Synthesis and evaluation of chlorinated substrate analogues for farnesyl diphosphate synthase

Substrate analogues for isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where the C3 methyl groups were replaced by chlorine, were synthesized and evaluated as substrates for avian farnesyl diphosphate synthase (FPPase). The IPP analogue (3-ClIPP) was a cosubstrate when incubated with dimethylallyl diphosphate (DMAPP) or geranyl diphosphate (GPP) to give the corresponding chlorinated analogues of geranyl diphosphate (3-ClGPP) and farnesyl diphosphate (3-ClFPP), respectively. No products were detected in incubations of 3-ClIPP with 3-ClDMAPP. Incubation of IPP with 3-ClDMAPP gave 11-ClFPP as the sole product. Values of K(M)(3-ClIPP) (with DMAPP) and K(M)(3-ClDMAPP) (with IPP) were similar to those for IPP and DMAPP; however, values of k(cat) for both analogues were substantially lower. These results are consistent with a dissociative electrophilic alkylation mechanism where the rate-limiting step changes from heterolytic cleavage of the carbon-oxygen bond in the allylic substrate to alkylation of the double bond of the homoallylic substrate.     

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.     

Selectivity in substrate–enzyme complexation studied by surface forces measurement

The selectivity of substrate in substrate–enzyme complexation of heptaprenyl diphosphate synthase was directly investigated using colloidal probe atomic force microscopy (AFM). This enzyme is composed of two dissociable subunits, which exhibits a catalytic activity only when they are associated together in the presence of a cofactor, Mg²⁺, and a substrate, farnesyl diphosphate (FPP). We have recently succeeded to directly demonstrate a specific interaction involved in this enzyme reaction and obtain new insights into the molecular mechanism of the reaction using the approach based on the colloidal probe AFM. The AFM measurement showed the adhesive force between the subunits only in the presence of both Mg²⁺ and FPP. In this study, we studied the substrate selectivity in the complexation by monitoring the adhesive force. The substrates studied are pyrophosphate (PPi), isopentenyl diphosphate (IPP), geranyl diphosphate (GPP), farnesyl monophosphate (FP), and farnesyl geranyl diphosphate (FGPP). No adhesion was observed in the case of PPi, IPP, and GPP. On the other hand, the significant adhesion was observed for phosphate derivatives, which bear prenyl units longer than three. This is in good agreement with the selectivity of the substrates by this enzyme, which catalyzes the condensation reaction of four IPP molecules with FPP to give heptaprenyl (C₃₅) diphosphates. Our study showed a useful methodology for examining the elemental processes of biological reactions.     

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.     

Proton exchange in type II isopentenyl diphosphate isomerase

[reaction: see text] Type II isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase from Synechocystis PCC 6803 catalyzes the interconversion of IPP and DMAPP. Upon incubation of the enzyme with IPP or DMAPP in 2H2O, one deuterium is incorporated into the C2 methylene of IPP, two deuteriums are incorporated at C4, and three deuteriums are incorporated into the (E)-methyl of DMAPP.     

Diversity of the biosynthesis of the isoprene units

This review covers the biosynthesis of the starter units of terpenoids, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) via the nonmevalonate pathway together with a new enzyme involved in the conversion of IPP and DMAPP, i.e type 2 IPP isomerase. The biosynthesis of terpenoids produced by actinomycetes is also reviewed. 117 references are cited.     

Study of IspH, a key enzyme in the methylerythritol phosphate pathway using fluoro-substituted substrate analogues

IspH, a [4Fe-4S]-cluster-containing enzyme, catalyzes the reductive dehydroxylation of 4-hydroxy-3-methyl-butenyl diphosphate (HMBPP) to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the methylerythritol phosphate pathway. Studies of IspH using fluoro-substituted substrate analogues to dissect the contributions of several factors to IspH catalysis, including the coordination of the HMBPP C(4)-OH group to the iron-sulfur cluster, the H-bonding network in the active site, and the electronic properties of the substrates, are reported.     

