase are summarized.     

A cell-free system for Streptococcus faecium for studies on the biosynthesis of triterpenoid carotenoids

A cell-free enzyme extract from Streptococcus faecium UNH 564P has been prepared. The extract incorporates either [2-14C]mevalonic acid (MVA) or [1-14C]isopentenyl pyrophosphate (IPP) into squalene and the carotenoids of the bacterium. ATP and manganese ion were found to be absolute requirements for MVA incorporation by the extract. Only manganese ion was found to be an absolute requirement for IPP incorporation by the extract. Other cofactors including magnesium ion, glutathione, potassium fluoride, NADP, and FAD significantly increase the incorporation of both substrates into the S. faecium terpenoids. Isolation and purification of the radioactive terpenoids from the cell-free system confirmed that the carotenoids of S. faecium are triterpenoids.     

Metabolism of plastid terpenoids. II. Regulation of phytoene synthesis in plastid stroma isolated from higher plants

In plastid stroma the synthesis of phytoene was regulated by several factors, (i) MgCl₂ or MnCl₂ alone and the combination of both oriented selectively the incorporation of isopentenyl pyrophosphate (IPP) between phytoene and other isoprenoids including geranylgeraniol, geranyllinalool, and solanesol (ii) neutral detergents (Tween-80, Triton X-100), polyethyleneglycol and digalactosyldiglyceride liposomes stimulated the incorporation of IPP into phytoene (iii) ATP or different glycolytic intermediates enhanced the synthesis of phytoene from IPP.     

Magnesium ion regulation of in vitro rubber biosynthesis by Parthenium argentatum Gray

Natural rubber is produced by a rubber transferase (a cis-prenyltransferase). Rubber transferase uses allylic pyrophosphate to initiate the rubber molecule and isopentenyl pyrophosphate (IPP) to form the polymer. Rubber biosynthesis also requires a divalent metal cation. Understanding how molecular weight is regulated is important because high molecular weight is required for high quality rubber. We characterized the in vitro effects of Mg(2+) on the biosynthetic rate of rubber produced by an alternative natural rubber crop, Parthenium argentatum (guayule). The affinity of the rubber transferase from P. argentatum for IPP.Mg was shown to depend on the Mg(2+) concentration in a similar fashion to the H. brasiliensis rubber transferase, although to a less extreme degree. Also, in vitro Mg(2+) concentration significantly affects rubber molecular weight of both species, but molecular weight is less sensitive to Mg(2+) concentration in P. argentatum than in H. brasiliensis.     

Monoterpene formation by leucoplasts of Citrofortunella mitis and Citrus unshiu

Leucoplasts of immature calamondin and satsuma fruits were incubated with [1-¹⁴C] isopentenyl pyrophosphate under various conditions. Optimal incorporation of the tracer into geranyl pyrophosphate and monoterpene hydrocarbons occurred in the presence of exogenous dimethylallyl pyrophosphate and Mn²⁺ which was more effective than Mg²⁺. The dependence of dimethylallyl pyrophosphate showed that about 10 moles were required for 1 mole of isopentenyl pyrophosphate for the best recovery in monoterpene hydrocarbon biosynthesis. A time-course incorporation of isopentenyl pyrophosphate revealed that the C₁₀ hydrocarbon elaboration was dependent on the geranyl pyrophosphate production and at no time neryl pyrophosphate was synthesized by leucoplasts. The amount of labelled farnesyl pyrophosphate was rather low whatever the conditions used in the experiments and sesquiterpene hydrocarbon biosynthesis was never observed.Abbreviations DMAPP dimethylallyl pyrophosphate - FPP farnesyl pyrophosphate - GPP geranyl pyrophosphate - IPP isopentenyl pyrophosphate - LPP linalyl pyrophosphate - NPP neryl pyrophosphate     

Correlation between time-dependent inhibition of human farnesyl pyrophosphate synthase and blockade of mevalonate pathway by nitrogen-containing bisphosphonates in cultured cells

