Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the basic five-carbon 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. In contrast, the type II enzyme (IDI-2) requires reduced flavin, raising the possibility that the reaction catalyzed by IDI-2 involves the net addition/abstraction of a hydrogen atom. As part of our studies of the mechanism of isomerization for IDI-2, we synthesized allene and alkyne substrate analogues for the enzyme. These molecules are predicted to be substantially less reactive toward proton addition than IPP and DMAPP, but have similar reactivities toward hydrogen atom addition. This prediction was verified by calculations of gas phase heats of reaction for addition of a proton and of a hydrogen atom to 1-butyne (3) and 1,2-butadiene (4) to form the 1-buten-2-yl carbocation and radical, respectively, and related affinities for 2-methyl-1-butene (5) and 2-methyl-2-butene (6) using G3MP2B3 and CBS-QB3 protocols. Alkyne 1-OPP and allene 2-OPP were not substrates for Thermus thermophilus IDI-2 or Escherichia coli IDI-1, but instead were competitive inhibitors. The experimental and computational results are consistent with a protonation-deprotonation mechanism for the enzyme-catalyzed isomerization of IPP and DMAPP.
Isopentenyl diphosphate isomerase catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In eukaryotes, archaebacteria, and some bacteria, IPP is synthesized from acetyl coenzyme A by the mevalonate pathway. The subsequent isomerization of IPP to DMAPP activates the five-carbon isoprene unit for subsequent prenyl transfer reactions. In Escherichia coli, the isoprene unit is synthesized from pyruvate and glyceraldehyde-3-phosphate by the recently discovered nonmevalonate pathway. An open reading frame (ORF696) encoding a putative IPP isomerase was identified in the E. coli chromosome at 65.3 min. ORF696 was cloned into an expression vector; the 20.5 kDa recombinant protein was purified in three steps, and its identity as an IPP isomerase was established biochemically. The gene for IPP isomerase, idi, is not clustered with other known genes for enzymes in the isoprenoid pathway. E. coli FH12 was constructed by disruption of the chromosomal idi gene with the aminoglycoside 3′-phosphotransferase gene and complemented by the wild-type idi gene on plasmid pFMH33 with a temperature-sensitive origin of replication. FH12/pFMH33 was able to grow at the restrictive temperature of 44°C and FH12 lacking the plasmid grew on minimal medium, thereby establishing that idi is a nonessential gene. Although the Vmax of the bacterial protein was 20-fold lower than that of its yeast counterpart, the catalytic efficiencies of the two enzymes were similar through a counterbalance in Kms. The E. coli protein requires Mg2+ or Mn2+ for activity. The enzyme contains conserved cysteine and glutamate active-site residues found in other IPP isomerases.
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 D2O without concomitant isomerization of the double bond. These results suggest that the enzyme-catalyzed isomerization reaction is not concerted.
The type II isopentenyl diphosphate/dimethylallyl diphosphate isomerase (IDI-2) is a flavin mononucleotide (FMN)-dependent enzyme that catalyzes the reversible isomerization of isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP), a reaction with no net change in redox state of the coenzyme or substrate. Here, UV-vis spectral analysis of the IDI-2 reaction revealed the accumulation of a reduced neutral dihydroflavin intermediate when the reduced enzyme was incubated with IPP or DMAPP. When IDI-2 was reconstituted with 1-deazaFMN and 5-deazaFMN, similar reduced neutral forms of the deazaflavin analogues were observed in the presence of IPP. Single turnover stopped-flow absorbance experiments indicated that this flavin intermediate formed and decayed at kinetically competent rates in the pre-steady-state and, thus, most likely represents a true intermediate in the catalytic cycle. UV-vis spectra of the reaction mixtures reveal trace amounts of a neutral semiquinone, but evidence for the presence of IPP-based radicals could not be obtained by EPR spectroscopy. Rapid-mix chemical quench experiments show no burst in DMAPP formation, suggesting that the rate determining step in the forward direction (IPP to DMAPP) occurs prior to DMAPP formation. A solvent deuterium kinetic isotope effect (D2OVmax = 1.5) was measured on vo in steady-state kinetic experiments at saturating substrate concentrations. A substrate deuterium kinetic isotope effect was also measured on the initital velocity (DVmax = 1.8) and on the decay rate of the flavin intermediate (Dks = 2.3) in single-turnover stopped-flow experiments using (R)-[2-2H]-IPP. Taken together, these data suggest that the C2–H bond of IPP is cleaved in the rate determining step and that general acid/base catalysis may be involved during turnover. Possible mechanisms for the IDI-2 catalyzed reaction are presented and discussed in terms of the available X-ray crystal structures.
