Many pathogenic bacteria utilize the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate, two major building blocks of isoprenoid compounds. The fifth enzyme in the MEP pathway, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (ME-CPP) synthase (IspF), catalyzes the conversion of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-ME2P) to ME-CPP with a corresponding release of cytidine 5-monophosphate (CMP). Since there is no ortholog of IspF in human cells IspF is of interest as a potential drug target. However, study of IspF has been hindered by a lack of enantiopure CDP-ME2P. Herein, we report the first synthesis of enantiomerically pure CDP-ME2P from commercially available D-arabinose. Cloned, expressed, and purified M. tuberculosis IspF was able to utilize the synthetic CDP-ME2P as a substrate, a result confirmed by mass spectrometry. A convenient, sensitive, in vitro IspF assay was developed by coupling the CMP released during production of ME-CPP to mononucleotide kinase, which can be used for high throughput screening.
The conversion of 2C-methyl-d-erythritol 4-phosphate (MEP) to 2C-methyl-d-erythritol 2,4-cyclodiphosphate (cMEDP) in the MEP entry into the isoprenoid biosynthetic pathway occurs in three consecutive steps catalyzed by the IspD, IspE, and IspF enzymes, respectively. In Agrobacterium tumefaciens the ispD and ispF genes are fused to encode a bifunctional enzyme that catalyzes the first (synthesis of 4-diphosphocytidyl-2-C-methyl d-erythritol) and third (synthesis of 2C-methyl-d-erythritol 2,4-cyclodiphosphate) steps. Sedimentation velocity experiments indicate that the bifunctional IspDF enzyme and the IspE protein associate in solution raising the possibility of substrate channeling among the active sites in these two proteins. Kinetic evidence for substrate channeling was sought by measuring the time courses for product formation during incubations of MEP, CTP, and ATP with the IspDF and IspE proteins with and without an excess of the inactive IspE (D152A) mutant in presence or absence of 30% (v/v) glycerol. The time dependencies indicate that the enzyme-generated intermediates are not transferred from the IspD active site in IspDF to the active site of IspE or from the active site in IspE to the active site in the IspF module of IspDF.
bifunctional; IspDF; IspE; non-channeling
Many bacterial pathogens utilize the 2-C-methyl-D-erythritol 4-phosphate pathway for biosynthesizing isoprenoid precursors, a pathway that is vital for bacterial survival and absent from human cells, providing a potential source of drug targets. However, the characterization of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase (IspE) has been hindered due to a lack of enantiopure CDP-ME and difficulty in obtaining pure IspE. Here, enantiopure CDP-ME was chemically synthesized and recombinant IspE from bacterial pathogens were purified and characterized. Although gene disruption was not possible in Mycobacterium tuberculosis, IspE is essential in Mycobacterium smegmatis. The biochemical and kinetic characteristics of IspE provide the basis for development of a high throughput screen and structural characterization.
The 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (IspE) from M. tuberculosis H37Rv was overexpressed in E. coli, purified and crystallized. Diffraction data for the native enzyme were collected to 2.1 Å resolution.
The 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (IspE) from Mycobacterium tuberculosis, an enzyme from the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, is crucial and essential for the survival of this pathogenic bacterium. IspE catalyzes the conversion of 4-diphosphocytidyl-2-C-methyl-d-erythritol (CDP-ME) to 4-diphosphocytidyl-2-C-methyl-d-erythritol 2-phosphate (CDP-ME2P) in an ATP-dependent manner. Solving the crystal structure of M. tuberculosis IspE will shed light on its structural details and mechanism of action and may provide the basis for the future design of drugs for the treatment of multidrug-resistant and extremely drug-resistant M. tuberculosis strains. Recombinant M. tuberculosis IspE was crystallized at 291 K using NaCl or Li2SO4 as a precipitant. A 2.1 Å resolution native data set was collected from a single flash-cooled crystal (100 K) belonging to space group P212121, with unit-cell parameters a = 52.5, b = 72.3, c = 107.3 Å. One molecule was assumed per asymmetric unit, which gives a Matthews coefficient of 3.4 Å3 Da−1 with 63% solvent content.
