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1.  Crystal structures of IspF from Plasmodium falciparum and Burkholderia cenocepacia: comparisons inform antimicrobial drug target assessment 
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1472-6807-14-1
PMCID: PMC3927217  PMID: 24410837
Antimicrobial drug target; Isoprenoid biosynthesis; X-ray crystallography; Zn2+-dependent enzyme
2.  Synthesis of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate and kinetic studies of Mycobacterium tuberculosis IspF, a potential drug target 
Chemistry & biology  2010;17(2):117-122.
SUMMARY
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.
doi:10.1016/j.chembiol.2010.01.013
PMCID: PMC2837070  PMID: 20189102
3.  A Structure-Based Approach to Ligand Discovery for 2C-Methyl-d-erythritol-2,4-cyclodiphosphate Synthase: A Target for Antimicrobial Therapy† 
Journal of Medicinal Chemistry  2009;52(8):2531-2542.
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.
doi:10.1021/jm801475n
PMCID: PMC2669732  PMID: 19320487
4.  2C-Methyl-d-erythritol 4-phosphate enhances and sustains cyclodiphosphate synthase IspF activity 
ACS chemical biology  2012;7(10):1702-1710.
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.
doi:10.1021/cb300243w
PMCID: PMC3477264  PMID: 22839733
cyclodiphosphate synthase; IspF; methylerythritol phosphate; MEP pathway regulation
5.  Absence of Substrate Channeling between Active Sites in the Agrobacterium tumefaciens IspDF and IspE Enzymes of the Methyl Erythritol Phosphate Pathway† 
Biochemistry  2006;45(11):3548-3553.
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.
doi:10.1021/bi0520075
PMCID: PMC2516919  PMID: 16533036
bifunctional; IspDF; IspE; non-channeling
6.  Characterization of Aquifex aeolicus 4-diphosphocytidyl-2C-methyl-d-erythritol kinase – ligand recognition in a template for antimicrobial drug discovery 
The Febs Journal  2008;275(11):2779-2794.
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.
doi:10.1111/j.1742-4658.2008.06418.x
PMCID: PMC2655357  PMID: 18422643
enzyme–ligand complex; GHMP kinase; isoprenoid biosynthesis; molecular recognition; non-mevalonate pathway
7.  A triclinic crystal form of Escherichia coli 4-diphosphocytidyl-2C-methyl-d-erythritol kinase and reassessment of the quaternary structure 
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 2.7.1.148) 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.
doi:10.1107/S1744309109054591
PMCID: PMC2833027  PMID: 20208151
mevalonate-independent pathway; isoprenoid biosynthesis; kinases
8.  Expression and characterization of soluble 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase from bacterial pathogens 
Chemistry & biology  2009;16(12):1230-1239.
Summary
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.
doi:10.1016/j.chembiol.2009.10.014
PMCID: PMC4020808  PMID: 20064433
9.  Crystallization and preliminary X-ray analysis of 4-­diphosphocytidyl-2-C-methyl-d-erythritol kinase (IspE) from Mycobacterium tuberculosis  
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.
doi:10.1107/S1744309111019567
PMCID: PMC3144805  PMID: 21795803
Mycobacterium tuberculosis; IspE; drug discovery
10.  Characterization of the Depletion of 2-C-Methyl-d-Erythritol-2,4-Cyclodiphosphate Synthase in Escherichia coli and Bacillus subtilis 
Journal of Bacteriology  2002;184(20):5609-5618.
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.
doi:10.1128/JB.184.20.5609-5618.2002
PMCID: PMC139617  PMID: 12270818
11.  Characterization of the Mycobacterium tuberculosis 4-Diphosphocytidyl-2-C-Methyl-d-Erythritol Synthase: Potential for Drug Development▿  
Journal of Bacteriology  2007;189(24):8922-8927.
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.
doi:10.1128/JB.00925-07
PMCID: PMC2168624  PMID: 17921290
12.  IspE Inhibitors Identified by a Combination of In Silico and In Vitro High-Throughput Screening 
PLoS ONE  2012;7(4):e35792.
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.
doi:10.1371/journal.pone.0035792
PMCID: PMC3340893  PMID: 22563402
13.  1-Deoxy-d-Xylulose 5-Phosphate Reductoisomerase (IspC) from Mycobacterium tuberculosis: towards Understanding Mycobacterial Resistance to Fosmidomycin 
Journal of Bacteriology  2005;187(24):8395-8402.