A class of drugs successfully used for treatment of metabolic bone diseases is the nitrogen-containing bisphosphonates (N-BPs), which act by inhibiting the vital enzyme, farnesyl pyrophosphate synthase (FPPS), of the mevalonate pathway. Inhibition of FPPS by N-BPs results in the intracellular accumulation of isopentenyl pyrophosphate (IPP) and consequently induces the biosynthesis of a cytotoxic ATP analog (ApppI). Previous cell-free data has reported that N-BPs inhibit FPPS by time-dependent manner as a result of the conformational change. This associated conformational change can be measured as an isomerization constant (K_isom) and reflects the binding differences of the N-BPs to FPPS. In the present study, we tested the biological relevance of the calculated K_isom values of zoledronic acid, risedronate and five experimental N-BP analogs in the cell culture model. We used IPP/ApppI formation as a surrogate marker for blocking of FPPS in the mevalonate pathway.As a result, a correlation between the time-dependent inhibition of FPPS and IPP/ApppI formation by N-BPs was observed. This outcome indicates that the time-dependent inhibition of FPPS enzyme is a biologically significant mechanism and further supports the use of the K_isom calculations for evaluation of the overall potency of the novel FPPS inhibitors. Additionally, data illustrates that IPP/ApppI analysis is a useful method to monitor the intracellular action of drugs and drug candidates based on FPPS inhibition.     

Crystal structures of human IPP isomerase: new insights into the catalytic mechanism

Type I isopentenyl diphosphate (IPP): dimethylally diphosphate (DMAPP) isomerase is an essential enzyme in human isoprenoid biosynthetic pathway. It catalyzes isomerization of the carbon-carbon double bonds in IPP and DMAPP, which are the basic building blocks for the subsequent biosynthesis. We have determined two crystal structures of human IPP isomerase I (hIPPI) under different crystallization conditions. High similarity between structures of human and Escherichia coli IPP isomerases proves the conserved catalytic mechanism. Unexpectedly, one of the hIPPI structures contains a natural substrate analog ethanol amine pyrophosphate (EAPP). Based on this structure, a water molecule is proposed to be the direct proton donor for IPP and different conformations of IPP and DMAPP bound in the enzyme are also proposed. In addition, structures of human IPPI show a flexible N-terminal alpha-helix covering the active pocket and blocking the entrance, which is absent in E. coli IPPI. Besides, the active site conformation is not the same in the two hIPPI structures. Such difference leads to a hypothesis that substrate binding induces conformational change in the active site. The inhibition mechanism of high Mn(2+) concentrations is also discussed.     

Prenyl transferases from Micrococcus luteus: Characterization of undecaprenyl pyrophosphate synthetase

Three prenyl transferases in Micrococcus luteus were recovered in the soluble fraction following cell disruption. Undecaprenyl pyrophosphate (C₅₅-PP) synthetase chromatographed on DEAE-cellulose independently from geranylgeranyl-PP and octaprenyl-PP synthetases. Further purification of C₅₅-PP synthetase resulted in an approximate 250-fold purification over the crude lysate. The molecular weight of the synthetase was estimated to be between 47,000 and 49,000 by Sephadex G-100 chromatography. The enzyme had a broad specificity toward the allylic pyrophosphate substrate. The reactivities of the allylic substrates increased with chain length, C₁₀ < C₁₅ < C₂₀, except for trans-solanesyl-PP, which was unreactive. Moreover, the enzyme was active on allylic substrates having both cis- and trans-stereochemistry. Although C₅₅-PP and C₅₀-PP were the major products, some shorter chain products were also produced, when t,t-farnesyl pyrophosphate and Δ³sopentenyl pyrophosphate (IPP) were used as substrates. The stereochemistries of the products formed with C₅₅-PP synthetase were established, using [¹⁴C]IPP and 2R-[2-³H] and 2S-[2-³H]IPP. Each new isoprene unit added had a cis-configuration. The enzyme was inactive in the absence of added effectors. It was stimulated by Triton X-100, egg lecithin, and a whole phospholipid extract from M. luteus. Cardiolipin and deoxycholate were poor activators of the enzyme. The product chain length distribution observed with the phospholipid-activated enzyme showed highly favored production of the C₅₅-PP product over the C₅₀-PP product.     

Prenyltransferase: the mechanism of the reaction.