Mycobacterium tuberculosis; IspE; drug discovery
Mycobacterium tuberculosis utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate and its isomer, dimethylallyl diphosphate, precursors of all isoprenoid compounds. This pathway is of interest as a source of new drug targets, as it is absent from humans and disruption of the responsible genes has shown a lethal phenotype for Escherichia coli. In the MEP pathway, 4-diphosphocytidyl-2-C-methyl-d-erythritol is formed from 2-C-methyl-d-erythritol 4-phosphate (MEP) and CTP in a reaction catalyzed by a 4-diphosphocytidyl-2-C-methyl-d-erythritol synthase (IspD). In the present work, we demonstrate that Rv3582c is essential for M. tuberculosis: Rv3582c has been cloned and expressed, and the encoded protein has been purified. The purified M. tuberculosis IspD protein was capable of catalyzing the formation of 4-diphosphocytidyl-2-C-methyl-d-erythritol in the presence of MEP and CTP. The enzyme was active over a broad pH range (pH 6.0 to 9.0), with peak activity at pH 8.0. The activity was absolutely dependent upon divalent cations, with 20 mM Mg2+ being optimal, and replacement of CTP with other nucleotide 5′-triphosphates did not support activity. Under the conditions tested, M. tuberculosis IspD had Km values of 58.5 μM for MEP and 53.2 μM for CTP. Calculated kcat and kcat/Km values were 0.72 min−1 and 12.3 mM−1 min−1 for MEP and 1.0 min−1 and 18.8 mM−1 min−1 for CTP, respectively.
4-Diphosphocytidyl-2C-methyl-d-erythritol kinase (IspE) catalyses the ATP-dependent conversion of 4-diphosphocytidyl-2C-methyl-d-erythritol (CDPME) to 4-diphosphocytidyl-2C-methyl-d-erythritol 2-phosphate with the release of ADP. This reaction occurs in the non-mevalonate pathway of isoprenoid precursor biosynthesis and because it is essential in important microbial pathogens and absent from mammals it represents a potential target for anti-infective drugs. We set out to characterize the biochemical properties, determinants of molecular recognition and reactivity of IspE and report the cloning and purification of recombinant Aquifex aeolicus IspE (AaIspE), kinetic data, metal ion, temperature and pH dependence, crystallization and structure determination of the enzyme in complex with CDP, CDPME and ADP. In addition, 4-fluoro-3,5-dihydroxy-4-methylpent-1-enylphosphonic acid (compound 1) was designed to mimic a fragment of the substrate, a synthetic route to 1 was elucidated and the complex structure determined. Surprisingly, this ligand occupies the binding site for the ATP α-phosphate not the binding site for the methyl-d-erythritol moiety of CDPME. Gel filtration and analytical ultracentrifugation indicate that AaIspE is a monomer in solution. The enzyme displays the characteristic α/β galacto-homoserine-mevalonate-phosphomevalonate kinase fold, with the catalytic centre positioned in a deep cleft between the ATP- and CDPME-binding domains. Comparisons indicate a high degree of sequence conservation on the IspE active site across bacterial species, similarities in structure, specificity of substrate recognition and mechanism. The biochemical characterization, attainment of well-ordered and reproducible crystals and the models resulting from the analyses provide reagents and templates to support the structure-based design of broad-spectrum antimicrobial agents.
enzyme–ligand complex; GHMP kinase; isoprenoid biosynthesis; molecular recognition; non-mevalonate pathway
2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (IspF) catalyzes the conversion of 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate to 2C-methyl-D-erythritol-2,4-cyclodiphosphate and cytidine monophosphate in production of isoprenoid-precursors via the methylerythritol phosphate biosynthetic pathway. IspF is found in the protozoan Plasmodium falciparum, a parasite that causes cerebral malaria, as well as in many Gram-negative bacteria such as Burkholderia cenocepacia. IspF represents a potential target for development of broad-spectrum antimicrobial drugs since it is proven or inferred as essential in these pathogens and absent from mammals. Structural studies of IspF from these two important yet distinct pathogens, and comparisons with orthologues have been carried out to generate reagents, to support and inform a structure-based approach to early stage drug discovery.