Type-2 isopentenyl diphosphate isomerase: evidence for a stepwise mechanism

Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). These two molecules are the building blocks for construction of isoprenoid carbon skeletons in nature. Two structurally unrelated forms of IDI are known. A variety of studies support a proton addition/proton elimination mechanism for both enzymes. During studies with Thermus thermophilus IDI-2, we discovered that the olefinic hydrogens of a vinyl thiomethyl analogue of isopentenyl diphosphate exchanged with solvent when the enzyme was incubated with D(2)O without concomitant isomerization of the double bond. These results suggest that the enzyme-catalyzed isomerization reaction is not concerted.     

Investigation of the catalytic mechanism of farnesyl pyrophosphate synthase by computer simulation

Farnesyl pyrophosphate synthase (FPPS) catalyses the formation of a key cellular intermediate in isoprenoid metabolic pathways, farnesyl pyrophosphate, by the sequential head-to-tail condensation of two molecules of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP). Recently, FPPS has been shown to represent an important target for the treatment of parasitic diseases such as Chagas disease and African trypanosomiasis. Bisphosphonates, pyrophosphate analogues in which the oxygen bridge between the two phosphorus atoms has been replaced by a carbon substituted with different side chains, are able to inhibit the FPPS enzyme. Moreover, nitrogen-containing bisphosphonates have been proposed as carbocation transition state analogues of FPPS. On the basis of structural and kinetic data, different catalytic mechanisms have been proposed for FPPS. By analyzing different reaction coordinates we propose that the reaction occurs in one step through a carbocationic transition state and the subsequent transfer of a hydrogen atom from IPP to the pyrophosphate moiety of DMAPP. Moreover, we have analyzed the role of the active site amino acids on the activation barrier and the reaction mechanism. The structure of the active site is well conserved in the isoprenyl diphosphate synthase family; thus, our results are relevant for the understanding of this important class of enzymes and for the design of more potent and specific inhibitors for the treatment of parasitic diseases.     

Isopentenyl diphosphate isomerase. Mechanism-based inhibition by diene analogues of isopentenyl diphosphate and dimethylallyl diphosphate

Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This is an essential step in the mevalonate entry into the isoprenoid biosynthetic pathway. The isomerization catalyzed by type I IDI involves protonation of the carbon-carbon double bond in IPP or DMAPP to form a tertiary carbocation, followed by deprotonation. Diene analogues for DMAPP (E-2-OPP and Z-2-OPP) and IPP (4-OPP) were synthesized and found to be potent active-site-directed irreversible inhibitors of the enzyme. X-ray analysis of the E.I complex between Escherichia coli IDI and 4-OPP reveals the presence of two isomers that differ in the stereochemistry of the newly formed C3-C4 double bond in the hydrocarbon chain of the inhibitor. In both adducts C5 of the inhibitor is joined to the sulfur of C67. In these structures the methyl group formed upon protonation of the diene moiety in 4-OPP is located near E116, implicating that residue in the protonation step.     

Type II isopentenyl diphosphate isomerase: irreversible inactivation by covalent modification of flavin

Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the basic building blocks of isoprenoid molecules. Two structurally unrelated classes of IDI are known. Type I IPP isomerase (IDI-1) utilizes a divalent metal in a protonation-deprotonation reaction; whereas, the type II enzyme (IDI-2) requires reduced flavin. Epoxy, diene, and fluorinated substrate analogues, irreversible inhibitors of IDI-1, were analyzed as mechanistic probes for IDI-2. 3,4-Oxido-3-methyl-1-butyl diphosphate (eIPP), 3-methylene-4-penten-1-yl diphosphate (vIPP), and 3-(fluoromethyl)-3-buten-1-yl diphosphate (fmIPP) inactivate IDI-2 through formation of covalent adducts with the reduced flavin. UV-visible spectra of the inactivated complexes are consistent with modification of the isoalloxazine ring at position N5. vIPP and fmIPP are also alternate substrates with isomerization competing with alkylation of the flavin cofactor. (Z)-3-(Fluoromethyl)-2-buten-1-yl diphosphate ((Z)-fmDMAPP) and (Z)-3-(difluoromethyl)-2-buten-1-yl diphosphate ((Z)-dfmDMAPP) are alternate substrates, which are isomerized to the corresponding IPP derivatives. The rates of isomerization of fmIPP and (Z)-fmDMAPP are approximately 50-fold less than IPP and DMAPP, respectively. dfmIPP is not an irreversible inhibitor. These studies indicate that the irreversible inhibitors inactivate the reduced flavin required for catalysis by electrophilic alkylation and are consistent with a protonation-deprotonation mechanism for the isomerization catalyzed by IDI-2.     