The enzyme, prenyltransferase, which normally catalyzes the addition of an allylic pyrophosphate to isopentenyl pyrophosphate, has been found to catalyze the hydrolysis of its allylic substrate. The rate of this hydrolysis is markedly stimulated by inorganic pyrophosphate. Competition experiments with 2-fluoroisopentenyl pyrophosphate and inorganic pyrophosphate demonstrated that inorganic pyrophosphate stimulated hydrolysis by binding at the isopentenyl pyrophosphate site. Hydrolysis carried out in H218O or with (1S)-[1-3H]geranyl pyrophosphate show the C-O bond is broken and the C1 carbon of geranyl pyrophosphate is inverted in the process. These results are interpreted to favor a carbonium ion mechanism for the prenyltransferase reaction.     

Substrate Binding of avian liver prenyltransferase.

Prenyltransferase (farnesyl pyrophosphate synthetase) was purified from avian liver and characterized by Sephadex and sodium dodecyl sulfate gel chromatography, peptide mapping, and end-group analysis. The enzyme is 85 800 +/- 4280 daltons and consists of two identical subunits as judged by sodium dodecyl sulfate gel electrophoresis, peptide mapping, and end-group analysis. Chemical analysis of the protein revealed no lipid or carbohydrate components. Avian prenyltransferase synthesizes farnesyl pyrophosphate from either dimethylallyl or geranyl pyrophosphate and isopentenyl pyrophosphate. A lower rate of geranylgeranyl pyrophosphate synthesis from farnesyl pyrophosphate and isopentenyl pyrophosphate was also demonstrated. Michaelis constants for farnesyl pyrophosphate synthesis are 0.5 muM for both isopentenyl pyrophosphate and geranyl pyrophosphate. The V max for the reaction is 1990 nmol min-1 mg-1 (170 mol min-1 mol-1 enzyme). Substrate inhibition by isopentenyl pyrophosphate is evident at high isopentenyl pyrophosphate and low geranyl pyrophosphate concentrations. Michaelis constants for geranylgeranyl pyrophosphate synthesis are 9 muM for farnesyl pyrophosphate and 20 muM for isopentenyl pyrophosphate. The Vmax is 16 nmol min-1 mg-1 (1.4 mol min-1 mol-1 enzyme). Two moles of each of the allylic substrates is bound per mol of enzyme. The apparent dissociation constants for dimethylallyl, geranyl, and farnesyl pyrophosphates are 1.8, 0.17, and 0.73 muM, respectively. Dimethylallyl and geranyl pyrophosphates bound competitively to prenyltransferase with one-for-one displacement. Four moles of isopentenyl pyrophosphate was bound per mole of enzyme. Citronellyl pyrophosphate, an analogue of geranyl pyrophosphate, was competitive with the binding of 2 of the 4 mol of isopentenyl pyrophosphate bound. The data are interpreted to indicate that each subunit of avian liver prenyltransferase has a single allylic binding site accommodating dimethylallyl, geranyl, and farnesyl pyrophosphates, and one binding site for isopentenyl pyrophosphate. In the absence of an allylic pyrophosphate or analogue, isopentenyl pyrophosphate also can bind to the allylic site.     

Indirect stimulation of human Vγ2Vδ2 T cells through alterations in isoprenoid metabolism

Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the 2-C-methyl-d-erythritol-4-phosphate pathway used by microbes, and isopentenyl pyrophosphate (IPP), an intermediate in the mevalonate pathway used by humans. Aminobisphosphonates and alkylamines indirectly stimulate Vγ2Vδ2 cells by inhibiting farnesyl diphosphate synthase (FDPS) in the mevalonate pathway, thereby increasing IPP/triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester that directly stimulate. In this study, we further characterize stimulation by these compounds and define pathways used by new classes of compounds. Consistent with FDPS inhibition, stimulation of Vγ2Vδ2 cells by aminobisphosphonates and alkylamines was much more sensitive to statin inhibition than stimulation by prenyl pyrophosphates; however, the continuous presence of aminobisphosphonates was toxic for T cells and blocked their proliferation. Aminobisphosphonate stimulation was rapid and prolonged, independent of known Ag-presenting molecules, and resistant to fixation. New classes of stimulatory compounds-mevalonate, the alcohol of HMBPP, and alkenyl phosphonates-likely stimulate differently. Mevalonate, a rate-limiting metabolite, appears to enter cells to increase IPP levels, whereas the alcohol of HMBPP and alkenyl phosphonates are directly recognized. The critical chemical feature of bisphosphonates is the amino moiety, because its loss switched aminobisphosphonates to direct Ags. Transfection of APCs with small interfering RNA downregulating FDPS rendered them stimulatory for Vγ2Vδ2 cells and increased cellular IPP. Small interfering RNAs for isopentenyl diphosphate isomerase functioned similarly. Our results show that a variety of manipulations affecting isoprenoid metabolism lead to stimulation of Vγ2Vδ2 T cells and that pulsing aminobisphosphonates would be more effective for the ex vivo expansion of Vγ2Vδ2 T cells for adoptive cancer immunotherapy.     