Efficient recombinant protein production and crystallization protocols were developed, and high-resolution crystal structures of IspF from P. falciparum (Emphasis/Emphasis>IspF) and B. cenocepacia (BcIspF) in complex with cytidine nucleotides determined. Comparisons with orthologues, indicate a high degree of order and conservation in parts of the active site where Zn2+ is bound and where recognition of the cytidine moiety of substrate occurs. However, conformational flexibility is noted in that area of the active site responsible for binding the methylerythritol component of substrate. Unexpectedly, one structure of BcIspF revealed two molecules of cytidine monophosphate in the active site, and another identified citrate coordinating to the catalytic Zn2+. In both cases interactions with ligands appear to help order a flexible loop at one side of the active site. Difficulties were encountered when attempting to derive complex structures with other ligands.
High-resolution crystal structures of IspF from two important human pathogens have been obtained and compared to orthologues. The studies reveal new data on ligand binding, with citrate coordinating to the active site Zn2+ and when present in high concentrations cytidine monophosphate displays two binding modes in the active site. Ligand binding appears to order a part of the active site involved in substrate recognition. The high degree of structural conservation in and around the IspF active site suggests that any structural model might be suitable to support a program of structure-based drug discovery.
Antimicrobial drug target; Isoprenoid biosynthesis; X-ray crystallography; Zn2+-dependent enzyme
The structure of a triclinic crystal form of 4-diphosphocytidyl-2C-methyl-d-erythritol kinase has been determined. Comparisons with a previously reported monoclinic crystal form raise questions about our knowledge of the quaternary structure of this enzyme.
4-Diphosphocytidyl-2C-methyl-d-erythritol kinase (IspE; EC 126.96.36.199) contributes to the 1-deoxy-d-xylulose 5-phosphate or mevalonate-independent biosynthetic pathway that produces the isomers isopentenyl diphosphate and dimethylallyl diphosphate. These five-carbon compounds are the fundamental building blocks for the biosynthesis of isoprenoids. The mevalonate-independent pathway does not occur in humans, but is present and has been shown to be essential in many dangerous pathogens, i.e. Plasmodium species, which cause malaria, and Gram-negative bacteria. Thus, the enzymes involved in this pathway have attracted attention as potential drug targets. IspE produces 4-diphosphosphocytidyl-2C-methyl-d-erythritol 2-phosphate by ATP-dependent phosphorylation of 4-diphosphocytidyl-2C-methyl-d-erythritol. A triclinic crystal structure of the Escherichia coli IspE–ADP complex with two molecules in the asymmetric unit was determined at 2 Å resolution and compared with a monoclinic crystal form of a ternary complex of E. coli IspE also with two molecules in the asymmetric unit. The molecular packing is different in the two forms. In the asymmetric unit of the triclinic crystal form the substrate-binding sites of IspE are occluded by structural elements of the partner, suggesting that the ‘triclinic dimer’ is an artefact of the crystal lattice. The surface area of interaction in the triclinic form is almost double that observed in the monoclinic form, implying that the dimeric assembly in the monoclinic form may also be an artifact of crystallization.
mevalonate-independent pathway; isoprenoid biosynthesis; kinases
The biosyntheses of isoprenoids is essential for the survival in all living organisms, and requires one of the two biochemical pathways: (a) Mevalonate (MVA) Pathway or (b) Methylerythritol Phosphate (MEP) Pathway. The latter pathway, which is used by all Gram-negative bacteria, some Gram-positive bacteria and a few apicomplexan protozoa, provides an attractive target for the development of new antimicrobials because of its absence in humans. In this report, we describe two different approaches that we used to identify novel small molecule inhibitors of Escherichia coli and Yersinia pestis 4-diphosphocytidyl-2-C-methyl D-erythritol (CDP-ME) kinases, key enzymes of the MEP pathway encoded by the E. coli ispE and Y. pestis ipk genes, respectively. In the first approach, we explored existing inhibitors of the GHMP kinases while in the second approach; we performed computational high-throughput screening of compound libraries by targeting the CDP-ME binding site of the two bacterial enzymes. From the first approach, we identified two compounds with 6-(benzylthio)-2-(2-hydroxyphenyl)-4-oxo-3,4-dihydro-2H-1,3-thiazine-5-carbonitrile and (Z)-3-methyl-4-((5-phenylfuran-2-yl)methylene)isoxazol-5(4H)-one scaffolds which inhibited Escherichia coli CDP-ME kinase in vitro. We then performed substructure search and docking experiments based on these two scaffolds and identified twenty three analogs for structure-activity relationship (SAR) studies. Three new compounds from the isoxazol-5(4H)-one series have shown inhibitory activities against E. coli and Y. pestis CDP-ME kinases with the IC50 values ranging from 7μM to 13μM. The second approach by computational high-throughput screening (HTS) of two million drug-like compounds yielded two compounds with benzenesulfonamide and acetamide moieties which, at a concentration of 20μM, inhibited 80% and 65%, respectively, of control CDP-ME kinase activity.
