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Targeting essential bacterial processes beyond cell wall, protein, nucleotide, and folate syntheses holds promise to reveal new antimicrobial agents and expand the potential drugs available for combination therapies. The synthesis of isoprenoid precursors, isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), is vital for all organisms; however, humans use the mevalonate pathway for production of IDP/DMADP while many pathogens, including Plasmodium falciparum and Mycobacterium tuberculosis, use the orthogonal methylerythritol phosphate (MEP) pathway. Toward developing novel antimicrobial agents, we have designed and synthesized a series of phosphonyl analogues of MEP and evaluated their abilities to interact with IspD, both as inhibitors of the natural reaction and as antimetabolite alternative substrates that could be processed enzymatically to form stable phosphonyl analogues as potential inhibitors of downstream MEP pathway intermediates. In this compound series, the S-monofluoro MEP analogue displays the most potent inhibitory activity against Escherichia coli IspD and is the best substrate for both the E. coli and P. falciparum IspD orthologues with a K m approaching that of the natural substrate for the E. coli enzyme. This work represents a first step toward the development of phosphonyl MEP antimetabolites to modulate early isoprenoid biosynthesis in human pathogens.

Neutron diffraction analysis studies reported an isolated hydronium ion (H 3 O + ) in the interior of d-xylose isomerase (XI) and phycocyanobilin-ferredoxin oxidoreductase (PcyA). H 3 O + forms hydrogen bonds (H-bonds) with two histidine side-chains and a backbone carbonyl group in PcyA, whereas H 3 O + forms H-bonds with three acidic residues in XI. Using a quantum mechanical/molecular mechanical (QM/MM) approach, we analyzed stabilization of H 3 O + by the protein environment. QM/MM calculations indicated that H 3 O + was unstable in the PcyA crystal structure, releasing a proton to an H-bond partner His88, producing H 2 O and protonated His88. On the other hand, H 3 O + was stable in the XI crystal structure. H-bond partners of isolated H 3 O + would be practically limited to acidic residues such as aspartic and glutamic acids in the protein environment., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Carbohydrates are much more than just a source of energy as they also mediate a variety of recognition processes that are central to human health. As such, saccharides can be applied in the food and pharmaceutical industries to stimulate our immune system (e.g., prebiotics), to control diabetes (e.g., low-calorie sweeteners), or as building blocks for anticancer and antiviral drugs (e.g., L: -nucleosides). Unfortunately, only a small number of all possible monosaccharides are found in nature in sufficient amounts to allow their commercial exploitation. Consequently, so-called rare sugars have to be produced by (bio)chemical processes starting from cheap and widely available substrates. Three enzyme classes that can be used for rare sugar production are keto-aldol isomerases, epimerases, and oxidoreductases. In this review, the recent developments in rare sugar production with these biocatalysts are discussed.

Most bacteria use the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the synthesis of their essential isoprenoid precursors. The absence of the MEP pathway in humans makes it a promising new target for the development of much needed new and safe antimicrobial drugs. However, bacteria show a remarkable metabolic plasticity for isoprenoid production. For example, the NADPH-dependent production of MEP from 1-deoxy-D-xylulose 5-phosphate in the first committed step of the MEP pathway is catalyzed by 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) in most bacteria, whereas an unrelated DXR-like (DRL) protein was recently found to catalyze the same reaction in some organisms, including the emerging human and animal pathogens Bartonella and Brucella. Here, we report the x-ray crystal structures of the Brucella abortus DRL enzyme in its apo form and in complex with the broad-spectrum antibiotic fosmidomycin solved to 1.5 and 1.8 Å resolution, respectively. DRL is a dimer, with each polypeptide folding into three distinct domains starting with the NADPH-binding domain, in resemblance to the structure of bacterial DXR enzymes. Other than that, DRL and DXR show a low structural relationship, with a different disposition of the domains and a topologically unrelated C-terminal domain. In particular, the active site of DRL presents a unique arrangement, suggesting that the design of drugs that would selectively inhibit DRL-harboring pathogens without affecting beneficial or innocuous bacteria harboring DXR should be feasible. As a proof of concept, we identified two strong DXR inhibitors that have virtually no effect on DRL activity.

A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize the essential isoprenoid precursor isopentenyl diphosphate via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway that is found in humans. As part of a structure-based drug-discovery program against tuberculosis, DXR, the enzyme that carries out the second step in the MEP pathway, has been investigated. This enzyme is the target for the antibiotic fosmidomycin and its active acetyl derivative FR-900098. The structure of DXR from Mycobacterium tuberculosis in complex with FR-900098, manganese and the NADPH cofactor has been solved and refined. This is a new crystal form that diffracts to a higher resolution than any other DXR complex reported to date. Comparisons with other ternary complexes show that the conformation is that of the enzyme in an active state: the active-site flap is well defined and the cofactor-binding domain has a conformation that brings the NADPH into the active site in a manner suitable for catalysis. The substrate-binding site is highly conserved in a number of pathogens that use this pathway, so any new inhibitor that is designed for the M. tuberculosis enzyme is likely to exhibit broad-spectrum activity.

Isoprene biosynthesis is an essential component of metabolism. Two pathways are known for the production of five-carbon (isoprene) intermediates: the mevalonate and nonmevalonate pathways. As many pathogenic organisms rely exclusively on the nonmevalonate pathway (NMP) for isoprenoids and humans do not, the enzymes of this route have been recently explored as new therapeutic targets. The second and first-committed step in the NMP is catalyzed by 1-deoxy-Dxylulose- 5-phosphate reductoisomerase (Dxr) and has received significant attention as a novel drug target. This review describes the biochemistry and crystal structures of Dxr and the synthesis and biological activity of inhibitors to date, with a focus on compounds targeting E. coli, Plasmodium, and M. tuberculosis enzymes and intact cells. Most inhibitors for Dxr use natural products fosmidomycin and FR900098 as starting points. The review discusses several families of fosmidomycinrelated analogs including α-substituted, 'reverse' and modified hydroxamate, spacer-modified, and hydroxy-amide analogs. Also discussed are non-fosmidomycin-like inhibitors, the aryl phosphonates, and lipophilic prodrugs of fosmidomycin and FR900098 designed to increase cell penetration. A comprehensive SAR of inhibitors is presented.