Zoledronic acid-induced IPP/ApppI production in vivo

Bisphosphonates are currently the most important class of anti-resorptive drugs used for the treatment of diseases involving excess bone resorption. Recently we discovered a new mechanism of action for bisphosphonates. Previously it has been shown that nitrogen-containing bisphosphonates (N-BPs) are not metabolized. However, our studies revealed that N-BPs induce formation of a novel pro-apoptotic ATP analog (ApppI), as a consequence of the inhibition of FPP synthase in the mevalonate pathway, and the subsequent accumulation of isopentenyl pyrophosphate (IPP) in vitro. The primary aim of the current study was to determine whether zoledronic acid (a N-BP) induces IPP/ApppI formation in vivo. Mass spectrometry was used to identify whether in vivo administration of zoledronic acid-induced IPP/ApppI production by mouse peritoneal macrophages or bone marrow cells. IPP/ApppI could be detected in extracts from peritoneal macrophages isolated from zoledronic acid-treated animals. Increasing IPP/ApppI accumulation was determined up to 7 days after drug injection, indicating prolonged FPP synthase inhibition by zoledronic acid. Importantly, this is the first report of in vivo production of ApppI, supporting the biological significance of this molecule.     

Molecular regulation of santalol biosynthesis in Santalum album L.

Santalum album L. commonly known as East-Indian sandal or chandan is a hemiparasitic tree of family santalaceae. Santalol is a bioprospecting molecule present in sandalwood and any effort towards metabolic engineering of this important moiety would require knowledge on gene regulation. Santalol is a sesquiterpene synthesized through mevalonate or non-mevalonate pathways. First step of santalol biosynthesis involves head to tail condensation of isopentenyl pyrophosphate (IPP) with its allylic co-substrate dimethyl allyl pyrophosphate (DMAPP) to produce geranyl pyrophosphate (GPP; C10 — a monoterpene). GPP upon one additional condensation with IPP produces farnesyl pyrophosphate (FPP; C15 — an open chain sesquiterpene). Both the reactions are catalyzed by farnesyl diphosphate synthase (FDS). Santalene synthase (SS), a terpene cyclase catalyzes cyclization of open ring FPP into a mixture of cyclic sesquiterpenes such as α-santalene, epi-β-santalene, β-santalene and exo bergamotene, the main constituents of sandal oil. The objective of the present work was to generate a comprehensive knowledge on the genes involved in santalol production and study their molecular regulation. To achieve this, sequences encoding farnesyl diphosphate synthase and santalene synthase were isolated from sandalwood using suppression subtraction hybridization and 2D gel electrophoresis technology. Functional characterization of both the genes was done through enzyme assays and tissue-specific expression of both the genes was studied. To our knowledge, this is the first report on studies on molecular regulation, and tissue-specific expression of the genes involved in santalol biosynthesis.     