Enantiomerically pure 2-C-methyl-D-erythritol 2,4-cyclodiphosphate 1 (ME-CPP) is synthesized from 1,2-O-isopropylidene-α-D-xylofuranose with facile phosphorylation in good yield. Subsequently, the synthesized enantiomerically pure 1 can be used as a substrate in IspG assays to identify inhibitors that may be developed into antibacterial drug leads.
The prevalence of tuberculosis, the prolonged and expensive treatment that this disease requires and an increase in drug resistance indicate an urgent need for new treatments. The 1-deoxy-D-xylulose 5-phosphate pathway of isoprenoid precursor biosynthesis is an attractive chemotherapeutic target because it occurs in many pathogens, including Mycobacterium tuberculosis, and is absent from humans. To underpin future drug development it is important to assess which enzymes in this biosynthetic pathway are essential in the actual pathogens and to characterize them.
The fifth enzyme of this pathway, encoded by ispF, is 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (IspF). A two-step recombination strategy was used to construct ispF deletion mutants in M. tuberculosis but only wild-type double crossover strains were isolated. The chromosomal copy could be deleted when a second functional copy was provided on an integrating plasmid, demonstrating that ispF is an essential gene under the conditions tested thereby confirming its potential as a drug target. We attempted structure determination of the M. tuberculosis enzyme (MtIspF), but failed to obtain crystals. We instead analyzed the orthologue M. smegmatis IspF (MsIspF), sharing 73% amino acid sequence identity, at 2.2 Å resolution. The high level of sequence conservation is particularly pronounced in and around the active site. MsIspF is a trimer with a hydrophobic cavity at its center that contains density consistent with diphosphate-containing isoprenoids. The active site, created by two subunits, comprises a rigid CDP-Zn2+ binding pocket with a flexible loop to position the 2C-methyl-D-erythritol moiety of substrate. Sequence-structure comparisons indicate that the active site and interactions with ligands are highly conserved.
Our study genetically validates MtIspF as a therapeutic target and provides a model system for structure-based ligand design.
Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fragment binders. Testing by surface plasmon resonance (SPR) found one molecule in the series to possess binding affinity equal to that of cytidine diphosphate, despite lacking any metal-coordinating phosphate groups. Close inspection of the SPR data suggest different binding stoichiometries between IspF and test compounds. Crystallographic analysis shows important variations between the binding mode of one synthesized compound and the pose of the bound fragment from which it was designed. The binding modes of these molecules add to our structural knowledge base for IspF and suggest future refinements in this compound series.
Fragment screening; MEP pathway; IspF; Non-mevalonate; Anti-infective; SPR
There is significant progress toward understanding catalysis throughout the essential MEP pathway to isoprenoids in human pathogens; however, little is known about pathway regulation. The present study begins by testing the hypothesis that isoprenoid biosynthesis is regulated via feedback inhibition of the fifth enzyme cyclodiphosphate IspF by downstream isoprenoid diphosphates. Here, we demonstrate recombinant E. coli IspF is not inhibited by downstream metabolites and isopentenyl diphosphate (IDP), dimethylallyl diphosphate (DMADP), geranyl diphosphate (GDP) and farnesyl diphosphate (FDP) under standard assay conditions. However, 2C-methyl-d-erythritol 4-phosphate (MEP), the product of reductoisomerase IspC and first committed MEP pathway intermediate, activates and sustains this enhanced IspF activity, and the IspF-MEP complex is inhibited by FDP. We further show that the methylerythritol scaffold itself, which is unique to this pathway, drives the activation and stabilization of active IspF. Our results suggest a novel feed-forward regulatory mechanism for 2Cmethyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) production and support an isoprenoid biosynthesis regulatory mechanism via feedback inhibition of the IspF-MEP complex by FDP. The results have important implications for development of inhibitors against the IspF-MEP complex, which may be the physiologically relevant form of the enzyme.