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.
doi:10.1128/JB.187.24.8395-8402.2005
PMCID: PMC1316992  PMID: 16321944
14.  The Nonmevalonate Pathway of Isoprenoid Biosynthesis in Mycobacterium tuberculosis Is Essential and Transcriptionally Regulated by Dxs ▿ †  
Journal of Bacteriology  2010;192(9):2424-2433.
Mycobacterium tuberculosis synthesizes isoprenoids via the nonmevalonate or DOXP pathway. Previous work demonstrated that three enzymes in the pathway (Dxr, IspD, and IspF) are all required for growth in vitro. We demonstrate the essentiality of the key genes dxs1 and gcpE, confirming that the pathway is of central importance and that the second homolog of the synthase (dxs2) cannot compensate for the loss of dxs1. We looked at the effect of overexpression of Dxr, Dxs1, Dxs2, and GcpE on viability and on growth in M. tuberculosis. Overexpression of dxs1 or dxs2 was inhibitory to growth, whereas overexpression of dxr or gcpE was not. Toxicity is likely to be, at least partially, due to depletion of pyruvate from the cells. Overexpression of dxs1 or gcpE resulted in increased flux through the pathway, as measured by accumulation of the metabolite 4-hydroxy-3-methyl-but-2-enyl pyrophosphate. We identified the functional translational start site and promoter region for dxr and demonstrated that it is expressed as part of a polycistronic mRNA with gcpE and two other genes. Increased expression of this operon was seen in cells overexpressing Dxs1, indicating that transcriptional control is effected by the first enzyme of the pathway via an unknown regulator.
doi:10.1128/JB.01402-09
PMCID: PMC2863480  PMID: 20172995
15.  Identification of Novel Small Molecule Inhibitors of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase of Gram-negative bacteria 
Bioorganic & medicinal chemistry  2011;19(19):5886-5895.
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.
doi:10.1016/j.bmc.2011.08.012
PMCID: PMC3188437  PMID: 21903402
16.  Utilizing a Dynamical Description of IspH to Aid in the Development of Novel Antimicrobial Drugs 
PLoS Computational Biology  2013;9(12):e1003395.
The nonmevalonate pathway is responsible for isoprenoid production in microbes, including H. pylori, M. tuberculosis and P. falciparum, but is nonexistent in humans, thus providing a desirable route for antibacterial and antimalarial drug discovery. We coordinate a structural study of IspH, a [4Fe-4S] protein responsible for converting HMBPP to IPP and DMAPP in the ultimate step in the nonmevalonate pathway. By performing accelerated molecular dynamics simulations on both substrate-free and HMBPP-bound [Fe4S4]2+ IspH, we elucidate how substrate binding alters the dynamics of the protein. Using principal component analysis, we note that while substrate-free IspH samples various open and closed conformations, the closed conformation observed experimentally for HMBPP-bound IspH is inaccessible in the absence of HMBPP. In contrast, simulations with HMBPP bound are restricted from accessing the open states sampled by the substrate-free simulations. Further investigation of the substrate-free simulations reveals large fluctuations in the HMBPP binding pocket, as well as allosteric pocket openings – both of which are achieved through the hinge motions of the individual domains in IspH. Coupling these findings with solvent mapping and various structural analyses reveals alternative druggable sites that may be exploited in future drug design efforts.
Author Summary
Drug resistance has recently entered into media conversations through the lens of MRSA (methicillin-resistant Staphylococcus aureus) infections, but conventional therapies are also failing to address resistance in cases of malaria and other bacterial infections, such as tuberculosis. To address these problems, we must develop new antibacterial and antimalarial medications. Our research focuses on understanding the structure and dynamics of IspH, an enzyme whose function is necessary for the survival of most bacteria and malaria-causing protozoans. Using computer simulations, we track how the structure of IspH changes in the presence and absence of its natural substrate. By inspecting the pockets that form in the normal motions of IspH, we propose a couple new routes by which new molecules may be developed to disrupt the function of IspH. It is our hope that these structural studies may be precursors to the development of novel therapies that may add to our current arsenal against bacterial and malarial infections.
doi:10.1371/journal.pcbi.1003395
PMCID: PMC3868525  PMID: 24367248
17.  A second target of the antimalarial and antibacterial agent fosmidomycin revealed by cellular metabolic profiling† 
Biochemistry  2011;50(17):3570-3577.