The type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) catalyzes the reversible isomerization of the two ubiquitous isoprene units, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are required to initiate the biosynthesis of all isoprenoid compounds found in nature. The overall chemical transformation catalyzed by IDI-2 involves a net 1,3-proton addition/elimination reaction. Surprisingly, IDI-2 requires a reduced flavin mononucleotide (FMN) coenzyme to carry out this redox neutral isomerization. The exact function of FMN in catalysis has not yet been clearly defined. To provide mechanistic insight into the role of the reduced flavin in IDI-2 catalysis, several FMN analogues with altered electronic properties were chemoenzymatically prepared, and their effects on the kinetic properties of the IDI-2 catalyzed reaction were investigated. Linear free energy relationships (LFERs) between the electronic properties of the flavin and the steady state kinetic parameters of the IDI-2 catalyzed reaction were observed. The LFER studies are complemented with kinetic isotope effect studies and kinetic characterization of an active site mutant enzyme (Q154N). Cumulatively, the data presented in this work (and in other studies) suggest that the reduced FMN coenzyme of IDI-2 functions as an acid/base catalyst, with the N5 atom of the flavin likely playing a critical role in the deprotonation of IPP en route to DMAPP formation. Several potential chemical mechanisms involving the reduced flavin as an acid/base catalyst are presented and discussed.
The E. coli isopentenyl diphosphate isomerase (IDI) catalyzed reaction of isopentenyl diphosphate (IPP) in D2O 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-mole of deuterium at C-2 (d-IPP). This shows that the release to D2O 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 kdis ≈ kcat = 0.08 s−1. The data provide an estimated rate constant of kas = 6 × 103 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. Biochemistry
1986, 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 Mg2+ to form the Michaelis complex where the metal cation bridges the protein and the substrate diphosphate group.
Isoprenoid compounds constitute an immensely diverse group of acyclic, monocyclic and polycyclic compounds that play important roles in all living organisms. Despite the diversity of their structures, this plethora of natural products arises from only two 5-carbon precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This review will discuss the enzymes in the mevalonate (MVA) and methylerythritol phosphate (MEP) biosynthetic pathways leading to IPP and DMAPP with a particular focus on MEP synthase (DXR) and IPP isomerase (IDI), which are potential targets for the development of antibiotic compounds. DXR is the second enzyme in the MEP pathway and the only one for which inhibitors with antimicrobial activity at pharmaceutically relevant concentrations are known. All of the published DXR inhibitors are fosmidomycin analogues, except for a few bisphosphonates with moderate inhibitory activity. These far, there are no other candidates that target DXR. IDI was first identified and characterised over 40 years ago (IDI-1) and a second convergently evolved isoform (IDI-2) was discovered in 2001. IDI-1 is a metalloprotein found in Eukarya and many species of Bacteria. Its mechanism has been extensively studied. In contrast, IDI-2 requires reduced flavin mononucleotide as a cofactor. The mechanism of action for IDI-2 is less well defined. This review will describe how lead inhibitors are being improved by structure-based drug design and enzymatic assays against DXR to lead to new drug families and how mechanistic probes are being used to address questions about the mechanisms of the isomerases.
DXR; IDI; isomerase; isopentenyl; isoprenoid; MEP; mevalonate; MVA; reductoisomerase
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 co-substrate 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 KM3-ClIPP (with DMAPP) and KM3-ClDMAPP (with IPP) were similar to those for IPP and DMAPP, however values of kcat 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.