cyclodiphosphate synthase; IspF; methylerythritol phosphate; MEP pathway regulation
2-C-methyl-D-erythritol-4-phosphate (MEP) is a key chemical intermediate of the non-mevalonate pathway for isoprenoid biosynthesis employed by many pathogenic microbes. MEP is also the precursor for the synthesis of 4-diphosphocytidyl-2-C-methyl D-erythritol (CDP-ME), another key intermediate of the non-mevalonate pathway. As this pathway is non-existent in higher animals, including humans, it represents great opportunities for novel antimicrobial development. To facilitate the in-depth studies of this pathway, we reported here a formal synthesis of CDP-ME through a new synthesis of 2-C-Methyl-D-erythritol-4-phosphoric acid from D-(+)-arabitol.
MEP; CDP-ME; selective phosphorylation; dioxanone; monophosphate
1-Deoxy-D-xylulsose-5-phosphate (DXP) is a key intermediate in the non-mevalonate or methyl erythritol phosphate (MEP) pathway for the biosynthesis of isoprenoid, which are essential building blocks involved in the construction of pathogens growth. Since the homologous enzymes of this pathway are not present in vertebrates, including humans, the MEP pathway presents a viable source for antimicrobial drug targets. However, an insight into the features of the enzymes involved in this pathway has been plagued by lack of chirally pure substrates. Here in, we report an efficient synthesis of enantiomerically pure 1-deoxy-D-xylulose-5-phosphate from commercially available 1,2-O-isopropylidene-α-D-xylofuranose through Weinreb amide formation in shorter route.
Isopentenols, such as prenol and isoprenol, are promising advanced biofuels because of their higher energy densities and better combustion efficiencies compared with ethanol. Microbial production of isopentenols has been developed recently via metabolically engineered E. coli. However, current yields remain low and the underlying pathways require systematic optimization.
In this study, we targeted the E. coli native 2-methyl-(D)-erythritol-4-phosphate (MEP) pathway and its upstream glycolysis pathway for the optimization of isopentenol production. Two codon optimized genes, nudF and yhfR from Bacillus subtilis, were synthesized and expressed in E. coli W3110 to confer the isopentenol production of the strain. Two key enzymes (IspG and Dxs) were then overexpressed to optimize the E. coli native MEP pathway, which led to a significant increase (3.3-fold) in isopentenol production. Subsequently, the glycolysis pathway was tuned to enhance the precursor and NADPH supplies for the MEP pathway by activating the pentose phosphate pathway (PPP) and Entner-Doudoroff pathway (ED), which resulted in additional 1.9 folds of increase in isopentenol production. A 5 L-scale batch cultivation experiment was finally implemented, showing a total of 61.9 mg L−1 isopentenol production from 20 g L−1 of glucose.
The isopentenol production was successfully increased through multi-step optimization of the MEP and its upstream glycolysis pathways. It demonstrated that the total fluxes and their balance of the precursors of the MEP pathway are of critical importance in isopentenol production. In the future, an elucidation of the contribution of PPP and ED to MEP is needed for further optimization of isopentenol production.
Electronic supplementary material
The online version of this article (doi:10.1186/s12934-014-0135-y) contains supplementary material, which is available to authorized users.
Isopentenol; MEP pathway; Entner-Doudoroff pathway; Pentose phosphate pathway (PPP); Escherichia coli; Metabolic engineering
CDP-ME kinase (IspE) contributes to the non-mevalonate or deoxy-xylulose phosphate (DOXP) pathway for isoprenoid precursor biosynthesis found in many species of bacteria and apicomplexan parasites. IspE has been shown to be essential by genetic methods and since it is absent from humans it constitutes a promising target for antimicrobial drug development. Using in silico screening directed against the substrate binding site and in vitro high-throughput screening directed against both, the substrate and co-factor binding sites, non-substrate-like IspE inhibitors have been discovered and structure-activity relationships were derived. The best inhibitors in each series have high ligand efficiencies and favourable physico-chemical properties rendering them promising starting points for drug discovery. Putative binding modes of the ligands were suggested which are consistent with established structure-activity relationships. The applied screening methods were complementary in discovering hit compounds, and a comparison of both approaches highlights their strengths and weaknesses. It is noteworthy that compounds identified by virtual screening methods provided the controls for the biochemical screens.