Emerging resistance of human pathogens to anti-infective agents make it necessary to develop new agents to treat infection. The methylerythritol phosphate pathway has been identified as an anti-infective target, as this essential isoprenoid biosynthetic pathway is widespread in human pathogens but absent in humans. The first enzyme of the pathway, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase, catalyzes the formation of DXP via condensation of D-glyceraldehyde 3-phosphate (D-GAP) and pyruvate in a thiamine diphosphate-dependent manner. Structural analysis has revealed a unique domain arrangement suggesting opportunities for the selective targeting of DXP synthase; however, reports on the kinetic mechanism are conflicting. Here, we present the results of tryptophan fluorescence binding and kinetic analyses of DXP synthase and propose a new model for substrate binding and mechanism. Our results are consistent with a random sequential kinetic mechanism, which is unprecedented in this enzyme class.

Reverse hydroxamate-based inhibitors of IspC, a key enzyme of the non-mevalonate pathway of isoprenoid biosynthesis and a validated antimalarial target, were synthesized and biologically evaluated. The binding mode of one derivative in complex with EcIspC and a divalent metal ion was clarified by X-ray analysis. Pilot experiments have demonstrated in vivo potential.

The natural antibiotic fosmidomycin acts via inhibition of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), an essential enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. Fosmidomycin is active on Mycobacterium tuberculosis DXR (MtDXR), but it lacks antibacterial activity probably because of poor uptake. α-Aryl substituted fosmidomycin analogues have more favorable physicochemical properties and are also more active in inhibiting malaria parasite growth. We have solved crystal structures of MtDXR in complex with 3,4-dichlorophenyl substituted fosmidomycin analogues; these show important differences compared to our previously described forsmidomycin-DXR complex. Our best inhibitor has an IC(50) = 0.15 μM on MtDXR but still lacked activity in a mycobacterial growth assay (MIC > 32 μg/mL). The combined results, however, provide insights into how DXR accommodates the new inhibitors and serve as an excellent starting point for the design of other novel and more potent inhibitors, particularly against pathogens where uptake is less of a problem, such as the malaria parasite.

1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is a novel target for developing new antibacterial (including antituberculosis) and antimalaria drugs. Forty-one lipophilic phosphonates, representing a new class of DXR inhibitors, were synthesized, among which 5-phenylpyridin-2-ylmethylphosphonic acid possesses the most activity against E. coli DXR (EcDXR) with a K(i) of 420 nM. Structure-activity relationships (SAR) are discussed, which can be rationalized using our EcDXR:inhibitor structures, and a predictive quantitative SAR (QSAR) model is also developed. Since inhibition studies of DXR from Mycobacterium tuberculosis (MtDXR) have not been performed well, 48 EcDXR inhibitors with a broad chemical diversity were found, however, to generally exhibit considerably reduced activity against MtDXR. The crystal structure of a MtDXR:inhibitor complex reveals the flexible loop containing the residues 198-208 has no strong interactions with the 3,4-dichlorophenyl group of the inhibitor, representing a structural basis for the reduced activity. Overall, these results provide implications in the future design and development of potent DXR inhibitors.

H(2)(18)O under the bridge: Recently, the deoxyxylulose phosphate (DXP) pathway was discovered to be a second pathway supplying isoprenoid biosynthetic precursors. One of steps is an IspG-catalyzed reductive deoxygenation of methylerythritol cyclodiphosphate (MEcPP) to 4-hydroxyl-3-methyl-2-(E)-1-diphosphate (HMBPP). Using [2-(13) C,(18) O]-MEcPP, we detected the positional isotopic exchange for the bridging oxygen in MEcPP., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Isopentenyl diphosphate is a precursor of various isoprenoids and is produced by the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in plastids of plants, protozoa and many eubacteria. A key enzyme in the MEP pathway, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), has been shown to be the target of fosmidomycin, which works as an antimalarial, antibacterial and herbicidal compound. In this paper, we report studies of kinetics and the crystal structures of the thermostable DXR from the hyperthermophile Thermotoga maritima. Unlike the mesophilic DXRs, Thermotoga DXR (tDXR) showed activity only with Mg(2+) at its growth temperature. We solved the crystal structures of tDXR with and without fosmidomycin. The structure without fosmidomycin but unexpectedly bound with 2-methyl-2,4-pentanediol (MPD), revealing a new extra space available for potential drug design. This structure adopted the closed form by rigid domain rotation but without the flexible loop over the active site, which was considered as a novel conformation. Further, the conserved Asp residue responsible for cation binding seemed to play an important role in adjusting the position of fosmidomycin. Taken together, our kinetic and the crystal structures illustrate the binding mode of fosmidomycin that leads to its slow, tight binding according to the conformational changes of DXR.

Isoprenoids are a highly diverse and important group of natural compounds. The enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) catalyzes a key regulatory step in the non-mevalonate isoprenoid biosynthetic pathway in eubacteria and in plant plastids. For example, in Artemisia annua DXR participates in regulation of the biosynthesis of artemisinin, an important antimalarial drug. We performed phylogenetic analysis using DXR protein sequences from a model prokaryote, Escherichia coli, a picoplanktonic alga, Ostreococcus lucimarinus, and higher plants. The functional domain of DXR was conserved, allowing molecular evolutionary comparisons of both prokaryotic and eukaryotic sequences of DXR. Despite this conservation, for some plant species such as Campthoteca acuminata and Arabidopsis thaliana, phylogenetic relationships of their lineages were consistently violated. Our analysis revealed that plant DXR has an N-terminal transit domain that is likely bipartite, consisting of a chloroplast transit peptide (cTP) and a lumen transit peptide (lTP). Several features observed in the lTP suggest that, while DXR is targeted to the chloroplast, it is localized to the thylakoid lumen. These features include a twin arginine motif, a hydrophobic region, and a proline-rich region. The transit peptide also showed putative motifs for a 14-3-3 binding site with a chaperone phosphorylation site at Thr.

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 A 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 A, beta = 117.8 degrees. Structural analysis by molecular replacement is in progress.

A three-dimensional model of the malarial drug target protein PfDXR was generated, and validated using structure-checking programs and protein docking studies. Structural and functional features unique to PfDXR were identified using the model and comparative sequence analyses with apicomplexan and non-apicomplexan DXR proteins. Furthermore, we have used the model to develop an efficient approach to screen for potential tool compounds for use in the rational design of novel DXR inhibitors.