Antimicrobial drug resistance is an urgent problem in control and treatment of many of the world's most serious infections, including Plasmodium falciparum malaria, tuberculosis, and healthcare-associated infections with Gram-negative bacteria. Because the non-mevalonate pathway of isoprenoid biosynthesis is essential in eubacteria and P. falciparum, and this pathway is not present in humans, there is great interest in targeting the enzymes of non-mevalonate metabolism for antibacterial and antiparasitic drug development. Fosmidomycin is a broad-spectrum antimicrobial agent currently in clinical trials of combination therapies to treat malaria. In vitro, fosmidomycin is known to inhibit the deoxyxylulose phosphate reductoisomerase (DXR) enzyme of isoprenoid biosynthesis from multiple pathogenic organisms. To define the in vivo metabolic response to fosmidomycin, we developed a novel mass spectrometry method to quantitate six metabolites of non-mevalonate isoprenoid metabolism from complex biological samples. Using this technique, we validate that the biological effects of fosmidomycin are mediated through blockade of de novo isoprenoid biosynthesis in both P. falciparum malaria parasites and E. coli bacteria: in both organisms, metabolic profiling demonstrated a block in isoprenoid metabolism following fosmidomycin treatment, and growth inhibition due to fosmidomycin was rescued by media supplemented with isoprenoid metabolites. Isoprenoid metabolism proceeded through DXR even in the presence of fosmidomycin, but was inhibited at the level of the downstream enzyme, methylerythritol phosphate cytidyltransferase (IspD). Overexpression of IspD in E. coli conferred fosmidomycin resistance, and fosmidomycin was found to inhibit IspD in vitro. This work has validated fosmidomycin as a biological reagent to block non-mevalonate isoprenoid metabolism, and suggests a second in vivo target for fosmidomycin within isoprenoid biosynthesis, in two evolutionarily diverse pathogens.
doi:10.1021/bi200113y
PMCID: PMC3082593  PMID: 21438569
18.  Inhibition of IspH, a [4Fe-4S]2+ enzyme involved in the biosynthesis of isoprenoids via the MEP pathway 
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.
doi:10.1021/ja309557s
PMCID: PMC3644560  PMID: 23316732
19.  A double mutation of Escherichia coli 2C-methyl-d-erythritol-2,4-cyclodiphosphate synthase disrupts six hydrogen bonds with, yet fails to prevent binding of, an isoprenoid diphosphate 
A double mutation designed to disrupt binding of isoprenoid diphosphate to an enzyme involved in isoprenoid biosynthesis was made and the structure determined. Despite the removal of six hydrogen-bonding interactions, the ligand, acquired during production in E. coli, remains bound. The reasons for this are discussed.
The essential enzyme 2C-methyl-d-erythritol-2,4-cyclodiphosphate (MECP) synthase, found in most eubacteria and the apicomplexan parasites, participates in isoprenoid-precursor biosynthesis and is a validated target for the development of broad-spectrum antimicrobial drugs. The structure and mechanism of the enzyme have been elucidated and the recent exciting finding that the enzyme actually binds diphosphate-containing isoprenoids at the interface formed by the three subunits that constitute the active protein suggests the possibility of feedback regulation of MECP synthase. To investigate such a possibility, a form of the enzyme was sought that did not bind these ligands but which would retain the quaternary structure necessary to create the active site. Two amino acids, Arg142 and Glu144, in Escherichia coli MECP synthase were identified as contributing to ligand binding. Glu144 interacts directly with Arg142 and positions the basic residue to form two hydrogen bonds with the terminal phosphate group of the isoprenoid diphosphate ligand. This association occurs at the trimer interface and three of these arginines interact with the ligand phosphate group. A dual mutation was designed (Arg142 to methionine and Glu144 to leucine) to disrupt the electrostatic attractions between the enzyme and the phosphate group to investigate whether an enzyme without isoprenoid diphosphate could be obtained. A low-resolution crystal structure of the mutated MECP synthase Met142/Leu144 revealed that geranyl diphosphate was retained despite the removal of six hydrogen bonds normally formed with the enzyme. This indicates that these two hydrophilic residues on the surface of the enzyme are not major determinants of isoprenoid binding at the trimer interface but rather that hydrophobic interactions between the hydrocarbon tail and the core of the enzyme trimer dominate ligand binding.
doi:10.1107/S1744309105018762
PMCID: PMC1952448  PMID: 16511114
MECP synthase; site-directed mutagenesis; isoprenoid biosynthesis
20.  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
21.  A Novel Family of Toxoplasma IMC Proteins Displays a Hierarchical Organization and Functions in Coordinating Parasite Division 
PLoS Pathogens  2010;6(9):e1001094.
