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1.  Two distinct pathways for essential metabolic precursors for isoprenoid biosynthesis 
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.
doi:10.2183/pjab.88.41
PMCID: PMC3365244  PMID: 22450534
biosynthesis; inhibitor; isoprenoid; MEP pathway; mevalonate pathway; terpenoid
2.  Inhibition Studies on Enzymes Involved in Isoprenoid Biosynthesis: Focus on Two Potential Drug Targets: DXR and IDI-2 Enzymes 
Current enzyme inhibition  2011;7(2):10.2174/157340811796575317.
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.
doi:10.2174/157340811796575317
PMCID: PMC3856697  PMID: 24339799
DXR; IDI; isomerase; isopentenyl; isoprenoid; MEP; mevalonate; MVA; reductoisomerase
3.  Isoprenoid Biosynthesis in Synechocystis sp. Strain PCC6803 Is Stimulated by Compounds of the Pentose Phosphate Cycle but Not by Pyruvate or Deoxyxylulose-5-Phosphate 
Journal of Bacteriology  2002;184(18):5045-5051.
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.
doi:10.1128/JB.184.18.5045-5051.2002
PMCID: PMC135332  PMID: 12193620
4.  Escherichia coli Open Reading Frame 696 Is idi, a Nonessential Gene Encoding Isopentenyl Diphosphate Isomerase 
Journal of Bacteriology  1999;181(15):4499-4504.
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.
PMCID: PMC103578  PMID: 10419945
5.  Isopentenyl Diphosphate Isomerase. Mechanism-Based Inhibition by Diene Analogues of Isopentenyl Diphosphate and Dimethylallyl Diphosphate 
Journal of the American Chemical Society  2005;127(49):17433-17438.
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 analogs 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 E. 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.
doi:10.1021/ja056187h
PMCID: PMC2528281  PMID: 16332094
6.  Metabolic engineering of Escherichia coli for high-specificity production of isoprenol and prenol as next generation of biofuels 
Background
The isopentenols, including isoprenol and prenol, are excellent alternative fuels. However, they are not compounds largely accumulated in natural organism. The need for the next generation of biofuels with better physical and chemical properties impels us to develop biosynthetic routes for the production of isoprenol and prenol from renewable sugar. In this study, we use the heterogenous mevalonate-dependent (MVA) isoprenoid pathway for the synthesis of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) intermediates, and then convert IPP and DMAPP to isoprenol and prenol, respectively.
Results
A mevalonate titer of 1.7 g/L was obtained by constructing an efficient MVA upper pathway in engineered E. coli. Different phosphatases and pyrophosphatases were investigated for their abilities in hydrolyzing the IPP and DMAPP. Consequently, ADP-ribose pyrophosphatase was found to be an efficient IPP and DMAPP hydrolase. Moreover, ADP-ribose pyrophosphatase from Bacillus subtilis (BsNudF) exhibited a equivalent substrate specificity towards IPP and DMAPP, while ADP-ribose pyrophosphatase from E. coli (EcNudF) presented a high substrate preference for DMAPP. Without the expression of any phosphatases or pyrophosphatases, a background level of isopentenols was synthesized. When the endogenous pyrophosphatase genes (EcNudF and yggV) that were capable of enhancing the hydrolyzation of the IPP and DMAPP were knocked out, the background level of isopentenols was still obtained. Maybe the synthesized IPP and DMAPP were hydrolyzed by some unknown hydrolases of E. coli. Finally, 1.3 g/L single isoprenol was obtained by blocking the conversion of IPP to DMAPP and employing the BsNudF, and 0.2 g/L ~80% prenol was produced by employing the EcNudF. A maximal yield of 12% was achieved in both isoprenol and prenol producing strains.