Deliberate and natural outbreaks of infectious disease underscore the necessity of effective vaccines and antimicrobial/antiviral therapeutics. The prevalence of antibiotic resistant strains and the ease by which antibiotic resistant bacteria can be intentionally engineered further highlights the need for continued development of novel antibiotics against new bacterial targets. Isoprenes are a class of molecules fundamentally involved in a variety of crucial biological functions. Mammalian cells utilize the mevalonic acid pathway for isoprene biosynthesis, whereas many bacteria utilize the methylerythritol phosphate (MEP) pathway, making the latter an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP synthase, a MEP pathway enzyme and potential target for antibiotic development. In vitro growth-inhibition assays using fosmidomycin, an inhibitor of MEP synthase, illustrates the effectiveness of MEP pathway inhibition with F. tularensis. To facilitate drug development, F. tularensis MEP synthase was cloned, expressed, purified, and characterized. Enzyme assays produced apparent kinetic constants (K(M)(DXP) = 104 microM, K(M)(NADPH) = 13 microM, k(cat)(DXP) = 2 s(-1), k(cat)(NADPH) = 1.3 s(-1)), an IC(50) for fosmidomycin of 247 nM, and a K(i) for fosmidomycin of 99 nM. The enzyme exhibits a preference for Mg(+2) as a divalent cation. Titanium dioxide chromatography-tandem mass spectrometry identified Ser177 as a site of phosphorylation. S177D and S177E site-directed mutants are inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP synthase is an excellent target for the development of novel antibiotics against F. tularensis.

1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) in the non-mevalonate pathway found in most bacteria is a validated anti-infective drug target. Fosmidomycin, a potent DXR inhibitor, is active against Gram-negative bacteria. A coordination chemistry and structure based approach was used to discover a novel, lipophilic DXR inhibitor with an IC(50) of 1.4 microM. It exhibited a broad spectrum of activity against Gram-negative and -positive bacteria with minimal inhibition concentrations of 20-100 microM (or 3.7-19 microg/mL).

Pinus densiflora Siebold et Zucc. is the major green canopy species in the mountainous area of Korea. To assess the response of resin acid biosynthetic genes to mechanical and chemical stimuli, we cloned cDNAs of genes encoding enzymes involved in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway (1-deoxy-d-xylulose 5-phosphate synthase (PdDXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (PdDXR) and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (PdHDR)) by the rapid amplification of cDNA ends (RACE) technique. In addition, we cloned the gene encoding abietadiene synthase (PdABS) as a marker for the site of pine resin biosynthesis. PdHDR and PdDXS occurred as two gene families. In the phylogenetic trees, PdDXSs, PdDXR and PdHDRs each formed a separate clade from their respective angiosperm homologs. PdDXS2, PdHDR2 and PdDXR were most actively transcribed in stem wood, whereas PdABS was specifically transcribed. The abundance of PdDXS2 transcripts in wood in the resting state was generally 50-fold higher than the abundance of PdDXS1 transcripts, and PdHDR2 transcripts were more abundant by an order of magnitude in wood than in other tissues, with the ratio of PdHDR2 to PdHDR1 transcripts in wood being about 1. Application of 1 mM methyl jasmonate (MeJA) selectively enhanced the transcript levels of PdDXS2 and PdHDR2 in wood. The ratios of PdDXS2 to PdDXS1 and PdHDR2 to PdHDR1 reached 900 and 20, respectively, on the second day after MeJA treatment, whereas the transcript level of PdABS increased twofold by 3 days after MeJA treatment. Wounding of the stem differentially enhanced the transcript ratios of PdDXS2 to PdDXS1 and PdHDR2 to PdHDR1 to 300 and 70, respectively. The increase in the transcript levels of the MEP pathway genes in response to wounding was accompanied by two orders of magnitude increase in PdABS transcripts. These observations indicated that resin acid biosynthesis activity, represented by PdABS transcription, was correlated with the selective transcriptions of PdDXS2 and PdHDR2. Introduction of PdDXS2, PdHDR1 and PdHDR2 rescued their respective knockout Escherichia coli mutants, confirming that at least these three genes were functionally active. Intracellular targeting of the green fluorescent protein fused to the N-terminal 100 amino acid residues of these genes in the Arabidopsis transient expression system showed that the proteins were all targeted to the chloroplasts. Our results suggest that the MEP pathway regulates resin biosynthesis in the wood of P. densiflora by differential transcription of the multiple PdDXS and PdHDR genes.

1-Deoxy-d-xylulose 5-phosphate (DXP) reductoisomerase (DXR, also known as methyl-d-erythritol 4-phosphate (MEP) synthase) is a NADPH-dependent enzyme, which catalyzes the conversion of DXP to MEP in the nonmevalonate pathway of isoprene biosynthesis. Two mechanisms have been proposed for the DXR-catalyzed reaction. In the alpha-ketol rearrangement mechanism, the reaction begins with deprotonation of the C-3 hydroxyl group followed by a 1,2-migration to give methylerythrose phosphate, which is then reduced to MEP by NADPH. In the retroaldol/aldol rearrangement mechanism, DXR first cleaves the C3-C4 bond of DXP in a retroaldol manner to generate a three-carbon and a two-carbon phosphate bimolecular intermediate. These two species are then reunited by an aldol reaction to form a new C-C bond, yielding an aldehyde intermediate. Subsequent reduction by NADPH affords MEP. To differentiate these mechanisms, we have prepared [3-(2)H]- and [4-(2)H]-DXP and carried out a competitive secondary kinetic isotope effect (KIE) study of the DXR reaction. The normal 2 degrees KIEs observed for [3-(2)H]- and [4-(2)H]-DXP provide compelling evidence supporting a retroaldol/aldol mechanism for the rearrangement catalyzed by DXR, with the rate-limiting step being cleavage of the C3-C4 bond of DXP.

In mycobacteria, the biosynthesis of the precursors to the essential isoprenoids, isopentenyl diphosphate and dimethylallyl pyrophosphate is carried out by the methylerythritol phosphate pathway. This route of synthesis is absent in humans, who utilize the alternative mevalonate acid route, thus making the enzymes of the methylerythritol phosphate pathway of chemotherapeutic interest. One such identified target is the second enzyme of the pathway, 1-deoxy-D-xylulose 5-phosphate reductoisomerase. Only limited information is currently available concerning the catalytic mechanism and structural dynamics of this enzyme, and only recently has a crystal structure of Mycobacterium tuberculosis species of this enzyme been resolved including all factors required for binding. Here, the dynamics of the enzyme is studied in complex with NADPH, Mn2+, in the presence and absence of the fosmidomycin inhibitor using conventional molecular dynamics and an enhanced sampling technique, reversible digitally filtered molecular dynamics. The simulations reveal significant differences in the conformational dynamics of the vital catalytic loop between the inhibitor-free and inhibitor-bound enzyme complexes and highlight the contributions of conserved residues in this region. The substantial fluctuations observed suggest that 1-deoxy-D-xylulose 5-phosphate reductoisomerase may be a promising target for computer-aided drug discovery through the relaxed complex method.