On the Early Steps of Cineol Biosynthesis in Eucalyptus globulus

Samples of [4‐²H₁]‐1‐deoxyxylulose ( 17a ) and [2‐¹³C, 4‐²H₁]‐1‐deoxyxylulose ( 17b ), have been prepared by modification of known procedures and fed in aqueous solution to twiglets of Eucalyptus globulus. The probes of cineol ( 6 ) isolated from these experiments were analyzed by GC/MS, ²H‐ and ¹³C‐NMR techniques. In the experiments with 17b , the formation of five isotopomers of 6 could be detected. Their structure and relative abundance demonstrate that the ¹³C‐label is incorporated to the same extent into the two C₅‐units of 6 , and that the ²H label is retained to an extent of 57% in the starter dimethylallyl‐diphosphate unit (DMAPP; 12 ), but completely or almost completely lost in the unit derived from isopentenyl diphosphate (IPP; 11 ), in the elongation step which leads to geranyl diphosphate (GPP; 1 ). These results confirm that the recently discovered mevalonate‐independent pathway to IPP and DMAPP is operative in the biosynthesis of cineol, and indicate, together with previous finding, that, within this pathway, formation of IPP and DMAPP occurs in independent rather than in sequential steps. In addition, the demonstration of different metabolic origins for the olefinic H‐atoms of GPP ( 1 ), the aliphatic C₁₀‐precursor of 6 , paves the way for a realistic interpretation of the strikingly consistent but hitherto unexplained anomalies detected in the natural‐abundance ²H‐NMR spectra of (+)‐ and (−)‐α‐pinene and of (+)‐limonene.     

Escherichia coli type I isopentenyl diphosphate isomerase: structural and catalytic roles for divalent metals

Isopentenyl diphosphate isomerase (IDI) catalyzes the essential conversion of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) in the mevalonate entry into the isoprenoid biosynthetic pathway. Two convergently evolved forms of IDI are known. Type I IDI, which is found in Eukarya and many Bacteria, catalyzes the isomerization of IPP and DMAPP by a protonation-deprotonation mechanism. The enzyme requires two divalent metal ions for activity. An X-ray structure of type I IDI from crystals soaked with (N,N-dimethylamino)-1-ethyl diphosphate (NIPP), a potent transition-state analogue for the carbocationic intermediate in the isomerization reaction, shows one of the metals in a His(3)Glu(2) hexacoordinate binding site, while the other forms a bridge between the diphosphate moiety of the substrate and the enzyme (Wouters, J.; et al. J. Biol. Chem. 2003, 278, 11903). Reconstitution of metal-free recombinant Escherichia coli type I IDI with several divalent metals-Mg(2+), Mn(2+), Zn(2+), Co(2+), Ni(2+), and Cd(2+)-generated active enzyme. Freshly purified IDI contained substoichiometric levels of a single metal ion, presumably bound in the hexacoordinate site. When NIPP was added to the disruption and purification buffers of enzyme, the purified protein contained 0.72 equiv of Mg(2+), 0.92 equiv of Zn(2+), and 0.10 equiv of Mn(2+). These results are consistent with a structure in which Mg(2+) facilitates diphosphate binding and Zn(2+) or Mn(2+) occupies the hexacoordinate site.     