Conclusions
To the best of our knowledge, this is the first successful report on high-specificity production of isoprenol and prenol by microbial fermentation. Over 1.3 g/L isoprenol achieved in shake-flask experiments represents a quite encouraging titer of higher alcohols. In addition, the substrate specificities of ADP-ribose pyrophosphatases were determined and successfully applied for the high-specificity synthesis of isoprenol and prenol. Altogether, this work presents a promising strategy for high-specificity production of two excellent biofuels, isoprenol and prenol.
doi:10.1186/1754-6834-6-57
PMCID: PMC3654967  PMID: 23618128
Isoprenol; Prenol; Metabolic engineering; Escherichia coli; Biofuel
7.  Current Development in Isoprenoid Precursor Biosynthesis and Regulation 
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.
doi:10.1016/j.cbpa.2013.06.020
PMCID: PMC4068245  PMID: 23891475
isoprenoids; MVA; MEP; methylthioadenosine; regulation; biosynthesis
8.  Characterization of Thermophilic Archaeal Isopentenyl Phosphate Kinases 
Biochemistry  2010;49(1):10.1021/bi9017957.
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.
doi:10.1021/bi9017957
PMCID: PMC3856865  PMID: 19928876
9.  Identification of an Archaeal Type II Isopentenyl Diphosphate Isomerase in Methanothermobacter thermautotrophicus 
Journal of Bacteriology  2004;186(6):1811-1817.
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.
doi:10.1128/JB.186.6.1811-1817.2004
PMCID: PMC355898  PMID: 14996812
10.  Characterization and Mechanistic Studies of Type II Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase from Staphylococcus aureus 
Biochemistry  2007;46(28):8401-8413.
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.
doi:10.1021/bi700286a
PMCID: PMC2515275  PMID: 17585782
11.  Evidence of a Role for LytB in the Nonmevalonate Pathway of Isoprenoid Biosynthesis 
Journal of Bacteriology  2000;182(20):5841-5848.
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.
PMCID: PMC94708  PMID: 11004185
12.  Discovery of a metabolic alternative to the classical mevalonate pathway 
eLife  2013;2:e00672.
Eukarya, Archaea, and some Bacteria encode all or part of the essential mevalonate (MVA) metabolic pathway clinically modulated using statins. Curiously, two components of the MVA pathway are often absent from archaeal genomes. The search for these missing elements led to the discovery of isopentenyl phosphate kinase (IPK), one of two activities necessary to furnish the universal five-carbon isoprenoid building block, isopentenyl diphosphate (IPP). Unexpectedly, we now report functional IPKs also exist in Bacteria and Eukarya. Furthermore, amongst a subset of species within the bacterial phylum Chloroflexi, we identified a new enzyme catalyzing the missing decarboxylative step of the putative alternative MVA pathway. These results demonstrate, for the first time, a functioning alternative MVA pathway. Key to this pathway is the catalytic actions of a newly uncovered enzyme, mevalonate phosphate decarboxylase (MPD) and IPK. Together, these two discoveries suggest that unforeseen variation in isoprenoid metabolism may be widespread in nature.
DOI: http://dx.doi.org/10.7554/eLife.00672.001
eLife digest
Living things make thousands of chemicals that are vital for life, and are also useful as medicines, perfumes, and food additives. The largest family of these natural chemicals is called the isoprenoids, and members of this family are found in all three domains of life: the eukaryotes (such as plants and animals), the Archaea (an ancient group of single-celled microbes), and bacteria.
The isoprenoids are made from a smaller building block called isopentenyl diphosphate, IPP for short, that contains five carbon atoms and two phosphate groups. IPP can be produced in two ways. The classical mevalonate pathway is found in most eukaryotes, including humans; statin drugs are used to inhibit this pathway to treat those with high cholesterol and reduce the risk of heart disease. The second pathway does not use the compound mevalonate and is found in many, but not all, bacteria as well as the chloroplasts of plants. Until recently, however, the enzymes needed for the last two steps of the classical mevalonate pathway appeared to be missing in the Archaea and in some bacteria.