Fosmidomycin and its homologue FR900098 are inhibitors of 1-deoxy-D-xylulose-5-phosphate reductoisomerase, which is part of the mevalonate-independent isoprenoid biosynthetic pathway. Replacement of the phosphonate moiety by uncharged sulfone or sulfonamide partial structures resulted in complete loss of activity. Dropping one of the two negative charges resulted in a marked decrease in activity. Through occupation of a hydrophobic binding site, some activity could be regained, leading to compounds with micromolar activity against cultured malaria parasites.

2C-Methyl-D-erythritol-4-phosphate synthase, encoded by the ispC gene (also designated dxr), catalyzes the first committed step in the nonmevalonate isoprenoid biosynthetic pathway. The reaction involves the isomerization of 1-deoxy-D-xylulose 5-phosphate, giving a branched-chain aldose derivative that is subsequently reduced to 2C-methyl-D-erythritol 4-phosphate. The isomerization step has been proposed to proceed as an intramolecular rearrangement or a retroaldol-aldol sequence. We report the preparation of (13)C-labeled substrate isotopologs that were designed to optimize the detection of an exchange of putative cleavage products that might occur in the hypothetical retroaldol-aldol reaction sequence. In reaction mixtures containing large amounts of 2C-methyl-D-erythritol-4-phosphate synthase from Escherichia coli, Mycobacterium tuberculosis or Arabidopsis thaliana, and a mixture of [1-(13)C(1)]-2C-methyl-D-erythritol 4-phosphate and [3-(13)C(1)]2C-methyl-D-erythritol 4-phosphate, the reversible reaction could be followed over thousands of reaction cycles. No fragment exchange could be detected by NMR spectroscopy, and the frequency of exchange, if any, is less than 5 p.p.m. per catalytic cycle. Hydroxyacetone, the putative second fragment expected from the retroaldol cleavage, was not incorporated into the enzyme product. In contrast to other reports, IspC did not catalyze the isomerisation of 1-deoxy-D-xylulose 5-phosphate to give 1-deoxy-L-ribulose 5-phosphate under any conditions tested. However, we could show that the isomerization reaction proceeds at room temperature without a requirement for enzyme catalysis. Although a retroaldol-aldol mechanism cannot be ruled out conclusively, the data show that a retroldol-aldol reaction sequence would have to proceed with very stringent fragment containment that would apply to the enzymes from three genetically distant organisms.

1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR, EC: 1.1.1.267) is the second enzyme in the 2C-methyl-d-erythritol 4-phosphate (MEP) pathway, one of the two pathways in plants that can produce isoprenoids. The MEP pathway is the source of isoprene emitted from leaves, but rubber production is believed to result primarily from the mevalonic acid (MVA) pathway. Two cDNAs for DXR designated HbDXR1 and HbDXR2 were isolated from leaves and latex of rubber tree using RT-PCR based methods. Both cDNAs contain an open reading frame (ORF) of 1416bp encoding 471 amino acids with a molecular mass of about 51kDa. The deduced HbDXRs show extensive sequence similarities to that of other plant DXRs (73-87% identity). Molecular modeling revealed that the two HbDXRs contain all typical characteristics of DXR and share spatial structures, which are very similar to that of Escherichia coli DXR. Phylogenetic and DNA gel blot analyses suggested that a duplication of the DXR gene has occurred in the rubber tree. Semi-quantitative RT-PCR analysis showed that the HbDXR genes are differentially regulated in various tissues of the rubber tree. The HbDXR2 was more highly expressed in clone RRIM 600 than in the wild type, and this is consistent with higher rubber content of this clone. While 2-chloroethane phosphonic acid (ethephon) significantly increased latex yield, it only transiently induced the HbDXR2 gene. The expression of HbDXR2 in the latex suggests its important role in isoprenoid biosynthesis by substrate molecules, indicating that the MEP pathway may have some indirect roles in the biosynthesis of rubber.

As the second enzyme of the non-mevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis, DXP reductoisomerase (DXR, EC: 1.1.1.267) catalyzes a committed step of the MEP pathway for camptothecin (CPT) biosynthesis. In order to understand more about the role of DXR involved in the CPT biosynthesis at the molecular level, the full-length DXR cDNA sequence (designated as CaDXR) was isolated and characterized for the first time from a medicinal Nyssaceae plant species, Camptotheca acuminata. The full-length cDNA of CaDXR was 1823 bp containing a 1416 bp open reading frame (ORF) encoding a polypeptide of 472 amino acids. Comparative and bioinformatic analyses revealed that CaDXR showed extensive homology with DXRs from other plant species and contained a conserved transit peptide for plastids, an extended Pro-rich region and a highly conserved NADPH binding motif in its N-terminal region owned by all plant DXRs. Phylogenetic analysis indicated that CaDXR was more ancient than other plant DXRs. Tissue expression pattern analysis revealed that CaDXR expressed strongly in stem, weak in leaf and root. CaDXR was found to be an elicitor-responsive gene, which could be induced by exogenous elicitor of methyl jasmonate. The functional color complementation assay indicated that CaDXR could accelerate the biosynthesis of carotenoids in the Escherichia coli transformant, demonstrating that DXP reductoisomerase plays an influential step in isoprenoid biosynthesis.

Isopentenyl diphosphate is the precursor of various isoprenoids that are essential to all living organisms. It is produced by the mevalonate pathway in humans but by an alternate route in plants, protozoa, and many bacteria. 1-deoxy-D-xylulose-5-phosphate reductoisomerase catalyzes the second step of this non-mevalonate pathway, which involves an NADPH-dependent rearrangement and reduction of 1-deoxy-D-xylulose 5-phosphate to form 2-C-methyl-D-erythritol 4-phosphate. The use of different pathways, combined with the reported essentiality of the enzyme makes the reductoisomerase a highly promising target for drug design. Here we present several high resolution structures of the Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase, representing both wild type and mutant enzyme in various complexes with Mn(2+), NADPH, and the known inhibitor fosmidomycin. The asymmetric unit corresponds to the biological homodimer. Although crystal contacts stabilize an open active site in the B molecule, the A molecule displays a closed conformation, with some differences depending on the ligands bound. An inhibition study with fosmidomycin resulted in an estimated IC(50) value of 80 nm. The double mutant enzyme (D151N/E222Q) has lost its ability to bind the metal and, thereby, also its activity. Our structural information complemented with molecular dynamics simulations and free energy calculations provides the framework for the design of new inhibitors and gives new insights into the reaction mechanism. The conformation of fosmidomycin bound to the metal ion is different from that reported in a previously published structure and indicates that a rearrangement of the intermediate is not required during catalysis.