Crystal structures of undecaprenyl pyrophosphate synthase in complex with magnesium, isopentenyl pyrophosphate, and farnesyl thiopyrophosphate: roles of the metal ion and conserved residues in catalysis

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the consecutive condensation reactions of a farnesyl pyrophosphate (FPP) with eight isopentenyl pyrophosphates (IPP), in which new cis-double bonds are formed, to generate undecaprenyl pyrophosphate that serves as a lipid carrier for peptidoglycan synthesis of bacterial cell wall. The structures of Escherichia coli UPPs were determined previously in an orthorhombic crystal form as an apoenzyme, in complex with Mg(2+)/sulfate/Triton, and with bound FPP. In a further search of its catalytic mechanism, the wild-type UPPs and the D26A mutant are crystallized in a new trigonal unit cell with Mg(2+)/IPP/farnesyl thiopyrophosphate (an FPP analogue) bound to the active site. In the wild-type enzyme, Mg(2+) is coordinated by the pyrophosphate of farnesyl thiopyrophosphate, the carboxylate of Asp(26), and three water molecules. In the mutant enzyme, it is bound to the pyrophosphate of IPP. The [Mg(2+)] dependence of the catalytic rate by UPPs shows that the activity is maximal at [Mg(2+)] = 1 mm but drops significantly when Mg(2+) ions are in excess (50 mm). Without Mg(2+), IPP binds to UPPs only at high concentration. Mutation of Asp(26) to other charged amino acids results in significant decrease of the UPPs activity. The role of Asp(26) is probably to assist the migration of Mg(2+) from IPP to FPP and thus initiate the condensation reaction by ionization of the pyrophosphate group from FPP. Other conserved residues, including His(43), Ser(71), Asn(74), and Arg(77), may serve as general acid/base and pyrophosphate carrier. Our results here improve the understanding of the UPPs enzyme reaction significantly.     

Probing the conformational change of Escherichia coli undecaprenyl pyrophosphate synthase during catalysis using an inhibitor and tryptophan mutants.

Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation reactions of eight isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate C(55) undecaprenyl pyrophosphate (UPP). In the present study, site-directed mutagenesis, fluorescence quenching, and stopped-flow methods were utilized to examine the substrate binding and the protein conformational change. (S)-Farnesyl thiopyrophosphate (FsPP), a FPP analogue, was synthesized to probe the enzyme inhibition and events associated with the protein fluorescence change. This compound with a much less labile thiopyrophosphate shows K(i) value of 0.2 microm in the inhibition of Escherichia coli UPPS and serves as a poor substrate, with the k(cat) value (3.1 x 10(-7) s(-1)) 10(7) times smaller than using FPP as the substrate. Reduction of protein intrinsic fluorescence was observed upon addition of FPP (or FsPP) to the UPPS solution. Moreover, fluorescence studies carried out using W91F and other mutant UPPS with Trp replaced by Phe indicate that FPP binding mainly quenches the fluorescence of Trp-91, a residue in the alpha3 helix that moves toward the active site during substrate binding. Using stopped-flow apparatus, a three-phase protein fluorescence change with time was observed by mixing the E.FPP complex with IPP in the presence of Mg(2+). However, during the binding of E.FsPP with IPP, only the fastest phase was observed. These results suggest that the first phase is due to the IPP binding to E.FPP complex, and the other two slow phases are originated from the protein conformational change. The two slow phases coincide with the time course of FPP chain elongation from C(15) to C(55) and product release.     

Crystal structures of ligand‐bound octaprenyl pyrophosphate synthase from Escherichia coli reveal the catalytic and chain‐length determining mechanisms

Octaprenyl pyrophosphate synthase (OPPs) catalyzes consecutive condensation reactions of one allylic substrate farnesyl pyrophosphate (FPP) and five homoallylic substrate isopentenyl pyrophosphate (IPP) molecules to form a C₄₀ long‐chain product OPP, which serves as a side chain of ubiquinone and menaquinone. OPPs belongs to the trans‐prenyltransferase class of proteins. The structures of OPPs from Escherichia coli were solved in the apo‐form as well as in complexes with IPP and a FPP thio‐analog, FsPP, at resolutions of 2.2–2.6 Å, and revealed the detailed interactions between the ligands and enzyme. At the bottom of the active‐site tunnel, M123 and M135 act in concert to form a wall which determines the final chain length. These results represent the first ligand‐bound crystal structures of a long‐chain trans‐prenyltransferase and provide new information on the mechanisms of catalysis and product chain elongation. Proteins 2015; 83:37–45. © 2014 Wiley Periodicals, Inc.