Apicomplexans employ a peripheral membrane system called the inner membrane complex (IMC) for critical processes such as host cell invasion and daughter cell formation. We have identified a family of proteins that define novel sub-compartments of the Toxoplasma gondii IMC. These IMC Sub-compartment Proteins, ISP1, 2 and 3, are conserved throughout the Apicomplexa, but do not appear to be present outside the phylum. ISP1 localizes to the apical cap portion of the IMC, while ISP2 localizes to a central IMC region and ISP3 localizes to a central plus basal region of the complex. Targeting of all three ISPs is dependent upon N-terminal residues predicted for coordinated myristoylation and palmitoylation. Surprisingly, we show that disruption of ISP1 results in a dramatic relocalization of ISP2 and ISP3 to the apical cap. Although the N-terminal region of ISP1 is necessary and sufficient for apical cap targeting, exclusion of other family members requires the remaining C-terminal region of the protein. This gate-keeping function of ISP1 reveals an unprecedented mechanism of interactive and hierarchical targeting of proteins to establish these unique sub-compartments in the Toxoplasma IMC. Finally, we show that loss of ISP2 results in severe defects in daughter cell formation during endodyogeny, indicating a role for the ISP proteins in coordinating this unique process of Toxoplasma replication.
Author Summary
Apicomplexans are the cause of important diseases in humans and animals including malaria (Plasmodium falciparum), which claims over a million human lives each year, and toxoplasmosis (Toxoplasma gondii), which causes birth defects and neurological disorders. These parasites possess a unique cortical system of membrane sacs arranged on a cytoskeletal meshwork, together referred to as the inner membrane complex (IMC). The IMC is the anchor point for the gliding motility machinery necessary for host invasion and also a scaffold around which new parasites are constructed during replication. Here we have uncovered new insights into the organization and function of this structure by identifying and characterizing ISP1-3, a family of proteins that define novel sub-compartments within the Toxoplasma IMC. Residues predicted for myristoylation and palmitoylation are critical in the membrane targeting of these proteins, suggesting that multiple palmitoyl acyltransferase activities reside within the IMC and dictate its organization. Surprisingly, ISP1 is required for proper sub-compartment sorting of ISP2 and 3, revealing a novel hierarchical targeting mechanism for the organization of this membrane system. Disruption of ISP2 results in defects during endodyogeny and a dramatic loss in parasite fitness, revealing that the ISP proteins play an important role in coordinating parasite replication.
doi:10.1371/journal.ppat.1001094
PMCID: PMC2936552  PMID: 20844581
22.  Chemoenzymatic synthesis of 4-diphosphocytidyl-2-C-methyl-D-erythritol: A substrate for IspE 
Tetrahedron letters  2008;49(29-30):4461-4463.
Enantiomerically pure 2-C-methyl-D-erythritol 4-phosphate 1 (MEP) is synthesized from 1,2-O-isopropylidene-α-D-xylofuranose via facile benzylation in good yield. Subsequently, 1 is used for enzymatic synthesis of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2 (CDP-ME) using 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD). The chemoenzymatically synthesized 2 can be used as substrate for assay of IspE and for high throughput screening to identify IspE inhibitors.
doi:10.1016/j.tetlet.2008.05.074
PMCID: PMC2832204  PMID: 19088853
23.  Sli2 (Ypk1), a Homologue of Mammalian Protein Kinase SGK, Is a Downstream Kinase in the Sphingolipid-Mediated Signaling Pathway of Yeast 
Molecular and Cellular Biology  2000;20(12):4411-4419.
