Enzyme In Methylerythritol Phosphate Pathway Research Articles

Positioning Bacillus subtilis as terpenoid cell factory.

SummaryIncreasing demands for bioactive compounds have motivated researchers to employ micro‐organisms to produce complex natural products. Currently, Bacillus subtilis has been attracting lots of attention to be developed into terpenoids cell factories due to its generally recognized safe status and high isoprene precursor biosynthesis capacity by endogenous methylerythritol phosphate (MEP) pathway. In this review, we describe the up‐to‐date knowledge of each enzyme in MEP pathway and the subsequent steps of isomerization and condensation of C5 isoprene precursors. In addition, several representative terpene synthases expressed in B. subtilis and the engineering steps to improve corresponding terpenoids production are systematically discussed. Furthermore, the current available genetic tools are mentioned as along with promising strategies to improve terpenoids in B. subtilis, hoping to inspire future directions in metabolic engineering of B. subtilis for further terpenoid cell factory development.

Apicoplast Metabolism: Parasite's Achilles' Heel.

Malaria continues to impinge heavily on mankind, with five continents still under its clasp. Widespread and rapid emergence of drug resistance in the Plasmodium parasite to current therapies accentuate the quest for novel drug targets and antimalarial compounds. Plasmodium parasites, maintain a non-photosynthetic relict organelle known as Apicoplast. Among the four major pathways of Apicoplast, biosynthesis of isoprenoids via Methylerythritol phosphate (MEP) pathway is the only indispensable function of Apicoplast that occurs during different stages of the malaria parasite. Moreover, the human host lacks MEP pathway. MEP pathway is a validated repertoire of novel antimalarial and antibacterial drug targets. Fosmidomycin, an efficacious antimalarial compound against IspC enzyme of MEP pathway is already in clinical trials as a combination drugs. Exploitation of other enzymes of MEP pathway would provide a much-needed impetus to the antimalarial drug discovery programs for the elimination of malaria. We outline the cardinal features of the MEP pathway enzymes and progress made towards the characterization of new inhibitors.

Structural modeling identifies Plasmodium vivax 4-diphosphocytidyl-2C-methyl-d-erythritol kinase (IspE) as a plausible new antimalarial drug target

Malaria parasites utilize Methylerythritol phosphate (MEP) pathway for synthesis of isoprenoid precursors which are essential for maturation and survival of parasites during erythrocytic and gametocytic stages. The absence of MEP pathway in the human host establishes MEP pathway enzymes as a repertoire of essential drug targets. The fourth enzyme, 4-diphosphocytidyl-2C-methyl-d-erythritol kinase (IspE) has been proved essential in pathogenic bacteria, however; it has not yet been studied in any Plasmodium species. This study was undertaken to investigate genetic polymorphism and concomitant structural implications of the Plasmodium vivax IspE (PvIspE) by employing sequencing, modeling and bioinformatics approach. We report that PvIspE gene displayed six non-synonymous mutations which were restricted to non-conserved regions within the gene from seven topographically distinct malaria-endemic regions of India. Phylogenetic studies reflected that PvIspE occupies unique status within Plasmodia genus and reflects that Plasmodium vivax IspE gene has a distant and non-conserved relation with human ortholog Mevalonate Kinase (MAVK). Structural modeling analysis revealed that all PvIspE Indian isolates have critically conserved canonical galacto-homoserine-mevalonate-phosphomevalonate kinase (GHMP) domain within the active site lying in a deep cleft sandwiched between ATP and CDPME-binding domains. The active core region was highly conserved among all clinical isolates, may be due to >60% β-pleated rigid architecture. The mapped structural analysis revealed the critically conserved active site of PvIspE, both sequence, and spacially among all Indian isolates; showing no significant changes in the active site. Our study strengthens the candidature of Plasmodium vivax IspE enzyme as a future target for novel antimalarials.

Genetic diversity and structural analysis of 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE) from Plasmodium falciparum.