(E)-4-Hydroxy-3-methyl-but-2-enyl diphosphate reductase (IspH or LytB) catalyzes the terminal step of the MEP/DOXP pathway where it converts (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) into the two products isopentenyl diphosphate and dimethylallyl diphosphate. The reaction involves the reductive elimination of the C4 hydroxyl group, using a total of two electrons. Here we show that the active form of IspH contains a [4Fe-4S] cluster and not the [3Fe-4S] form. Our studies show that the cluster is not only the direct electron source for the reaction but that a reaction intermediate is bound directly to the cluster. This active form, has been trapped in a state, dubbed FeSA, that was detected in EPR spectroscopy when one-electron-reduced IspH was incubated with HMBPP. In addition, three mutants of IspH protein have been prepared and studied, His42, His124 and Glu126 (Aquifex aeolicus numbering), with particular attention to the effects on the cluster properties and possible reaction intermediates. None of the mutants affected the properties of the [4Fe-4S]+ cluster significantly, but different effects were observed when one-electron-reduced forms were incubated with HMBPP. Replacing the His42 led to an increased Km value and much lower catalytic efficiency, confirming the role of this residue in substrate binding. Replacing the His124 also resulted in lower catalytic efficiency. In this case, however, enzyme showed the loss of the [4Fe-4S]+ EPR signal upon addition of HMBPP without the subsequent formation of the FeSA signal. Instead, a radical-type signal was observed in some of the samples indicating that this residue plays a role in the correct positioning of the substrate. The incorrect orientation in the mutant leads to the formation of substrate-based radicals instead of the cluster-bound-intermediate complex FeSA. Replacing the Glu126 also resulted in lower catalytic efficiency, with yet a third type of EPR signal being detected upon incubation with HMBPP. 31P- and 2H-ENDOR measurements on the FeSA species incubated with regular and 2H-C4-labeled HMBPP reveal that the substrate binds to the enzyme in close proximity of the active-site cluster with the C4 adjacent to the site of linkage between the FeS cluster and HMBPP. Comparison of the spectroscopic properties of this intermediate to those of intermediates detected in (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase and ferredoxin:thioredoxin reductase suggest that HMBPP binds to the FeS cluster via its hydroxyl group instead of a side-on binding as previously proposed for the species detected in the inactive Glu126 variant. Consequences for the IspH reaction mechanism are discussed.
(E)-4-Hydroxy-3-methyl-but-2-enyl diphosphate reductase; IspH; EPR; ENDOR; reaction intermediate
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.
The nonmevalonate route to isoprenoid biosynthesis is essential in Gram-negative bacteria and apicomplexan parasites. The enzymes of this pathway are absent from mammals, contributing to their appeal as chemotherapeutic targets. One enzyme, 2C-methyl-d-erythritol-2,4-cyclodiphosphate synthase (IspF), has been validated as a target by genetic approaches in bacteria. Virtual screening against Escherichia coli IspF (EcIspF) was performed by combining a hierarchical filtering methodology with molecular docking. Docked compounds were inspected and 10 selected for experimental validation. A surface plasmon resonance assay was developed and two weak ligands identified. Crystal structures of EcIspF complexes were determined to support rational ligand development. Cytosine analogues and Zn2+-binding moieties were characterized. One of the putative Zn2+-binding compounds gave the lowest measured KD to date (1.92 ± 0.18 μM). These data provide a framework for the development of IspF inhibitors to generate lead compounds of therapeutic potential against microbial pathogens.
Isoprenoids, which are a large group of natural and chemical compounds with a variety of applications as e.g. fragrances, pharmaceuticals and potential biofuels, are produced via two different metabolic pathways, the mevalonate (MVA) pathway and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we attempted to replace the endogenous MVA pathway in Saccharomyces cerevisiae by a synthetic bacterial MEP pathway integrated into the genome to benefit from its superior properties in terms of energy consumption and productivity at defined growth conditions. It was shown that the growth of a MVA pathway deficient S. cerevisiae strain could not be restored by the heterologous MEP pathway even when accompanied by the co-expression of genes erpA, hISCA1 and CpIscA involved in the Fe-S trafficking routes leading to maturation of IspG and IspH and E. coli genes fldA and fpr encoding flavodoxin and flavodoxin reductase believed to be responsible for electron transfer to IspG and IspH.