The crystal structure of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) from Escherichia coli complexed with Mg(2+), NADPH and fosmidomycin was solved at 2.2 A resolution. DXR is the key enzyme in the 2-C-methyl-D-erythritol 4-phosphate pathway and is an effective target of antimalarial drugs such as fosmidomycin. In the crystal structure, electron density for the flexible loop covering the active site was clearly observed, indicating the well ordered conformation of DXR upon substrate binding. On the other hand, no electron density was observed for the nicotinamide-ribose portion of NADPH and the position of Asp149 anchoring Mg(2+) was shifted by NADPH in the active site.

The recently discovered non-mevalonate pathway of isoprenoid biosynthesis serves as the unique source of terpenoids in numerous pathogenic eubacteria and in apicoplast-type protozoa, most notably Plasmodium, but is absent in mammalian cells. It is therefore an attractive target for anti-infective chemotherapy. The first committed step of the non-mevalonate pathway is catalyzed by 2C-methyl-D-erythritol 4-phosphate synthase (IspC). Using photometric and NMR spectroscopic assays, we screened extracts of Mediterranean plants for inhibitors of the enzyme. Strongest inhibitory activity was found in leaf extracts of Cercis siliquastrum.

The ispC gene of Arabidopsis thaliana was expressed in pseudomature form without the putative plastid-targeting sequence in a recombinant Escherichia coli strain. The recombinant protein was purified by affinity chromatography and was shown to catalyze the formation of 2C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate at a rate of 5.6 micromol x min(-1) x mg(-1) (k(cat) 4.4 s(-1)). The Michaelis constants for 1-deoxy-D-xylulose 5-phosphate and the cosubstrate NADPH are 132 and 30 microm, respectively. The enzyme has an absolute requirement for divalent metal ions, preferably Mn2+ and Mg2+, and is inhibited by fosmidomycin with a Ki of 85 nm. The pH optimum is 8.0. NADH can substitute for NADPH, albeit at a low rate (14% as compared to NADPH). The enzyme catalyzes the reverse reaction at a rate of 2.1 micromol x min(-1) x mg(-1).

1-deoxy-D-xylulose 5-phosphate reductoisomerase catalyzes the NADPH-dependent rearrangement and reduction of 1-deoxy-D-xylulose 5-phosphate to form 2-C-methyl-D-erythritol 4-phosphate, as the second step of the deoxyxylulose 5-phosphate/methylerythritol 4-phosphate pathway found in many bacteria and plants. The end product, isopentenyl diphosphate, is the precursor of various isoprenoids vital to all living organisms. The pathway is not found in humans; the mevalonate pathway is instead used for the formation of isopentenyl diphosphate. This difference, combined with its essentiality, makes the reductoisomerase an excellent drug target in a number of pathogenic organisms. The structure of 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Mycobacterium tuberculosis (Rv2870c) was solved by molecular replacement and refined to a resolution of 1.9 A. The enzyme exhibited an estimated kcat of 5.3 s-1 and Km and kcat/Km values of 7.2 microM and 7.4x10(5) M-1 s-1 for NADPH and 340 microM and 1.6x10(4) M-1 s-1 for 1-deoxy-D-xylulose 5-phosphate. In the structure, a sulfate is bound at the expected site of the phosphate moiety of the sugar substrate. The M. tuberculosis enzyme displays a similar fold to the previously published structures from Escherichia coli and Zymomonas mobilis. Comparisons offer suggestions for the design of specific drugs. Furthermore, the new structure represents an intermediate conformation between the open apo form and the closed holo form observed previously, giving insights into the conformational changes associated with catalysis.

The methylerythritol phosphate pathway to isoprenoids has been firmly established as an alternate to the mevalonate pathway in many bacteria, plants, algae, and the malaria parasite Plasmodium falciparum. The second enzyme in this pathway, deoxy-D-xylulose 5-phosphate reductoisomerase (DXR; E.C. 1.1.1.267), has been the focus of many investigations since it was found to be the target of the antibacterial and antimalarial compound, fosmidomycin. Several x-ray crystal structures of the Escherichia coli and Zymomonas mobilis DXR enzymes have provided important structural information about the residues potentially involved in substrate binding and catalysis. Site-directed mutagenesis studies can be used to complement the structural studies, providing kinetic data for specific changes of active site residues. Active site mutants were prepared of the recombinant Synechocystis sp. PCC6803 DXR, targeting residues D152, S153, E154, H155, M206, and E223. Alteration of the three acidic residues had major effects on catalysis, changes to S153 and M206 had variable effects on binding and catalysis, and a H155A mutation had only minimal effects on the kinetic parameters.

Diterpenoid ginkgolides having potent platelet-activating factor antagonist activity are major active ingredients of ginkgo extract. Class 2-type 1-deoxy-D-xylulose 5-phosphate synthase (GbDXS2) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (GbDXR), the first two enzymes in 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, operating in the earlier step of ginkgolide biosynthesis, were cloned from embryonic roots of Ginkgo biloba through a homology-based polymerase chain reaction for role assessment of the enzymes. Plasmids harboring each gene rescued the respective knockout E. coli mutants. The levopimaradiene synthase gene (LPS), responsible for the first committed step in ginkgolide biosynthesis, and GbDXS2 were transcribed exclusively in embryonic root, suggesting a specific role of GbDXS2 in ginkgolide biosynthesis. GbDXR retained a higher transcription level in roots than in leaves, whereas class 1 DXS (GbDXS1) showed 30 to 50 % higher level in leaves. Ginkgolides and bilobalide were found both in leaves and roots from an earlier stage of the embryo culture. Exclusive transcription of ginkgolide biosynthesis-specific LPS and GbDXS2 in roots and the appearance of ginkgolides in leaves was consistent with translocation of the compounds from roots to leaves.

