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.     

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.     

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.     

Zinc is an essential cofactor for type I isopentenyl diphosphate:dimethylallyl diphosphate isomerase

Isopentenyl diphosphate (IPP) isomerase catalyzes the interconversion of IPP and dimethylallyl diphosphate (DMAPP). This is an essential reaction in the mevalonate pathway for biosynthesis of isoprenoid compounds. A crystal structure of Escherichia coli type I IPP isomerase shows a his3glu2 octahedral metal binding site (Durbecq, V. et al. EMBO, 2001, 20, 1530-1537). A metal ion analysis of recombinant E. coli type I IPP isomerase purified from metal-free buffer or buffer containing 10 muM ZnCl2 and 10 muM MnCl2 indicated that the protein contained one atom of Zn2+ per molecule. The metal content and the activity of the enzyme did not change when dialyzed for 6 h against metal-free buffer but rapidly decreased upon dialysis against buffer containing o-phenanthroline. Structural and catalytic roles for Zn2+ are discussed.     

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.     

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.     

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.     

Structure‐Based Design and Synthesis of the First Weak Non‐Phosphate Inhibitors for IspF,an Enzyme in the Non‐Mevalonate Pathway of Isoprenoid Biosynthesis

In this paper, we describe the structure‐based design, synthesis, and biological evaluation of cytosine derivatives and analogues that inhibit IspF, an enzyme in the non‐mevalonate pathway of isoprenoid biosynthesis. This pathway is responsible for the biosynthesis of the C₅ precursors to isoprenoids, isopentenyl diphosphate (IPP, 1 ) and dimethylallyl diphosphate (DMAPP, 2 ; Scheme 1). The non‐mevalonate pathway is the sole source for 1 and 2 in the protozoan Plasmodium parasites. Since mammals exclusively utilize the alternative mevalonate pathway, the enzymes of the non‐mevalonate pathway have been identified as attractive new drug targets in the fight against malaria. Based on computer modeling (cf. Figs. 2 and 3), new cytosine derivatives and analogues (Fig. 1) were selected as potential drug‐like inhibitors of IspF protein, and synthesized (Schemes 2–5). Determination of the enzyme activity by ¹³C‐NMR spectroscopy in the presence of the new ligands showed inhibitory activities for some of the prepared cytosine and pyridine‐2,5‐diamine derivatives in the upper micromolar range (IC₅₀ values; Table). The data suggest that it is possible to inhibit IspF protein without binding to the polar diphosphate binding site and the side chain of Asp56′, which interacts with the ribose moiety of the substrate and substrate analogues. Furthermore, a new spacious sub‐pocket was discovered which accommodates aromatic spacers between cytosine derivatives or analogues (binding to ‘Pocket III’) and rings that occupy the flexible hydrophobic region of ‘Pocket II’. The proposed binding mode remains to be further validated by X‐ray crystallography.     

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.     

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.     

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.     

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.     

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.     

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.     

Synthesis of (S)-isoprenoid thiodiphosphates as substrates and inhibitors

Thiolo thiophosphate analogues of isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP) were synthesized. Inorganic thiopyrophosphate (SPP(i)) was prepared from trimethyl phosphate in four steps. The tris(tetra-n-butylammonium) salt was then used to convert isopentenyl tosylate to (S)-isopentenyl thiodiphosphate (ISPP). (S)-Dimethylallyl (DMASPP), (S)-geranyl (GSPP), (S)-farnesyl (FSPP), and (S)-geranylgeranyl thiodiphosphate (GGSPP) were prepared from the corresponding bromides in a similar manner. ISPP and GSPP were substrates for avian farnesyl diphosphate synthase (FPPase). Incubation of the enzyme with ISPP and GPP gave FSPP, whereas incubation with IPP and GSPP gave FPP. GSPP was a substantially less reactive than GPP in the chain elongation reaction and was an excellent competitive inhibitor, K(I)(GSPP) = 24.8 microM, of the enzyme. Thus, when ISPP and DMAPP were incubated with FPPase, GSPP accumulated and was only slowly converted to FSPP.     