ISP-1 is a new type of immunosuppressant, the structure of which is homologous to that of sphingosine. In a previous study, ISP-1 was found to inhibit mammalian serine palmitoyltransferase, the primary enzyme involved in sphingolipid biosynthesis, and to reduce the intracellular pool of sphingolipids. ISP-1 induces the apoptosis of cytotoxic T cells, which is triggered by decreases in the intracellular levels of sphingolipids. In this study, the inhibition of yeast (Saccharomyces cerevisiae) proliferation by ISP-1 was observed. This ISP-1-induced growth inhibition was also triggered by decreases in the intracellular levels of sphingolipids. In addition, DNA duplication without cytokinesis was detected in ISP-1-treated yeast cells on flow cytometry analysis. We have cloned multicopy suppressor genes of yeast which overcome the lethal sphingolipid depletion induced by ISP-1. One of these genes, SLI2, is synonymous with YPK1, which encodes a serine/threonine kinase. Kinase-dead mutants of YPK1 did not show any resistance to ISP-1, leading us to predict that the kinase activity of the Ypk1 protein should be essential for this resistance to ISP-1. Ypk1 protein overexpression had no effect on sphingolipid biosynthesis by the yeast. Furthermore, both the phosphorylation and intracellular localization of the Ypk1 protein were regulated by the intracellular sphingolipid levels. These data suggest that the Ypk1 protein is a downstream kinase in the sphingolipid-mediated signaling pathway of yeast. The Ypk1 protein was reported to be a functional homologue of the mammalian protein kinase SGK, which is a downstream kinase of 3-phosphoinositide-dependent kinase 1 (PDK1). PDK1 phosphotidylinositol (PI) is regulated by PI-3,4,5-triphosphate and PI-3,4-bisphosphate through the pleckstrin homology (PH) domain. Overexpression of mammalian SGK also overcomes the sphingolipid depletion in yeast. Taking both the inability to produce PI-3,4,5-triphosphate and PI-3,4-bisphosphate and the lack of a PH domain in the yeast homologue of PDK1, the Pkh1 protein, into account, these findings further suggest that yeast may use sphingolipids instead of inositol phospholipids as lipid mediators.
PMCID: PMC85808  PMID: 10825204
24.  Identification of isp, a locus encoding an immunogenic secreted protein conserved among group A streptococci. 
Infection and Immunity  1996;64(7):2548-2555.
The protein Mga (mga), which is required for transcription of several virulence genes of group A streptococci (GAS), including the antiphagocytic M protein, was suggested to act as the response regulator element of a bacterial two-component pathway. To investigate whether a gene encoding a cognate sensor protein is located upstream of mga, 3.1 kb of DNA 5' of the mga translational start site was cloned from serotype M6 GAS strain JRS4. Sequence analysis of this region revealed two adjacent open reading frames, a previously described orf and a new locus, isp (immunogenic secreted protein), which could encode proteins of 9 and 59 kDa, respectively. Inactivation of either open reading frame had no significant effect on transcription of the gene encoding M protein (emm) under normal growth conditions, suggesting that neither isp nor orf is involved in the Mga regulatory circuit. A protein migrating at an apparent molecular weight of 65,000 was produced when isp was transcribed and translated in vitro. The predicted isp product (Isp) contains an amino-terminal signal sequence region homologous to that found in bacterial secreted proteins, and expression of isp in Escherichia coli resulted in the presence of Isp in the periplasmic fraction. Convalescent-phase serum from a patient with an active GAS infection recognized forms of Isp both from the periplasm of E. coli and the supernatant of a GAS strain. Both isp and orf are highly conserved among strains of GAS, as shown by hybridization analyses.
PMCID: PMC174109  PMID: 8698478
25.  Coregulation of Terpenoid Pathway Genes and Prediction of Isoprene Production in Bacillus subtilis Using Transcriptomics 
PLoS ONE  2013;8(6):e66104.
The isoprenoid pathway converts pyruvate to isoprene and related isoprenoid compounds in plants and some bacteria. Currently, this pathway is of great interest because of the critical role that isoprenoids play in basic cellular processes, as well as the industrial value of metabolites such as isoprene. Although the regulation of several pathway genes has been described, there is a paucity of information regarding system level regulation and control of the pathway. To address these limitations, we examined Bacillus subtilis grown under multiple conditions and determined the relationship between altered isoprene production and gene expression patterns. We found that with respect to the amount of isoprene produced, terpenoid genes fall into two distinct subsets with opposing correlations. The group whose expression levels positively correlated with isoprene production included dxs, which is responsible for the commitment step in the pathway, ispD, and two genes that participate in the mevalonate pathway, yhfS and pksG. The subset of terpenoid genes that inversely correlated with isoprene production included ispH, ispF, hepS, uppS, ispE, and dxr. A genome-wide partial least squares regression model was created to identify other genes or pathways that contribute to isoprene production. These analyses showed that a subset of 213 regulated genes was sufficient to create a predictive model of isoprene production under different conditions and showed correlations at the transcriptional level. We conclude that gene expression levels alone are sufficiently informative about the metabolic state of a cell that produces increased isoprene and can be used to build a model that accurately predicts production of this secondary metabolite across many simulated environmental conditions.
doi:10.1371/journal.pone.0066104
PMCID: PMC3686787  PMID: 23840410

Results 1-25 (811630)