The second enzyme in the methylerythritol phosphate pathway to isoprenoids, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR; EC 1.1.1.267) mediates the transformation of 1-deoxy-D-xylulose 5-phosphate (DXP) into 2-C-methyl-D-erythritol 4-phosphate. Several DXR mutants have been prepared to study amino acid residues important in binding or catalysis, but in-depth studies of many conserved residues in the flexible loop portion of the enzyme have not been conducted. In the course of our studies of this enzyme, an analog of DXP, 1,2-dideoxy-D-threo-3-hexulose 6-phosphate (1-methyl-DXP), was found to be a weak competitive inhibitor. Using the X-ray crystal structures of DXR as a guide, a highly conserved tryptophan residue in the flexible loop was identified that potentially blocks the use of this analog as a substrate. To test this hypothesis, four mutants of the Synechocystis sp. PCC6803 DXR were prepared and a W204F mutant was found to utilize the analog as a substrate.

The inhibition of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) by fosmidomycin was studied by using a kinetic assay based on the consumption of NADPH and synthetic substrate. Fosmidomycin is a slow tight-binding inhibitor of DXR that shows strong negative cooperativity (absolute value(h) = 0.3) in binding. Cooperativity is displayed during the initial (weak, K0.5 = 10 microM) binding event and does not change as the binding tightens to the equilibrium value of 0.9 nM over a period of seconds to minutes. A series of fosmidomycin fragments was examined, but all showed much weaker inhibition, in the mM range. A series of cyclic fosmidomycin analogues was also synthesised and tested, but these showed high-microM binding at best. None of the synthetic compounds showed time-dependent inhibition. We concluded that the slow tight-binding behaviour, and perhaps also cooperativity, are mediated by significant reorganisation of the active site upon fosmidomycin binding. This makes the rational design of new inhibitors of DXR difficult at best.

The mechanism of the reaction catalyzed by 2-C-methyl-d-erythritol 4-phosphate (MEP) synthase from Escherichia coli has been studied by steady-state and single-turnover kinetic experiments for the 1-deoxy-d-xylulose 5-phosphoric acid (DXP) analogues, 1,1,1-trifluoro-1-deoxy-d-xylulose 5-phosphoric acid (CF(3)-DXP), 1,1-difluoro-1-deoxy-d-xylulose 5-phosphoric acid (CF(2)-DXP), 1-fluoro-1-deoxy-d-xylulose 5-phosphoric acid (CF-DXP), and 1,2-dideoxy-d-hexulose 6-phosphate (Et-DXP). CF(3)-DXP, CF(2)-DXP, and Et-DXP were poor inhibitors, most likely because of the increase in steric bulk at C1 of DXP. The three analogues were also poor substrates for the enzyme. In contrast, CF-DXP was a good substrate (k(cat)(CF)(-)(DXP) = 37 +/- 2 s(-)(1), K(m)(CF)(-)(DXP) = 227 +/- 25 microM) for MEP synthase when compared to DXP (k(cat)(DXP) = 29 +/- 1 s(-)(1), K(m)(DXP) = 45 +/- 4 microM). A primary deuterium isotope effect was observed under single-turnover conditions when CF-DXP was incubated with 4S-[(2)H]NADPH ((H)k/(D)k = 1.34 +/-0.01), whereas no isotope effect was observed upon incubation with DXP and 4S-[(2)H]NADPH ((H)k/(D)k = 1.02 +/- 0.02). The reaction did not exhibit burst kinetics for either substrate, indicating that product release is not rate-limiting. These studies suggest that positive charge does not develop at C2 of DXP during catalysis. In addition, the isotope effect with CF-DXP and 4S-[(2)H]NADPH but not DXP indicates that the rearrangement step, which precedes hydride transfer, is rate-limiting for DXP but becomes partially rate-limiting for CF-DXP. Thus, rearrangement appears to be enhanced by substitution of a hydrogen atom in the methyl group of DXP by fluorine. These observations are consistent with a retro-aldol/aldol mechanism for the rearrangement during conversion of DXP to MEP.

We present structure-activity relationships for 43 inhibitors of 1-deoxyxylulose-5-phosphate (DOXP)-reductoisomerase, derived from protein-based docking, ligand-based 3D QSAR, and a combination of both approaches as realized by AFMoC (adaptation of fields for molecular comparison). DOXP-reductoisomerase (DXR) is a key enzyme of the non-mevalonate pathway for isoprenoid building blocks. This target has been characterized as having potential in the treatment of malaria with fosmidomycin, an established DXR inhibitor, presently in clinical trials. As part of an effort to optimize the properties of fosmidomycin, analogues have been synthesized and tested to gain further insights into the primary determinants of structural affinity. These data have been used to create a predictive model for DXR inhibition applying data taken from several DXR X-ray structures. These structures still leave the active fosmidomycin conformation and detailed reaction mechanism undetermined. This together with the small inhibitor data set provides a major challenge for presently available docking programs and 3D QSAR tools. To overcome these difficulties we have applied the AFMoC protocol. AFMoC makes more efficient use of available modeling data by tailoring DrugScore knowledge-based potentials specifically toward a given protein using inhibitor potency data. While 3D QSAR methods achieved valid models which lack predictivity, AFMoC was found to provide superior performance, based both on cross-validation runs as well as for inhibitors not considered in the training set. In particular, AFMoC's ability to gradually transform between generally applicable unadapted interaction fields to case specifically adapted ones proved to be of major importance. Using 50% tailored fields was found to permit the precise prediction of binding affinities for related ligands without losing the capability to estimate the affinities of structurally distinct inhibitors.

[structure: see text] Four deoxyxylulose phosphate (DXP) analogues were synthesized and evaluated as substrates/inhibitors for methylerythritol phosphate (MEP) synthase. In analogues CF(3)-DXP (1), CF(2)-DXP (2), and CF-DXP (3), the three methyl hydrogens at C1 of DXP were sequentially replaced by fluorine. In the fourth analogue, Et-DXP (4), the methyl group in DXP was replaced by an ethyl moiety. Analogues 1, 2, and 4 were not substrates for MEP synthase under normal catalytic conditions and were instead modest inhibitors with IC(50) values of 2.0, 3.4, and 6.2 mM, respectively. In contrast, 3 was a good substrate (k(cat) = 38 s(-)(1), K(m) = 227 muM) with a turnover rate similar to that of the natural substrate. These results are consistent with a retro-aldol/aldol mechanism rather than an alpha-ketol rearrangement for the enzyme-catalyzed conversion of DXP to MEP.