Type II isopentenyl diphosphate (IPP) isomerase catalyzes the interconversion of IPP and dimethylallyl diphosphate (DMAPP). Although the reactions catalyzed by the type II enzyme and the well-studied type I IPP isomerase are identical, the type II protein requires reduced flavin for activity. The chemical mechanism, including the role of flavin, has not been established for type II IPP isomerase. Recombinant type II IPP isomerase from Thermus thermophilus HB27 was purified by Ni2+ affinity chromatography. Aerobically purified enzyme was inactive until the flavin cofactor was reduced by NADPH, dithionite, or photochemically. The inactive oxidized flavin-enzyme complex bound IPP in a Mg2+ dependent manner with KD ~ KmIPP, suggesting that the substrate binds to the inactive oxidized and active reduced forms of the protein with similar affinities. N,N-dimethyl-3-amino-1-propyl diphosphate (NIPP), a transition state analog for the type I isomerase, competitively inhibits the type II enzyme, but with much lower affinity. pH dependent spectral changes indicate that the binding of IPP, DMAPP, and a saturated analogue isopentyl diphosphate promotes protonation of anionic reduced flavin. Electron paramagnetic resonance (EPR) and UV-visible spectroscopy show a substrate-dependent accumulation of the neutral flavin semiquinone during both the flavoenzyme reduction and re-oxidation processes in the presence of IPP and related analogues. Redox potentials of IPP-bound enzyme indicate that the neutral semiquinone state of the flavin is stabilized thermodynamically relative to free FMN in solution.
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.
Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks required for biosynthesis of isoprenoid compounds. Two different isomerases have been reported. The type I enzyme, first characterized in the late 1950s, is widely distributed in eukaryota and eubacteria. The type II enzyme was recently discovered in Streptomyces sp. strain CL190. Open reading frame 48 (ORF48) in the archaeon Methanothermobacter thermautotrophicus encodes a putative type II IPP isomerase. A plasmid-encoded copy of the ORF complemented IPP isomerase activity in vivo in Salmonella enterica serovar Typhimurium strain RMC29, which contains chromosomal knockouts in the genes for type I IPP isomerase (idi) and 1-deoxy-d-xylulose 5-phosphate (dxs). The dxs gene was interrupted with a synthetic operon containing the Saccharomyces cerevisiae genes erg8, erg12, and erg19 allowing for the conversion of mevalonic acid to IPP by the mevalonate pathway. His6-tagged M. thermautotrophicus type II IPP isomerase was produced in Escherichia coli and purified by Ni2+ chromatography. The purified protein was characterized by matrix-assisted laser desorption ionization mass spectrometry. The enzyme has optimal activity at 70°C and pH 6.5. NADPH, flavin mononucleotide, and Mg2+ are required cofactors. The steady-state kinetic constants for the archaeal type II IPP isomerase from M. thermautotrophicus are as follows: Km, 64 μM; specific activity, 0.476 μmol mg−1 min−1; and kcat, 1.6 s−1.
The recently identified type II isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) isomerase (IDI-2) is a flavoenzyme that requires FMN and NAD(P)H for activity. IDI-2 is an essential enzyme for the biosynthesis of isoprenoids in several pathogenic bacteria including Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis, and thus is considered as a potential new drug target to battle bacterial infections. One notable feature of the IDI-2 reaction is that there is no net change in redox state between the substrate (IPP) and product (DMAPP), indicating that the FMN cofactor must start and finish each catalytic cycle in the same redox state. Here, we report the characterization and initial mechanistic studies of the S. aureus IDI-2. The steady-state kinetic analyses under aerobic and anaerobic conditions show that FMN must be reduced to be catalytically active and the overall IDI-2 reaction is O2 sensitive. Interestingly, our results demonstrate that NADPH is needed only in catalytic amounts to activate the enzyme for multiple turnovers of IPP to DMAPP. The hydride transfer from NAD(P)H to reduce FMN is determined to be pro-S stereospecific. Photoreduction and oxidation-reduction potential studies reveal that the S. aureus IDI-2 can stabilize significant amounts of the neutral FMN semiquinone. In addition, reconstitution of apo-IDI-2 with 5-deazaFMN resulted in a dead enzyme, whereas reconstitution with 1-deazaFMN led to the full recovery of enzyme activity. Taken together, these studies of S. aureus IDI-2 support a catalytic mechanism in which the reduced flavin coenzyme mediates a single electron transfer to and from the IPP substrate during catalysis.