1-Deoxy-d-xylulose 5-phosphate reductoisomerase (IspC) catalyzes the first committed step in the mevalonate-independent isopentenyl diphosphate biosynthetic pathway and is a potential drug target in some pathogenic bacteria. The antibiotic fosmidomycin has been shown to inhibit IspC in a number of organisms and is active against most gram-negative bacteria but not gram positives, including Mycobacterium tuberculosis, even though the mevalonate-independent pathway is the sole isopentenyl diphosphate biosynthetic pathway in this organism. Therefore, the enzymatic properties of recombinant IspC from M. tuberculosis were characterized. Rv2870c from M. tuberculosis converts 1-deoxy-d-xylulose 5-phosphate to 2-C-methyl-d-erythritol 4-phosphate in the presence of NADPH. The enzymatic activity is dependent on the presence of Mg2+ ions and exhibits optimal activity between pH 7.5 and 7.9; the Km for 1-deoxyxylulose 5-phosphate was calculated to be 47.1 μM, and the Km for NADPH was 29.7 μM. The specificity constant of Rv2780c in the forward direction is 1.5 × 106 M−1 min−1, and the reaction is inhibited by fosmidomycin, with a 50% inhibitory concentration of 310 nM. In addition, Rv2870c complements an inactivated chromosomal copy of IspC in Salmonella enterica, and the complemented strain is sensitive to fosmidomycin. Thus, M. tuberculosis resistance to fosmidomycin is not due to intrinsic properties of Rv2870c, and the enzyme appears to be a valid drug target in this pathogen.
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.
2-C-methyl-d-erythritol 4-phosphate is the first committed intermediate in the biosynthesis of the isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate. Supplementation of the growth medium with 2-C-methyl-d-erythritol has been shown to complement disruptions in the Escherichia coli gene for 1-deoxy-d-xylulose 5-phosphate synthase, the enzyme that synthesizes the immediate precursor of 2-C-methyl-d-erythritol 4-phosphate. In order to be utilized in isoprenoid biosynthesis, 2-C-methyl-d-erythritol must be phosphorylated. We describe the construction of Salmonella enterica serovar Typhimurium strain RMC26, in which the essential gene encoding 1-deoxy-d-xylulose 5-phosphate synthase has been disrupted by insertion of a synthetic mevalonate operon consisting of the yeast ERG8, ERG12, and ERG19 genes, responsible for converting mevalonate to isopentenyl diphosphate under the control of an arabinose-inducible promoter. Random mutagenesis of RMC26 produced defects in the sorbitol phosphotransferase system that prevented the transport of 2-C-methyl-d-erythritol into the cell. RMC26 and mutant strains of RMC26 unable to grow on 2-C-methyl-d-erythritol were incubated in buffer containing mevalonate and deuterium-labeled 2-C-methyl-d-erythritol. Ubiquinone-8 was isolated from these cells and analyzed for deuterium content. Efficient incorporation of deuterium was observed for RMC26. However, there was no evidence of deuterium incorporation into the isoprenoid side chain of ubiquinone Q8 in the RMC26 mutants.
The ispF gene product in Escherichia coli has been shown to catalyze the formation of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (MEC) in the deoxyxylulose (DOXP) pathway for isoprenoid biosynthesis. In this work, the E. coli gene ispF and its Bacillus subtilis orthologue, yacN, were deleted and conditionally complemented by expression of these genes from distant loci in the respective organisms. In E. coli, complementation was achieved through integration of ispF at the araBAD locus with control from the arabinose-inducible araBAD promoter, while in B. subtilis, yacN was placed at amyE under control of the xylose-inducible xylA promoter. In both cases, growth was severely retarded in the absence of inducer, consistent with these genes being essential for survival. E. coli cells depleted of MEC synthase revealed a filamentous phenotype. This was in contrast to the depletion of MEC synthase in B. subtilis, which resulted in a loss of rod shape, irregular septation, multicompartmentalized cells, and thickened cell walls. To probe the nature of the predominant deficiency of MEC synthase-depleted cells, we investigated the sensitivity of these conditionally complemented mutants, grown with various concentrations of inducer, to a wide variety antibiotics. Synthetic lethal behavior in MEC synthase-depleted cells was prevalent for cell wall-active antibiotics.