Researchers subsequently discovered that an enzyme called isopentenyl phosphate kinase, shortened to IPK, was responsible for one of these two missing steps—the addition of IPP’s second phosphate group. The way this enzyme worked also suggested that there was an alternative mevalonate pathway in which the order of the last two steps was reversed. However, the identity of the enzyme responsible for the other step—the removal of a molecule of carbon dioxide to make the starting material needed by IPK—remained mysterious.
Now Dellas et al. have discovered the enzyme responsible for this missing step in Green non-sulphur bacteria, confirming the existence of the alternative mevalonate pathway for the first time. Previously it had been thought that this enzyme acted in the classical mevalonate pathway; but in fact this enzyme has evolved a new function and is not involved in the classical pathway at all. Moreover, Dellas et al. show that Green non-sulphur bacteria, and some eukaryotes, also have functional IPK enzymes. This means that IPK has now unexpectedly been observed in all three domains of life, and hints at another target to medically control mevalonate pathways. The discovery of the missing enzyme in the alternative pathway opens the door to the re-examination of many other living things, to find which have the new pathway and to work out why.
DOI: http://dx.doi.org/10.7554/eLife.00672.002
doi:10.7554/eLife.00672
PMCID: PMC3857490  PMID: 24327557
Mevalonate pathway; Isopentenyl diphosphate; Archaea; Mevalonate phosphate decarboxylase; Chloroflexi; Plants; Arabidopsis; Other
13.  A Whole-Cell Phenotypic Screening Platform for Identifying Methylerythritol Phosphate Pathway-Selective Inhibitors as Novel Antibacterial Agents 
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.
doi:10.1128/AAC.00987-12
PMCID: PMC3421842  PMID: 22777049
14.  Synthesis and Evaluation of Chlorinated Substrate Analogues for Farnesyl Diphosphate Synthase 
The Journal of organic chemistry  2011;76(6):1838-1843.
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.
doi:10.1021/jo1024305
PMCID: PMC3055917  PMID: 21344952
15.  Triple subcellular targeting of isopentenyl diphosphate isomerases encoded by a single gene 
Plant Signaling & Behavior  2012;7(11):1495-1497.
Isopentenyl diphosphate isomerase (IDI) is a key enzyme of the isoprenoid pathway, catalyzing the interconversion of isopentenyl diphosphate and dimethylallyl diphosphate, the universal precursors of all isoprenoids. In plants, several subcellular compartments, including cytosol/ER, peroxisomes, mitochondria and plastids, are involved in isoprenoid biosynthesis. Here, we report on the unique triple targeting of two Catharanthus roseus IDI isoforms encoded by a single gene (CrIDI1). The triple localization of CrIDI1 in mitochondria, plastids and peroxisomes is explained by alternative transcription initiation of CrIDI1, by the specificity of a bifunctional N-terminal mitochondria/plastid transit peptide and by the presence of a C-terminal peroxisomal targeting signal. Moreover, bimolecular fluorescence complementation assays revealed self-interactions suggesting that the IDI likely acts as a multimer in vivo.
doi:10.4161/psb.21892
PMCID: PMC3548878  PMID: 22951398
isopentenyl diphosphate isomerase; isoprenoid; alkaloid; triple targeting; subcellular localization; Catharanthus roseus
16.  Characterization of an Isopentenyl Diphosphate Isomerase involved in the Juvenile Hormone pathway in Aedes aegypti 
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.
doi:10.1016/j.ibmb.2012.07.002
PMCID: PMC3438293  PMID: 22782071
Mosquito; IPP isomerase; juvenile hormone; corpora allata; Aedes
17.  X-ray structures of isopentenyl phosphate kinase 
ACS chemical biology  2010;5(5):517-527.
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.
doi:10.1021/cb100032g
PMCID: PMC2879073  PMID: 20402538
18.  Type II Isopentenyl Diphosphate Isomerase: Probing the Mechanism with Alkyne/Allene Diphosphate Substrate Analogues† 
Biochemistry  2010;49(29):6228-6233.