Synthesis of [1-C] and stereo-specifically [1-H] labeled fluorinated substrate analogues of IspH enzyme in the deoxyxylulose phosphate pathway

IspH in the deoxyxylulose phosphate (DXP) pathway catalyzes the reductive dehydration of (E)-4-hydroxy-3-methyl-2-butenyl diphosphate (HMBPP) to isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), which are the starting materials for the synthesis of thousands of isoprenoids. Several models have been proposed in the literature to account for this unique transformation, and most of them involve the formation of an allylic radical intermediate. To facilitate trapping and characterizing the proposed intermediates in the IspH-catalyzed reactions, in the present work, we report the synthesis of four isotopically labeled IspH substrate analogues. These isotopically labeled mechanistic probes will be utilized in the future for characterizing the proposed IspH reaction intermediates by the combination of bioorganic and biophysical approaches.     

Methylerythritol Phosphate Pathway: Enzymatic Evidence for a Rotation in the LytB/IspH-Catalyzed Reaction

IspH/LytB, an oxygen-sensitive [4Fe-4S] enzyme, catalyzes the last step of the methylerythritol phosphate (MEP) pathway, a target for the development of new antimicrobial agents. This metalloenzyme converts (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Here, the synthesis of (S)-[4-²H₁]HMBPP and (R)-[4-²H₁]HMBPP is reported together with a detailed NMR analysis of the products formed after their respective incubation with E. coli IspH/LytB in the presence of the biological reduction system used by E. coli to reduce the [4Fe-4S] center. (S)-[4-²H₁]HMBPP was converted into [4-²H₁]DMAPP and (E)-[4-²H₁]IPP, whereas (R)-[4-²H₁]HMBPP yielded [4-²H₁]DMAPP and (Z)-[4-²H₁]IPP, hence providing the direct enzymatic evidence that the mechanism catalyzed by IspH/LytB involves a rotation of the CH₂OH group of the substrate to display it away from the [4Fe-4S].     

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.     

Enzymatic Formation of a Skipped Methyl‐Substituted Octaprenyl Side Chain of Longestin (KS‐505a): Involvement of Homo‐IPP as a Common Extender Unit

Longestin (KS‐505a), a specific inhibitor of phosphodiesterase, is a meroterpenoid that consists of a unique octacyclic terpene skeleton with branched methyl groups at unusual positions (C1 and C12). Biochemical analysis of Lon23, a methyltransferase involved in the biosynthesis of longestin, demonstrated that it methylates homoisopentenyl diphosphate (homo‐IPP) to afford (3Z)‐3‐methyl IPP. This compound, along with IPP, is selectively accepted as extender units by Lon22, a geranylgeranyl diphosphate (GGPP) synthase homologue, to yield dimethylated GGPP (dmGGPP). The absolute configuration of dmGGPP was determined to be (4R,12R) by degradation and chiral GC analysis. These findings allowed us to propose an enzymatic sequence for key steps of the biosynthetic pathway of the unusual homoterpenoid longestin.     

Biosynthesis of the isoprenoid moieties of furanonaphthoquinone I and endophenazine A in Streptomyces cinnamonensis DSM 1042

Streptomyces cinnamonensis DSM 1042 produces the polyketide-isoprenoid compound furanonaphthoquinone I (FNQ I) and isoprenylated phenazines, predominantly endophenazine A. However, the recently identified biosynthetic gene cluster for these compounds only contains a single gene for a mevalonate pathway enzyme, that is, a putative mevalonate kinase gene. This is in strong contrast to all Streptomyces strains examined so far, where all six genes encoding the mevalonate pathway enzymes are clustered in a single operon of 6.8 kb and, thus, raised the question about the biosynthetic origin of the isoprenoid moieties of FNQ I and endophenazine A. In this study, we investigated the incorporation of [13C2]acetate and [2-13C]glycerol into FNQ I and endophenazine A. The results unequivocally prove that the isoprenoid building blocks of both compounds are predominantly formed via the mevalonate pathway (approximately 80%) but that the MEP pathway (approximately 20%) contributes to the biosynthesis of these molecules, too. In actinomycetes, this is the first experimentally proven example of the utilization of both biosynthetic routes for the formation of one single secondary metabolite. The incorporation pattern of [2-13C]glycerol was consistent with a "reverse" prenyl transfer, that is, with the formation of a C-C bond from C-3 of GPP to the polyketide nucleus of FNQ I.