Isoprenoid compounds are ubiquitous in nature, participating in important biological phenomena such as signal transduction, aerobic cellular respiration, photosynthesis, insect communication, and many others. They are derived from the 5-carbon isoprenoid substrates isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In Archaea and Eukarya, these building blocks are synthesized via the mevalonate pathway. However, the genes required to convert mevalonate phosphate (MP) to IPP are missing in several species of Archaea. An enzyme with isopentenyl phosphate kinase (IPK) activity was recently discovered in Methanocaldococcus jannaschii (MJ), suggesting a departure from the classical sequence of converting MP to IPP. We have determined the high-resolution crystal structures of isopentenyl phosphate kinases in complex with both substrates and products from Thermoplasma acidophilum (THA), as well as the IPK from Methanothermobacter thermautotrophicus (MTH), by means of single-wavelength anomalous diffraction (SAD) and molecular replacement. A histidine residue (His50) in THA IPK makes a hydrogen bond with the terminal phosphates of IP and IPP, poising these molecules for phosphoryl transfer through an in-line geometry. Moreover, a lysine residue (Lys14) makes hydrogen bonds with non-bridging oxygen atoms at Pα and Pγ and with the Pβ- Pγ bridging oxygen atom in ATP. These interactions suggest a transition state-stabilizing role for this residue. Lys14 is a part of a newly discovered “lysine triangle” catalytic motif in IPK’s that also includes Lys5 and Lys205. Moreover, His50, Lys5, Lys14, and Lys205 are conserved in all IPK’s and can therefore serve as fingerprints for identifying new homologues.
It is proposed that the lytB gene encodes an enzyme of the deoxyxylulose-5-phosphate (DOXP) pathway that catalyzes a step at or subsequent to the point at which the pathway branches to form isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A mutant of the cyanobacterium Synechocystis strain PCC 6803 with an insertion in the promoter region of lytB grew slowly and produced greenish-yellow, easily bleached colonies. Insertions in the coding region of lytB were lethal. Supplementation of the culture medium with the alcohol analogues of IPP and DMAPP (3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol) completely alleviated the growth impairment of the mutant. The Synechocystis lytB gene and a lytB cDNA from the flowering plant Adonis aestivalis were each found to significantly enhance accumulation of carotenoids in Escherichia coli engineered to produce these colored isoprenoid compounds. When combined with a cDNA encoding deoxyxylulose-5-phosphate synthase (dxs), the initial enzyme of the DOXP pathway, the individual salutary effects of lytB and dxs were multiplied. In contrast, the combination of lytB and a cDNA encoding IPP isomerase (ipi) was no more effective in enhancing carotenoid accumulation than ipi alone, indicating that the ratio of IPP and DMAPP produced via the DOXP pathway is influenced by LytB.
Isoprenoids are a diverse group of molecules found in all organisms, where they perform such important biological functions as hormone signaling (e.g., steroids) in mammals, antioxidation (e.g., carotenoids) in plants, electron transport (e.g., ubiquinone), and cell wall biosynthesis intermediates in bacteria. All isoprenoids are synthesized by the consecutive condensation of the five-carbon monomer isopentenyl diphosphate (IPP) to its isomer, dimethylallyl diphosphate (DMAPP). The biosynthetic pathway for the formation of IPP from acetyl-CoA (i.e., the mevalonate pathway) had been established mainly in mice and the budding yeast Saccharomyces cerevisiae. Curiously, most prokaryotic microorganisms lack homologs of the genes in the mevalonate pathway, even though IPP and DMAPP are essential for isoprenoid biosynthesis in bacteria. This observation provided an impetus to search for an alternative pathway to synthesize IPP and DMAPP, ultimately leading to the discovery of the mevalonate-independent 2-C-methyl-d-erythritol 4-phosphate pathway. This review article focuses on our significant contributions to a comprehensive understanding of the biosynthesis of IPP and DMAPP.