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.
doi:10.1021/bi100844e
PMCID: PMC2912430  PMID: 20560533
19.  Probing phosphorylation by non-mammalian isoprenoid biosynthetic enzymes using 1H–31P–31P correlation NMR spectroscopy†‡ 
Molecular bioSystems  2009;5(9):935-944.
The biogenesis of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) is accomplished by the methylerythritol phosphate (MEP) pathway in plants, bacteria and parasites, making it a potential target for the development of anti-infective agents and herbicides. The biosynthetic enzymes comprising this pathway catalyze intriguing chemical transformations on diphosphate scaffolds, offering an opportunity to generate novel analogs in this synthetically challenging compound class. Such a biosynthetic approach to generating new diphosphate analogs may involve transformation through discrete diphosphate species, presenting unique challenges in structure determination and characterization of unnatural enzyme-generated diphosphate products produced in tandem. We have developed 1H–31P–31P correlation NMR spectroscopy techniques for the direct characterization of crude MEP pathway enzyme products at low concentrations (200 μM to 5 mM) on a room temperature (non-cryogenic) NMR probe. Coupling the 100% natural abundance of the 31P nucleus with the high intrinsic sensitivity of proton NMR, 1H–31P–31P correlation spectroscopy is particularly useful for characterization of unnatural diphosphate enzyme products in the MEP pathway. As proof of principle, we demonstrate the rapid characterization of natural enzyme products of the enzymes IspD, E and F in tandem enzyme incubations. In addition, we have characterized several unnatural enzyme products using this technique, including new products of cytidyltransferase IspD bearing erythritol, glycerol and ribose components. The results of this study indicate that IspD may be a useful biocatalyst and highlight 1H–31P–31P correlation spectroscopy as a valuable tool for the characterization of other unnatural products in non-mammalian isoprenoid biosynthesis.
doi:10.1039/b903513c
PMCID: PMC3161243  PMID: 19668858
20.  Linear Free Energy Relationships Demonstrate a Catalytic Role for the Flavin Mononucleotide Coenzyme of the Type II Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase 
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.
doi:10.1021/ja104090m
PMCID: PMC2918422  PMID: 20593767
21.  Open reading frame 176 in the photosynthesis gene cluster of Rhodobacter capsulatus encodes idi, a gene for isopentenyl diphosphate isomerase. 
Journal of Bacteriology  1996;178(3):619-624.
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.
PMCID: PMC177703  PMID: 8550491
22.  Molecular characterization of farnesyl pyrophosphate synthase from Bacopa monniera by comparative modeling and docking studies 
Bioinformation  2012;8(22):1075-1081.
Farnesyl pyrophosphate synthase (FPS; EC 2.5.1.10) is a key enzyme in isoprenoid biosynthetic pathway and provides precursors for the biosynthesis of various pharmaceutically important metabolites. It catalyzes head to tail condensation of two isopentenyl pyrophosphate molecules with dimethylallyl pyrophosphate to form C15 compound farnesyl pyrophosphate. Recent studies have confirmed FPS as a molecular target of bisphosphonates for drug development against bone diseases as well as pathogens. Although large numbers of FPSs from different sources are known, very few protein structures have been reported till date. In the present study, FPS gene from medicinal plant Bacopa monniera (BmFPS) was characterized by comparative modeling and docking. Multiple sequence alignment showed two highly conserved aspartate rich motifs FARM and SARM (DDXXD). The 3-D model of BmFPS was generated based on structurally resolved FPS crystal information of Gallus gallus. The generated models were validated by various bioinformatics tools and the final model contained only α-helices and coils. Further, docking studies of modeled BmFPS with substrates and inhibitors were performed to understand the protein ligand interactions. The two Asp residues from FARM (Asp100 and Asp104) as well as Asp171, Lys197 and Lys262 were found to be important for catalytic activity. Interaction of nitrogen containing bisphosphonates (risedronate, alendronate, zoledronate and pamidronate) with modeled BmFPS showed competitive inhibition; where, apart from Asp (100, 104 and 171), Thr175 played an important role. The results presented here could be useful for designing of mutants for isoprenoid biosynthetic pathway engineering well as more effective drugs against osteoporosis and human pathogens.