The key enzyme in the non-mevalonate pathway of isoprenoid biosynthesis, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) has been shown to be the target enzyme of fosmidomycin, an antimalarial, antibacterial and herbicidal compound. Here we report the crystal structure of selenomethionine-labelled Escherichia coli DXR in a ternary complex with NADPH and fosmidomycin at 2.2 A resolution. The structure reveals a considerable conformational rearrangement upon fosmidomycin binding and provides insights into the slow, tight binding inhibition mode of the inhibitor. Although the inhibitor displays an unusual non-metal mediated mode of inhibition, which is an artefact most likely due to the low metal affinity of DXR at the pH used for crystallization, the structural data add valuable information for the rational design of novel DXR inhibitors. Using this structure together with the published structural data and the 1.9 A crystal structure of DXR in a ternary complex with NADPH and the substrate 1-deoxy-D-xylulose 5-phosphate, a model for the physiologically relevant tight-binding mode of inhibition is proposed. The structure of the substrate complex must be interpreted with caution due to the presence of a second diastereomer in the active site.

The methylerythritol phosphate pathway to isoprenoids, an alternate biosynthetic route present in many bacteria, algae, plants, and the malarial parasite Plasmodium falciparum, has become an attractive target for the development of new antimalarial and antibacterial compounds. The second enzyme in this pathway, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR; EC 1.1.1.267), has been shown to be the molecular target for fosmidomycin, a promising antimalarial drug. This enzyme converts 1-deoxy-D-xylulose 5-phosphate (DXP) into the branched compound 2-C-methyl-D-erythritol 4-phosphate (MEP). The transformation of DXP into MEP requires an isomerization, followed by a NADPH-dependent reduction. The discovery of DXR, its subsequent characterization, and the identification of inhibitors will be presented.

Deoxyxylulose 5-phosphate (DXP) analogs were synthesized and evaluated as alternative substrates and inhibitors of recombinant Synechocystis PCC6803 DXP reductoisomerase (DXR; EC 1.1.1.267). Five of the compounds tested (1,2-dideoxy-D-threo-3-hexulose 6-phosphate, 1-deoxy-l-ribulose 5-phosphate, 2S,3R-dihydroxybutyramide 4-phosphate, 4S-hydroxypentan-2-one 5-phosphate, and 3S-hydroxypentan-2-one 5-phosphate) acted as relatively weak competitive inhibitors when compared to fosmidomycin. A sixth compound, 3R,4S-dihydroxy-5-oxohexylphosphonic acid, served as an alternate substrate, as has recently been reported for the same compound with Escherichia coli DXR.

Isopentenyl pyrophosphate (IPP) is a universal building block for the ubiquitous isoprenoids that are essential to all organisms. The enzymes of the non-mevalonate pathway for IPP synthesis, which is unique to many pathogenic bacteria, have recently been explored as targets for antibiotic development. Several crystal structures of 2C-methyl-D-erythritol-2,4-cyclophosphate (MECDP) synthase, the fifth of seven enzymes involved in the non-mevalonate pathway for synthesis of IPP, have been reported; however, the composition of metal ions in the active site and the presence of a hydrophobic cavity along the non-crystallographic threefold symmetry axis has varied between the reported structures. Here, the structure of MEDCP from Shewanella oneidensis MR1 (SO3437) was determined to 1.6 A resolution in the absence of substrate. The presence of a zinc ion in the active-site cleft, tetrahedrally coordinated by two histidine side chains, an aspartic acid side chain and an ambiguous fourth ligand, was confirmed by zinc anomalous diffraction. Based on analysis of anomalous diffraction data and typical metal-to-ligand bond lengths, it was concluded that an octahedral sodium ion was 3.94 A from the zinc ion. A hydrophobic cavity was observed along the threefold non-crystallographic symmetry axis, filled by a well defined non-protein electron density that could be modeled as farnesyl pyrophosphate (FPP), a downstream product of IPP, suggesting a possible feedback mechanism for enzyme regulation. The high-resolution data clarified the FPP-binding mode compared with previously reported structures. Multiple sequence alignment indicated that the residues critical to the formation of the hydrophobic cavity and for coordinating the pyrophosphate group of FPP are present in the majority of MEDCP synthase enzymes, supporting the idea of a specialized biological function related to FPP binding in a subfamily of MEDCP synthase homologs.

We have investigated the docking of a variety of inhibitors and substrates to the isoprene biosynthesis pathway enzymes farnesyl diphosphate synthase (FPPS), isopentenyl diphosphate/dimethylallyl diphosphate isomerase (IPPI) and deoxyxylulose-5-phosphate reductoisomerase (DXR) using the Lamarckian genetic alogorithm program, AutoDock. The docked ligand structures are predicted with a approximately 0.8 A rms deviation from the structures determined crystallographically. The errors found are a function of the number of atoms in the ligand (R = 0.91, p < 0.0001) and, to a lesser extent, on the resolution of the crystallographic structure (R = 0.70, p < 0.008). The structures of three isoprenoid diphosphates docked to the FPPS enzyme reveal strong electrostatic interactions with Mg(2+), lysine and arginine active site residues. Similar results are obtained with the docking of four IPPI inhibitors to the IPPI enzyme. The DXR substrate, deoxyxylulose-5-phosphate, is found to dock to Mn(2+)-NADPH-DXR in an almost identical manner as does the inhibitor fosimdomycin to Mn(2+)-DXR (ligand heavy atom rms deviation = 0.90 A) and is poised to interact with NADPH. Bisphosphonate inhibitors are found to bind to the allylic binding sites in both eukaryotic and prokaryotic FPPSs, in good accord with recent crystallographic results (a 0.4 A rms deviation from the X-ray structure with the E. coli enzyme). Overall, these results show for the first time that the geometries of a broad variety of phosphorus-containing inhibitors and substrates of isoprene biosynthesis pathway enzymes can be well predicted by using computational methods, which can be expected to facilitate the design of novel inhibitors of these enzymes.

We have obtained the single-crystal X-ray crystallographic structures of the bisphosphonates [(1-isoquinolinylamino)methylene]-1,1-bisphosphonate and [[(5-chloro-2-pyridinyl)amino]methylene]-1,1-bisphosphonate, bound to the enzyme 1-deoxyxylulose-5-phosphate reductoisomerase (DXR, EC 1.1.1.267, also known as 2-C-methyl-d-erythritol-4-phosphate synthase), an important target for the development of antimalarial drugs. Our results indicate that both bisphosphonates bind into the fosmidomycin binding site. The aromatic groups are in a shallow hydrophobic pocket, and the phosphonate groups are involved in electrostatic interactions with Mg2+ or a cluster of carboxylic acid groups and lysine while the fosmidomycin phosphonate-binding site is occupied by a sulfate ion (as also observed in the DXR/NADP+ structure). The availability of these two new crystal structures opens up the possibility of the further development of bisphosphonates and related systems as DXR inhibitors and, potentially, as antiinfective agents.

