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1.  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
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  The structure of Mycobacteria 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, an essential enzyme, provides a platform for drug discovery 
Background
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.
Results
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.
Conclusion
Our study genetically validates MtIspF as a therapeutic target and provides a model system for structure-based ligand design.
doi:10.1186/1472-6807-7-68
PMCID: PMC2151065  PMID: 17956607
9.  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
10.  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
11.  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
12.  Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei 
Bioorganic & medicinal chemistry letters  2013;23(24):10.1016/j.bmcl.2013.09.101.
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.
doi:10.1016/j.bmcl.2013.09.101
PMCID: PMC3874807  PMID: 24157367
Fragment screening; MEP pathway; IspF; Non-mevalonate; Anti-infective; SPR
13.  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
14.  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
15.  Reconstruction and Evaluation of the Synthetic Bacterial MEP Pathway in Saccharomyces cerevisiae 
PLoS ONE  2012;7(12):e52498.
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.
doi:10.1371/journal.pone.0052498
PMCID: PMC3532213  PMID: 23285068
16.  The Sorbitol Phosphotransferase System Is Responsible for Transport of 2-C-Methyl-d-Erythritol into Salmonella enterica Serovar Typhimurium 
Journal of Bacteriology  2004;186(2):473-480.
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.
doi:10.1128/JB.186.2.473-480.2004
PMCID: PMC305747  PMID: 14702317
17.  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
18.  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
19.  Crystallization and preliminary X-ray crystallographic study of 1-deoxy-d-xylulose 5-­phosphate reductoisomerase from Plasmodium falciparum  
1-Deoxy-d-xylulose 5-phosphate reductoisomerase from P. falciparum has been crystallized in the presence of NADPH. Diffraction data to 1.85 Å resolution have been collected using synchrotron radiation.
The nonmevalonate pathway of isoprenoid biosynthesis present in Plasmodium falciparum is known to be an effective target for antimalarial drugs. The second enzyme of the nonmevalonate pathway, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), catalyzes the transformation of 1-deoxy-d-xylulose 5-phosphate (DXP) to 2-C-methyl-d-erythritol 4-phosphate (MEP). For crystallographic studies, DXR from the human malaria parasite P. falciparum (PfDXR) was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method in the presence of NADPH. X-ray diffraction data to 1.85 Å resolution were collected from a monoclinic crystal form belonging to space group C2 with unit-cell parameters a = 168.89, b = 59.65, c = 86.58 Å, β = 117.8°. Structural analysis by molecular replacement is in progress.
doi:10.1107/S1744309110001739
PMCID: PMC2833050  PMID: 20208174
1-deoxy-d-xylulose 5-phosphate reductoisomerase; malaria; nonmevalonate pathway
20.  Purification, crystallization and preliminary X-ray diffraction of human S100A15 
S100 proteins are differentially expressed during epithelial cell maturation, tumorigenesis and inflammation. The novel human S100A15 protein has been cloned, expressed, purified and crystallized in two crystal forms, a triclinic and a monoclinic form, which diffract to 1.7 and 2.0 Å, respectively.
Human S100A15 is a novel member of the S100 family of EF-hand calcium-binding proteins and was recently identified in psoriasis, where it is significantly upregulated in lesional skin. The protein is implicated as an effector in calcium-mediated signal transduction pathways. Although its biological function is unclear, the association of the 11.2 kDa S100A15 with psoriasis suggests that it contributes to the pathogenesis of the disease and could provide a molecular target for therapy. To provide insight into the function of S100A15, the protein was crystallized to visualize its structure and to further the understanding of how the many similar calcium-binding mediator proteins in the cell distinguish their cognate target molecules. The S100A15 protein has been cloned, expressed and purified to homogeneity and produced two crystal forms. Crystals of form I are triclinic, with unit-cell parameters a = 33.5, b = 44.3, c = 44.8 Å, α = 71.2, β = 68.1, γ = 67.8° and an estimated two molecules in the asymmetric unit, and diffract to 1.7 Å resolution. Crystals of form II are monoclinic, with unit-cell parameters a = 82.1, b = 33.6, c = 52.2 Å, β = 128.2° and an estimated one molecule in the asymmetric unit, and diffract to 2.0 Å resolution. This structural analysis of the human S100A15 will further aid in the phylogenic comparison between the other members of the S100 protein family, especially the highly homologous paralog S100A7.