biosynthesis; inhibitor; isoprenoid; MEP pathway; mevalonate pathway; terpenoid
The role of peroxisomes in isoprenoid metabolism, especially in plants, has been questioned in several reports. A recent study of Sapir-Mir et al.1 revealed that the two isoforms of isopentenyl diphosphate (IPP) isomerase, catalyzing the isomerisation of IPP to dimethylallyl diphosphate (DMAPP) are found in the peroxisome. In this addendum, we provide additional data describing the peroxisomal localization of 5-phosphomevalonate kinase and mevalonate 5-diphosphate decarboxylase, the last two enzymes of the mevalonic acid pathway leading to IPP.2 This finding was reinforced in our latest report showing that a short isoform of farnesyl diphosphate, using IPP and DMAPP as substrates, is also targeted to the organelle.3 Therefore, the classical sequestration of isoprenoid biosynthesis between plastids and cytosol/ER can be revisited by including the peroxisome as an additional isoprenoid biosynthetic compartment within plant cells.
5-phosphomevalonate kinase; Arabidopsis thaliana; Catharanthus roseus; farnesyl diphosphate synthase; isoprenoid; mevalonate 5-diphosphate decarboxylase; mevalonic acid pathway; peroxisome
Archaea synthesize isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the essential building blocks of isoprenoid compounds, from mevalonate (MVA). However, an analysis of the genomes of several members of the Archaea failed to identify genes for the enzymes required to convert phosphomevalonate (PM) to IPP in Eukaryotes. The recent discovery of an isopentenyl kinase (IPK) in Methanocaldococcus jannaschii (MJ) suggests a new variation of the MVA pathway where PM is decarboxylated to give isopentenyl phosphate (IP), which is phosphorylated to produce IPP. A blast search using the MJ protein as a probe revealed a subfamily of amino acid kinases that include the fosfomycin resistance protein fomA, which deactivates the antibiotic by phosphorylation of its phosphonate residue in a reaction similar to the conversion of IP to IPP. IPK genes were cloned from two organisms identified in the search, Methanothermobacter thermautotrophicus (MTH) and Thermoplasma acidophilum (THA), and the His-tagged recombinant proteins were purified by Ni-NTA chromatography. The enzymes catalyze the reversible phosphorylation of IP by ATP, Keq = 6.3 ± 1. The catalytic efficiencies (V/K) of the proteins were ~2 × 106 M−1s−1. In the reverse direction, ADP was a substrate inhibitor for THA IPK, KiADP = 58 ± 6 µM but not for MTH IPK. Both enzymes were active over a broad range of pH and temperature. Five compounds, dimethylallyl phosphate, isopentenyl thiolophosphate, 1-butyl phosphate, 3-buten-1-yl phosphate, and geranyl phosphate, were evaluated as alternative substrate for the MTH and THA IP kinases. All of the compounds were phosphorylated, although the catalytic efficiency was low for geranyl phosphate.
Type-2 isopentenyl diphosphate isomerase, which catalyzes the interconversion if isopentenyl diphosphate and dimethylallyl diphosphate, contains a tightly bound molecule of FMN. Incubation of the active enzyme•FMNH2 complex with an analog of isopentenyl diphosphate, where the methyl group has been replaced with a cyclopropane ring, results in isomerization of the analog to the corresponding allylic isomer without inactivation of the enzyme. In contrast, the related epoxide analog is a potent irreversible inhibitor that covalently modifies the flavin cofactor in a proton-initiated reaction. These results suggest that the mechanism for isomerization by the type-2 isopentenyl diphosphate isomerase may be similar to the protonation-deprotonation sequence of the type-1 enzyme and places limits on the lifetimes of radical intermediates in an alternative hydrogen atom addition/abstraction mechanism.