Plasmodium parasite harbours unique methylerythritol phosphate (MEP) pathway which is obligatory for the biosynthesis of isoprenoids. In malaria parasites, the isoprenoids are indispensable during hepatic, erythrocytic and gametocytic stages. Owing to the criticality of MEP pathway and the potential of its enzymes to act as antimalarial drug target, this study comprehensively investigated the genetic diversity and structural composition of 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE), fourth enzyme of MEP pathway in Indian Plasmodium falciparum (PfIspE). The study employed sequencing, modeling and bioinformatics approaches to examine the genetic diversity and associated structural polymorphism in the PfIspE gene amplified from the clinical blood samples collected from seven malaria endemic geographical regions of India. The sequence analysis showed that PfIspE gene is highly conserved with 100% sequence identity among all the P. falciparum Indian isolates as well as with the PfIspE gene of reference strain 3D7. Phylogenetic analysis suggested that PfIspE is highly evolved and differ sufficiently from human orthologue mevalonate kinase gene. Structural modeling studies revealed that PfIspE has conserved ATP and CDPME-binding domains. The active site was observed to be relatively rigid in architecture with >60% β-pleated sheets. The results of genetic, phylogeny and modeling studies strengthen the potential of PfIspE enzyme as a promising antimalarial drug target.

Computational analysis, structural modeling and ligand binding site prediction of Plasmodium falciparum 1-deoxy-d-xylulose-5-phosphate synthase

Malaria remains one of the most serious infectious diseases in the world. There are five human species of the Plasmodium genus, of which Plasmodium falciparum is the most virulent and responsible for the vast majority of malaria related deaths. The unique biochemical processes that exist in Plasmodium falciparum provide a useful way to develop novel inhibitors. One such biochemical pathway is the methyl erythritol phosphate pathway (MEP), required to synthesize isoprenoid precursors. In the present study, a detailed computational analysis has been performed for 1-deoxy-d-xylulose-5-phosphate synthase, a key enzyme in MEP. The protein is found to be stable and residues from 825 to 971 are highly conserved across species. The homology model of the enzyme is developed using three web-based servers and Modeller software. It has twelve disordered regions indicating its druggability. Virtual screening of ZINC database identifies ten potential compounds in thiamine diphosphate binding region of the enzyme.

Molecular cloning, characterization and expression analysis of the gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Aquilaria sinensis (Lour.) Gilg.

The major constituents of agarwood oils are sesquiterpenes that are obtained from isoprenoid precursors through the plastidial methylerythritol phosphate (MEP) pathway and the cytosolic mevalonate pathway. In this study, a novel full-length cDNA of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), which was the second key enzyme in the plastid MEP pathway of sesquiterpenes biosynthesis was isolated from the stem of Aquilaria sinensis (Lour.) Gilg by the methods of reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) technique for the first time, and named as AsDXR. The full-length cDNA of AsDXR was 1768 bp, containing a 1437 bp open reading frame (ORF) encoding a polypeptide of 478 amino acids with a molecular weight of 51.859 kD and the theoretical isoelectric point of 6.29. Comparative and bioinformatic analysis of the deduced AsDXR protein showed extensive homology with DXRs from other plant species, especially Theobroma cacao and Gossypium barbadense, and contained a conserved transit peptide for plastids, and extended pro-rich region and a highly conserved NADPH-binding motif owned by all plant DXRs. Southern blot analysis indicated that AsDXR belonged to a small gene family. Tissue expression pattern analysis revealed that AsDXR expressed strongly in root and stem, but weakly in leaf. Additionally, AsDXR expression was found to be activated by exogenous elicitor of MeJA (methyl jasmonate). The contents of three sesquiterpenes (α-guaiene, α-humulene and Δ-guaiene) were significantly induced by MeJA. This study enables us to further elucidate the role of AsDXR in the biosynthesis of agarwood sesquiterpenes in A. sinensis at the molecular level.

Enhanced C30 carotenoid production in Bacillus subtilis by systematic overexpression of MEP pathway genes.

Creating novel biosynthetic pathways and modulating the synthesis of important compounds are one of the hallmarks of synthetic biology. Understanding the key parameters controlling the flux of chemicals throughout a metabolic pathway is one of the challenges ahead. Isoprenoids are the most functionally and structurally diverse group of natural products from which numerous medicines and relevant fine chemicals are derived. The well-characterized and broadly used production organism Bacillus subtilis forms an ideal background for creating and studying novel synthetic routes. In comparison to other bacteria, B. subtilis emits the volatile compound isoprene, the smallest representative of isoprenoids, in high concentrations and thus represents an interesting starting point for an isoprenoid cell factory. In this study, the effect of systematic overexpression of the genes involved in the methylerythritol phosphate (MEP) pathway on isoprenoid production in B. subtilis was investigated. B. subtilis strains harboring a plasmid containing C30 carotenoid synthetic genes, crtM and crtN, were combined with pHCMC04G plasmids carrying various synthetic operons of the MEP pathway genes. The levels of produced carotenoids, diaponeurosporene and diapolycopene, were used as indication of the role of the various enzymes on the flux of the MEP pathway. It was shown that the production of carotenoids can be increased significantly by overexpressing the MEP pathway enzymes. More broadly, the strains developed in this study can be used as a starting point for various isoprenoid cell factories.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-6531-3) contains supplementary material, which is available to authorized users.