1-Deoxy-D-xylulose-5-phosphate (DXP) isomeroreductase catalyzes the isomerization and reduced nicotinamide adenine dinucleotide phosphate- (NADPH-) dependent reduction of DXP to generate 2-C-methylerythritol 4-phosphate (MEP) in the first committed step of the MEP pathway of isoprenoid biosynthesis. We have cloned the gene encoding the Mycobacterium tuberculosis DXP isomeroreductase, expressed the protein in Escherichia coli, and purified the enzyme to homogeneity using conventional column chromatography methods. DXP isomeroreductase is a metal ion-activated enzyme displaying superior specificity for Co(2+), good specificity for Mn(2+), and poor specificity for Mg(2+). Although NADPH is preferred over reduced nicotinamide adenine dinucleotide (NADH) about 100-fold as evaluated by the relative k(cat)/K(m) values, the maximum turnover numbers are similar, suggesting that the 2'-phosphate of NADPH contributes predominantly to binding and not to catalysis. While k(cat) was independent of pH in the region 6.0

1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) is the second enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. The structure of the apo-form of this enzyme from Zymomonas mobilis has been solved and refined to 1.9-A resolution, and that of a binary complex with the co-substrate NADPH to 2.7-A resolution. The subunit of DXR consists of three domains. Residues 1-150 form the NADPH binding domain, which is a variant of the typical dinucleotide-binding fold. The second domain comprises a four-stranded mixed beta-sheet, with three helices flanking the sheet. Most of the putative active site residues are located on this domain. The C-terminal domain (residues 300-386) folds into a four-helix bundle. In solution and in the crystal, the enzyme forms a homo-dimer. The interface between the two monomers is formed predominantly by extension of the sheet in the second domain. The adenosine phosphate moiety of NADPH binds to the nucleotide-binding fold in the canonical way. The adenine ring interacts with the loop after beta1 and with the loops between alpha2 and beta2 and alpha5 and beta5. The nicotinamide ring is disordered in crystals of this binary complex. Comparisons to Escherichia coli DXR show that the two enzymes are very similar in structure, and that the active site architecture is highly conserved. However, there are differences in the recognition of the adenine ring of NADPH in the two enzymes.

(3,4)-3,4-Dihydroxy-5-oxohexylphosphonic acid, an isosteric analogue of 1-deoxy-D-xylulose 5-phosphate (DXP), was obtained in enantiomerically pure form from (+)-2,3--benzylidene--threitol by a seven-step sequence. This phosphonate did not affect the growth of. It did not inhibit the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), but was converted by this enzyme into (3,4)-3,4,5-trihydroxy-3-methylpentylphosphonic acid, an isosteric analogue of 2-C-methyl-D-erythritol 4-phosphate. The enzyme was, however, less efficient with the methylene phosphonate analogue than with the natural substrate.

The dxr gene encoding the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) from the cyanobacterium Synechocystis sp. PCC6803 was expressed in Escherichia coli to produce both the native and N-terminal histidine-tagged forms of DXR. The enzymes were purified from the cell extracts using either anion exchange chromatography or metal affinity chromatography and gel filtration. The purified recombinant native and histidine-tagged enzymes each displayed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, corresponding to the calculated subunit molecular weights of 42,500 and 46,700, respectively. By native PAGE, both enzymes were dimers under reducing conditions. The kinetic properties for the enzymes were characterized and only minor variations were observed, demonstrating that the N-terminal histidine tag does not greatly affect the activity of the enzyme. Both enzymes had similar properties to previously characterized reductoisomerases from other sources. The K(m)'s for the metal ions Mn(2+), Mg(2+), and Co(2+) were determined for native DXR for the first time, with the K(m) for Mg(2+) being approximately 200-fold higher than the K(m)'s for Mn(2+) and Co(2+).

1-Deoxy-D-xylulose-5-phosphate (DXP) is a key intermediate in the non-mevalonate pathway to terpenoids in bacteria, and it is the substrate for the enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXP-R). In order to study the mechanism of DXP-R, we required a flexible synthesis of the substrate which would allow the incorporation of isotopic labels, and the variation of the two stereocentres. Thus 1,4-dihydroxypent-2-yne was selectively reduced to give the E-olefin, and selective phosphorylation of the primary alcohol followed by oxidation of the secondary alcohol gave a substrate suitable for dihydroxylation. Dihydroxylation using stoichiometric OsO4 in the presence of chiral ligands gave protected DXP in high ee. Final hydrogenolysis gave DXP in quantitative yield and high purity. DXP-R was produced by rapid cloning of the dxr gene from Escherichia coli through controlled expression and ion exchange chromatography. The synthetic DXP was fully active in enzyme assays catalysed by recombinant DXP-R.

2-C-Methyl-d-erythritol 4-phosphate synthase (IspC) is the first enzyme committed to isoprenoid biosynthesis in the methylerythritol phosphate pathway, which represents an alternative route to the classical mevalonate pathway. As it is present in many pathogens and plants, but not in man, this pathway has attracted considerable interest as a target for novel antibiotics and herbicides. Fosmidomycin represents a specific high-affinity inhibitor of IspC. Very recently, its anti-malaria activity in man has been demonstrated in clinical trials. Here, we present the crystal structure of Escherichia coli IspC in complex with manganese and fosmidomycin at 2.5 A resolution. The (N-formyl-N-hydroxy)amino group provides two oxygen ligands to manganese that is present in a distorted octahedral coordination, whereas the phosphonate group is anchored in a specific pocket by numerous hydrogen bonds. Both sites are connected by a spacer of three methylene groups. The substrate molecule, 1-d-deoxyxylulose 5-phosphate, can be superimposed onto fosmidomycin, explaining the stereochemical course of the reaction.

The key enzyme in the nonmevalonate pathway, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), has been shown to be an effective target of antimalarial drugs. Here we report the crystal structure of DXR complexed with NADPH and a sulfate ion from Escherichia coli at 2.2 A resolution. The structure showed the presence of an extra domain, which is absent from other NADPH-dependent oxidoreductases, in addition to the conformation of catalytic residues and the substrate binding site. A flexible loop covering the substrate binding site plays an important role in the enzymatic reaction and the determination of substrate specificity.