Isopentenyl diphosphate isomerase (IPPI) is an enzyme involved in the synthesis of juvenile hormone (JH) in the corpora allata (CA) of insects. IPPI catalyzes the conversion of isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP); afterwards IPP and DMAPP condense in a head-to-tail manner to produce geranyl diphosphate (GPP), this head-to-tail condensation can be repeated, by the further reaction of GPP with IPP, yielding the JH precursor farnesyl diphosphate. An IPPI expressed sequence tag (EST) was obtained from an Aedes aegypti corpora-allata + corpora cardiaca library. Its full-length cDNA encodes a 244-aa protein that shows a high degree of similarity with type I IPPIs from other organisms, particularly for those residues that have important roles in catalysis, metal coordination and interaction with the diphosphate moiety of the IPP. Heterologous expression produced a recombinant protein that metabolized IPP into DMAPP; treatment of DMAPP with phosphoric acid produced isoprene, a volatile compound that was measured with an assay based on a solid-phase micro extraction protocol and direct analysis by gas chromatography. A. aegypti IPPI (AaIPPI) required Mg2+ or Mn2+ but not Zn2+ for full activity and it was entirely inhibited by iodoacetamide. Real time PCR experiments showed that AaIPPI is highly expressed in the CA. Changes in AaIPPI mRNA levels in the CA in the pupal and adult female mosquito corresponded well with changes in JH synthesis (Li et al., 2003). This is the first molecular and functional characterization of an isopentenyl diphosphate isomerase involved in the production of juvenile hormone in the CA of an insect.
Mosquito; IPP isomerase; juvenile hormone; corpora allata; Aedes
The photosynthetic cyanobacterium Synechocystis sp. strain PCC6803 possesses homologs of known genes of the non-mevalonate 2-C-methyl-d-erythritol 2-phosphate (MEP) pathway for synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Isoprenoid biosynthesis in extracts of this cyanobacterium, measured by incorporation of radiolabeled IPP, was not stimulated by pyruvate, an initial substrate of the MEP pathway in Escherichia coli, or by deoxyxylulose-5-phosphate, the first pathway intermediate in E. coli. However, high rates of IPP incorporation were obtained with addition of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GA3P), as well as a variety of pentose phosphate cycle compounds. Fosmidomycin (at 1 μM and 1 mM), an inhibitor of deoxyxylulose-5-phosphate reductoisomerase, did not significantly inhibit phototrophic growth of the cyanobacterium, nor did it affect [14C]IPP incorporation stimulated by DHAP plus GA3P. To date, it has not been possible to unequivocally demonstrate IPP isomerase activity in this cyanobacterium. The combined results suggest that the MEP pathway, as described for E. coli, is not the primary path by which isoprenoids are synthesized under photosynthetic conditions in Synechocystis sp. strain PCC6803. Our data support alternative routes of entry of pentose phosphate cycle substrates derived from photosynthesis.
Isoprenoids are one of the largest classes of natural products and all of them are constructed from two precursors, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). For decades, the mevalonic acid (MVA) pathway was proposed to be the only IPP and DMAPP biosynthetic pathway. This review summarizes the newly discovered IPP and DMAPP production pathways since late 1990s, their distribution among different kingdoms, and their roles in secondary metabolite production. These new IPP and DMAPP production pathways include the methylerythritol phosphate (MEP) pathway, a modified MVA pathway, and the 5-Methylthioadenosine shunt pathway. Relative to the studies on the MVA pathway, information on the MEP pathway regulation is limited and the mechanistic details of several of its novel transformations remain to be addressed. Current status on both MEP pathway regulation and mechanistic issues are also presented.
isoprenoids; MVA; MEP; methylthioadenosine; regulation; biosynthesis
Isoprenoid biosynthesis is essential for survival of all living organisms. More than 50,000 unique isoprenoids occur naturally, with each constructed from two simple five-carbon precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Two pathways for the biosynthesis of IPP and DMAPP are found in nature. Humans exclusively use the mevalonate (MVA) pathway, while most bacteria, including all Gram-negative and many Gram-positive species, use the unrelated methylerythritol phosphate (MEP) pathway. Here we report the development of a novel, whole-cell phenotypic screening platform to identify compounds that selectively inhibit the MEP pathway. Strains of Salmonella enterica serovar Typhimurium were engineered to have separately inducible MEP (native) and MVA (nonnative) pathways. These strains, RMC26 and CT31-7d, were then used to differentiate MVA pathway- and MEP pathway-specific perturbation. Compounds that inhibit MEP pathway-dependent bacterial growth but leave MVA-dependent growth unaffected represent MEP pathway-selective antibacterials. This screening platform offers three significant results. First, the compound is antibacterial and is therefore cell permeant, enabling access to the intracellular target. Second, the compound inhibits one or more MEP pathway enzymes. Third, the MVA pathway is unaffected, suggesting selectivity for targeting the bacterial versus host pathway. The cell lines also display increased sensitivity to two reported MEP pathway-specific inhibitors, further biasing the platform toward inhibitors selective for the MEP pathway. We demonstrate development of a robust, high-throughput screening platform that combines phenotypic and target-based screening that can identify MEP pathway-selective antibacterials simply by monitoring optical density as the readout for cell growth/inhibition.