Molecular interactions of nitrogen-containing bisphosphonates within farnesyl diphosphate synthase

Bisphosphonates, known for their effectiveness in the treatment of osteoporosis, inhibit bone resorption via mechanisms that involve binding to bone mineral and cellular effects on osteoclasts. The major molecular target of nitrogen-containing bisphosphonates (N-BPs) in osteoclasts is farnesyl diphosphate synthase (FPPS). N-BPs likely inhibit this enzyme by mimicking one or more of the natural isoprenoid lipid substrates (GPP/DMAPP and IPP) but the mode of inhibition is not established. The active site of FPPS comprises a subsite for each substrate. Kinetic studies with recombinant human FPPS indicate that both potent (risedronate) and weak (NE-58051) enzyme inhibitors compete with GPP for binding to FPPS, however, binding to this site does not completely explain the difference in potency of the two inhibitors, suggesting that a second binding site may also be a target of bisphosphonate inhibition. Using the docking software suite Autodock, we explored a dual inhibitor binding mode for recombinant human FPPS. Experimental support for dual binding is suggested by Dixon plots for the inhibitors. N-BPs may inhibit by binding to both the GPP and a second site with differences in potency at least partly arising from inhibition at the second site.     

Enzymatic Synthesis of Variediene Analogs

Five analogs of dimethylallyl diphosphate (DMAPP) with additional or shifted Me groups were converted with isopentenyl diphosphate (IPP) and the fungal variediene synthase from Aspergillus brasiliensis (AbVS). These enzymatic reactions resulted in the formation of several new terpene analogs that were isolated and structurally characterised by NMR spectroscopy. Several DMAPP analogs showed a changed reactivity giving access to compounds with unusual skeletons. Their formation is mechanistically rationalised and the absolute configurations of all obtained compounds were determined through a stereoselective deuteration strategy, revealing absolute configurations that are analogous to that of the natural enzyme product variediene.     

Isopentenyl diphosphate isomerase catalyzed reactions in D2O: product release limits the rate of this sluggish enzyme-catalyzed reaction

The E. coli isopentenyl diphosphate isomerase (IDI) catalyzed reaction of isopentenyl diphosphate (IPP) in D(2)O gives a 66% yield of dimethylallyl diphosphate labeled with deuterium at the (E)-methyl group (d-DMAPP) and a 34% yield of IPP labeled with 1 mol of deuterium at C-2 (d-IPP). This shows that the release to D(2)O of the initial product of the IDI-catalyzed reaction (d-DMAPP) is slower than its conversion to d-IPP. Product dissociation is therefore rate determining for isomerization of IPP with a rate constant k(dis) ≈ k(cat) = 0.08 s(-1). The data provide an estimated rate constant of k(as) = 6 × 10(3) M(-1) s(-1) for binding of DMAPP to E. coli IDI that is similar to rate constants determined for the binding of N-protonated 2-amino ethyl diphosphate intermediate analogs to IDI from yeast [Reardon, J. E.; Abeles, R. H. Biochemistry1986, 25, 5609-5616]. We propose that ligand binding to IDI is relatively slow because there is a significant kinetic barrier to reorganization of the initial encounter complex between enzyme, substrate, and an essential Mg(2+) to form the Michaelis complex where the metal cation bridges the protein and the substrate diphosphate group.     