doi:10.1107/S1744309106012838
PMCID: PMC2219979  PMID: 16682778
S100A15; psoriasis; calcium binding; EF-hand
21.  A Closer Look at the Spectroscopic Properties of Possible Reaction Intermediates in WT and Mutant (E)-4-hydroxy-3-methyl-but-2-enyl Diphosphate Reductase (IspH/LytB)† 
Biochemistry  2012;51(24):4835-4849.
(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.
doi:10.1021/bi3001215
PMCID: PMC3426640  PMID: 22646150
(E)-4-Hydroxy-3-methyl-but-2-enyl diphosphate reductase; IspH; EPR; ENDOR; reaction intermediate
22.  A monoclinic polymorph of N-eth­oxy­carbonyl-N′-(3-phenyl-1H-1,2,4-triazol-5-yl)thio­urea 
The title compound, C12H13N5O2S {systematic name: ethyl N-[N-(3-phenyl-1H-1,2,4-triazol-5-yl)carbamothio­yl]carbamate}, is a monoclinic polymorph (space group P21/c) which crystallizes with three similar independent mol­ecules in the asymmetric unit. The triazole ring makes dihedral angles of 6.6 (2), 8.4 (2) and 10.6 (2)° with the phenyl ring in the three independent molecules. The structure was previously reported [Dolzhenko et al. (2010a ▶). Acta Cryst., E46, o425] as a triclinic polymorph crystallizing in space group P . Mol­ecules in both polymorphs possess two S(6) rings generated by intra­molecular N—H⋯S and N—H⋯O hydrogen bonds, resulting in similar mol­ecular geometries. However, the two polymorphs differ in the crystal packing. In contrast to the dimers of the triclinic polymorph, mol­ecules of the monoclinic polymorph are connected by inter­molecular N—H⋯S and N—H⋯N hydrogen bonds, forming pseudosymmetric trimers arranged in sheets parallel to (302).
doi:10.1107/S1600536810026164
PMCID: PMC3007582  PMID: 21588305
23.  Purification, crystallization and preliminary X-ray diffraction analysis of inner membrane complex (IMC) subcompartment protein 1 (ISP1) from Toxoplasma gondii  
To characterize the ISP family of proteins present in apicomplexan parasites, ISP1 from T. gondii was expressed, purified and crystallized. Two crystal forms (cubic and orthorhombic) were analyzed by X-ray diffraction and data were processed to 2.05 and 2.1 Å resolution, respectively.
The protozoan parasites of the Apicomplexa phylum are devastating global pathogens. Their success is largely due to phylum-specific proteins found in specialized organelles and cellular structures. The inner membrane complex (IMC) is a unique apicomplexan structure that is essential for motility, invasion and replication. The IMC subcompartment proteins (ISP) have recently been identified in Toxoplasma gondii and shown to be critical for replication, although their specific mechanisms are unknown. Structural characterization of TgISP1 was pursued in order to identify the fold adopted by the ISPs and to generate detailed insight into how this family of proteins functions during replication. An N-terminally truncated form of TgISP1 was purified from Escherichia coli, crystallized and subjected to X-ray diffraction analysis. Two crystal forms of TgISP1 belonging to space groups P4132 or P4332 and P212121 diffracted to 2.05 and 2.1 Å resolution, respectively.
doi:10.1107/S174430911202297X
PMCID: PMC3388934  PMID: 22750877
Toxoplasma gondii; inner membrane complex; ISP1
24.  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
25.  Formal Synthesis of 4-diphosphocytidyl-2-C-methyl D-erythritol From D-(+)-Arabitol 
Tetrahedron  2012;68(43):8937-8941.
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.
doi:10.1016/j.tet.2012.08.020
PMCID: PMC3462025  PMID: 23049145
MEP; CDP-ME; selective phosphorylation; dioxanone; monophosphate

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