The MEP pathway, which is absent in animals but present in most pathogenic bacteria, in the parasite responsible for malaria and in plant plastids, is a target for the development of antimicrobial drugs. IspH, an oxygen-sensitive [4Fe-4S] enzyme, catalyzes the last step of this pathway and converts (E)-4-hydroxy-2-methylbut-2-enyl 1-diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A crucial step in the mechanism of this enzyme is the binding of the C4 hydroxyl of HMBPP to the unique fourth iron site in the [4Fe-4S]2+ moiety. Here we report the synthesis and the kinetic investigations of two new extremely potent inhibitors of E. coli IspH where the OH group of HMBPP is replaced by an amino and a thiol group. (E)-4-Mercapto-3-methyl-but-2-en-1-yl diphosphate is a reversible tight-binding inhibitor of IspH with Ki = 20 ± 2 nM. A detailed kinetic analysis revealed that (E)-4-amino-3-methylbut-2-en-1-yl diphosphate is a reversible slow-binding inhibitor of IspH with Ki = 54 ± 19 nM. The slow binding behavior of this inhibitor is best described by a one-step mechanism with the slow step consisting in the formation of the enzyme-inhibitor (EI) complex.
Isopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. A database search based on probes from the highly conserved regions in three eukaryotic IPP isomerases revealed substantial similarity with ORF176 in the photosynthesis gene cluster in Rhodobacter capsulatus. The open reading frame was cloned into an Escherichia coli expression vector. The encoded 20-kDa protein, which was purified in two steps by ion exchange and hydrophobic interaction chromatography, catalyzed the interconversion of IPP and dimethylallyl diphosphate. Thus, the photosynthesis gene cluster encodes all of the enzymes required to incorporate IPP into the ultimate carotenoid and bacteriochlorophyll metabolites in R. capsulatus. More recent searches uncovered additional putative open reading frames for IPP isomerase in seed-bearing plants (Oryza sativa, Arabadopsis thaliana, and Clarkia breweri), a worm (Caenorhabiditis elegans), and another eubacterium (Escherichia coli). The R. capsulatus enzyme is the smallest of the IPP isomerases to be identified thus far and may consist mostly of a fundamental catalytic core for the enzyme.
In vitro synthesis of chemicals and pharmaceuticals using enzymes is of considerable interest as these biocatalysts facilitate a wide variety of reactions under mild conditions with excellent regio-, chemo- and stereoselectivities. A significant challenge in a multi-enzymatic reaction is the need to optimize the various steps involved simultaneously so as to obtain high-yield of a product. In this study, statistical experimental design was used to guide the optimization of a total synthesis of amorpha-4,11-diene (AD) using multienzymes in the mevalonate pathway. A combinatorial approach guided by Taguchi orthogonal array design identified the local optimum enzymatic activity ratio for Erg12:Erg8:Erg19:Idi:IspA to be 100∶100∶1∶25∶5, with a constant concentration of amorpha-4,11-diene synthase (Ads, 100 mg/L). The model also identified an unexpected inhibitory effect of farnesyl pyrophosphate synthase (IspA), where the activity was negatively correlated with AD yield. This was due to the precipitation of farnesyl pyrophosphate (FPP), the product of IspA. Response surface methodology was then used to optimize IspA and Ads activities simultaneously so as to minimize the accumulation of FPP and the result showed that Ads to be a critical factor. By increasing the concentration of Ads, a complete conversion (∼100%) of mevalonic acid (MVA) to AD was achieved. Monovalent ions and pH were effective means of enhancing the specific Ads activity and specific AD yield significantly. The results from this study represent the first in vitro reconstitution of the mevalonate pathway for the production of an isoprenoid and the approaches developed herein may be used to produce other isopentenyl pyrophosphate (IPP)/dimethylallyl pyrophosphate (DMAPP) based products.