A soluble form of phosphatase in Saccharomyces cerevisiae capable of converting farnesyl diphosphate into E,E-farnesol

After anion-exchange chromatography, the soluble fraction of a cell-free extract of Saccharomyces cerevisiae showed two phosphatase activity peaks when p-nitrophenyl phosphate (pNPP) was used as the substrate. However, only the second pNPP active peak demonstrated the ability to convert farnesyl diphosphate (FPP) into E,E-farnesol. N-terminal sequence analysis of the purified pNPP/FPP phosphatase revealed that it was a truncated form of alkaline phosphatase Pho8 lacking 62 amino acids from the N-terminus and was designated Pho8Delta62. Although other isoprenyl diphosphates such as geranyl diphosphate (GPP) and geranylgeranyl diphosphate (GGPP) could also be hydrolyzed by Pho8Delta62 to the corresponding alcohols, selectivity was observed among these substrates. The optimum pH was 7.0 for all three isoprenyl diphosphate substrates. Although lower hydrolytic activity was observed for FPP and GGPP at pH 6.0 and 8.5, hydrolysis of GPP was observed only at pH 7.0. Mg2+ and Mn2+ inhibited hydrolysis of FPP and GGPP, and GGPP was more sensitive to Mg2+ inhibition than FPP. The rate of FPP hydrolysis increased in the presence of Triton X-100.     

Stereochemical analysis of isopentenyl diphosphate isomerase type II from Staphylococcus aureus using chemically synthesized (S)- and (R)-[2-2H]isopentenyl diphosphates

[chemical reaction: see text]. To study the catalysis of isopentenyl diphosphate (IPP) isomerase type II from Staphylococcus aureus, which is a flavoprotein catalyzing the interconversion of IPP and dimethylallyl diphosphate, we have chemically synthesized (S)- and (R)-[2-2H]IPP and carried out stereochemical analysis of the reaction. Our results show that the C-2 deprotonation of IPP by this enzyme is pro-R stereospecific, suggesting a similar stereochemical course as the type I enzyme.     

Geranylgeranyl diphosphate synthases from Scoparia dulcis and Croton sublyratus. cDNA cloning, functional expression, and conversion to a farnesyl diphosphate synthase

cDNAs encoding geranylgeranyl diphosphate synthase (GGPPS) of two diterpene producing plants, Scoparia dulcis and Croton sublyratus, were isolated using the homology-based polymerase chain reaction method. Both cloned genes showed high amino acid sequence homology (60-70%) to other plant GGPPSs and contained highly conserved aspartate-rich motifs. The obtained clones were functionally expressed in Escherichia coli and showed sufficient GGPPS activity to catalyze the condensation of farnesyl diphosphate (FPP) and isopentenyl diphosphate to form geranylgeranyl diphosphate. To investigate the factor determining the product chain length of plant GGPPSs, S. dulcis GGPPS mutants in which either the small amino acids at the fourth and fifth positions before the first aspartate-rich motif (FARM) were replaced with aromatic amino acids or in which two additional amino acids in FARM were deleted were constructed. Both mutants behaved like FPPS-like enzymes and almost exclusively produced FPP when dimethylallyl diphosphate was used as a primer substrate, and failed to accept FPP as a primer substrate. These results indicate that both small amino acids at the fourth and fifth positions before FARM and the amino acid insertion in FARM play essential roles in product length determination in plant GGPPSs.     

Structure-based drug design targeting biosynthesis of isoprenoids: a crystallographic state of the art of the involved enzymes

Biosynthesis of the universal terpenoid precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), from three acetyl CoA moieties through mevalonate was studied extensively in the 1950s. For several decades, the mevalonate paradigm reigned supreme and a mevalonate origin was attributed to a growing number of natural products, in many cases erroneously. Besides this biosynthetic pathway, the existence of a second one leading to IPP and DMAPP through 1-deoxy-D-xylulose 5-phosphate and 2C-methyl-D-erythritol 4-phosphate was discovered more recently in plants and some eubacteria. This pathway is widely distributed in the bacterial kingdom including major human pathogens, such as Mycobacterium tuberculosis or Helicobacter pylori and is also essential in the malaria vector Plasmodium falciparum. During the last few years, the genes, enzymes, intermediates and mechanisms of the biosynthetic route have been elucidated by a combination of methods including comparative genomics, enzymology, advanced NMR technology and crystallography. The present crystallographic review of enzymes involved in isoprenoid biosynthesis will be useful for understanding the various catalytic mechanisms and could potentially help for structure-based drug design.