Abbreviations
IPP - Isopentenyl Pyrophosphate, DMAPP - Dimethylallyl Pyrophosphate, GPP - Geranyl Pyrophosphate, FPP - FPPFarnesyl Pyrophosphate, DOPE - Discrete Optimized Protein Energy, BmFPS - Bacopa monniera Farnesyl Pyrophosphate Synthase, RMSD - Root Mean square Deviation, OPLS-AA - Optimized Potentials for Liquid Simulations- All Atom, FARM - First Aspartate Rich Motif, SARM - Second Aspartate Rich Motif.
doi:10.6026/97320630081075
PMCID: PMC3523221  PMID: 23251041
Bacopa monniera; Bisphosphonates; Comparative modeling and docking; Farnesyl pyrophosphate synthase
23.  Inactivation of sll1556 in Synechocystis Strain PCC 6803 Impairs Isoprenoid Biosynthesis from Pentose Phosphate Cycle Substrates In Vitro 
Journal of Bacteriology  2004;186(14):4685-4693.
In cyanobacteria many compounds, including chlorophylls, carotenoids, and hopanoids, are synthesized from the isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate. Isoprenoid biosynthesis in extracts of the cyanobacterium Synechocystis strain PCC 6803 grown under photosynthetic conditions, stimulated by pentose phosphate cycle substrates, does not appear to require methylerythritol phosphate pathway intermediates. The sll1556 gene, distantly related to type 2 IPP isomerase genes, was disrupted by insertion of a Kanr cassette. The mutant was fully viable under photosynthetic conditions although impaired in the utilization of pentose phosphate cycle substrates. Compared to the parental strain the Δsll1556 mutant (i) is deficient in isoprenoid biosynthesis in vitro with substrates including glyceraldehyde-3-phosphate, fructose-6-phosphate, and glucose-6-phosphate; (ii) has smaller cells (diameter ca. 13% less); (iii) has fewer thylakoids (ca. 30% less); and (iv) has a more extensive fibrous outer wall layer. Isoprenoid biosynthesis is restored with pentose phosphate cycle substrates plus the recombinant Sll1556 protein in the Δsll1556 supernatant fraction. IPP isomerase activity could not be demonstrated for the purified Sll1556 protein under our in vitro conditions. The reduction of thylakoid area and the effect on outer wall layer components are consistent with an impairment of isoprenoid biosynthesis in the mutant, possibly via hopanoid biosynthesis. Our findings are consistent with an alternate metabolic shunt for biosynthesis of isoprenoids.
doi:10.1128/JB.186.14.4685-4693.2004
PMCID: PMC438581  PMID: 15231801
24.  Kinetic and Spectroscopic Characterization of Type II Isopentenyl Diphosphate Isomerase from Thermus thermophilus: Evidence for Formation of Substrate Induced Flavin Species† 
Biochemistry  2007;46(18):5437-5445.
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.
doi:10.1021/bi0616347
PMCID: PMC2516918  PMID: 17428035
25.  Indirect Stimulation of Human Vγ2Vδ2 T cells Through Alterations in Isoprenoid Metabolism1 
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/ApppI 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 antigen 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 antigens. Transfection of APC with siRNA downregulating FDPS rendered them stimulatory for Vγ2Vδ2 cells, and increased cellular IPP. siRNAs 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.
doi:10.4049/jimmunol.1002697
PMCID: PMC3326638  PMID: 22013129
gamma delta T cell; Vgamma2Vdelta2 T cells; human; bisphosphonate; antigen presentation; prenyl pyrophosphates; isopentenyl pyrophosphate; isoprenoid metabolism; farnesyl diphosphate synthase; siRNA

Results 1-25 (107295)