A sugar phosphatase regulates the methylerythritol phosphate (MEP) pathway in malaria parasites.

Isoprenoid biosynthesis through the methylerythritol phosphate (MEP) pathway generates commercially important products and is a target for antimicrobial drug development. MEP pathway regulation is poorly understood in microorganisms. We employ a forward genetics approach to understand MEP pathway regulation in the malaria parasite, Plasmodium falciparum. The antimalarial fosmidomycin inhibits the MEP pathway enzyme deoxyxylulose 5-phosphate reductoisomerase (DXR). Fosmidomycin-resistant P. falciparum are enriched for changes in the PF3D7_1033400 locus (hereafter referred to as PfHAD1), encoding a homologue of haloacid dehalogenase (HAD)-like sugar phosphatases. We describe the structural basis for loss-of-function PfHAD1 alleles and find that PfHAD1 dephosphorylates a variety of sugar phosphates, including glycolytic intermediates. Loss of PfHAD1 is required for fosmidomycin resistance. Parasites lacking PfHAD1 have increased MEP pathway metabolites, particularly the DXR substrate, deoxyxylulose 5-phosphate. PfHAD1 therefore controls substrate availability to the MEP pathway. Because PfHAD1 has homologs in plants and bacteria, other HAD proteins may be MEP pathway regulators.

Methylerythritol Phosphate Pathway of Isoprenoid Biosynthesis

Isoprenoids are a class of natural products with more than 55,000 members. All isoprenoids are constructed from two precursors, isopentenyl diphosphate and its isomer dimethylallyl diphosphate. Two of the most important discoveries in isoprenoid biosynthetic studies in recent years are the elucidation of a second isoprenoid biosynthetic pathway [the methylerythritol phosphate (MEP) pathway] and a modified mevalonic acid (MVA) pathway. In this review, we summarize mechanistic insights on the MEP pathway enzymes. Because many isoprenoids have important biological activities, the need to produce them in sufficient quantities for downstream research efforts or commercial application is apparent. Recent advances in both MVA and MEP pathway-based synthetic biology are also illustrated by reviewing the landmark work of artemisinic acid and taxadien-5α-ol production through microbial fermentations.

A Whole-Cell Phenotypic Screening Platform for Identifying Methylerythritol Phosphate Pathway-Selective Inhibitors as Novel Antibacterial Agents

Isoprenoid biosynthesis is essential for survival of all living organisms. More than 50,000 unique isoprenoids occur naturally, with each constructed from two simple five-carbon precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Two pathways for the biosynthesis of IPP and DMAPP are found in nature. Humans exclusively use the mevalonate (MVA) pathway, while most bacteria, including all Gram-negative and many Gram-positive species, use the unrelated methylerythritol phosphate (MEP) pathway. Here we report the development of a novel, whole-cell phenotypic screening platform to identify compounds that selectively inhibit the MEP pathway. Strains of Salmonella enterica serovar Typhimurium were engineered to have separately inducible MEP (native) and MVA (nonnative) pathways. These strains, RMC26 and CT31-7d, were then used to differentiate MVA pathway- and MEP pathway-specific perturbation. Compounds that inhibit MEP pathway-dependent bacterial growth but leave MVA-dependent growth unaffected represent MEP pathway-selective antibacterials. This screening platform offers three significant results. First, the compound is antibacterial and is therefore cell permeant, enabling access to the intracellular target. Second, the compound inhibits one or more MEP pathway enzymes. Third, the MVA pathway is unaffected, suggesting selectivity for targeting the bacterial versus host pathway. The cell lines also display increased sensitivity to two reported MEP pathway-specific inhibitors, further biasing the platform toward inhibitors selective for the MEP pathway. We demonstrate development of a robust, high-throughput screening platform that combines phenotypic and target-based screening that can identify MEP pathway-selective antibacterials simply by monitoring optical density as the readout for cell growth/inhibition.

Francisella tularensis 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase: kinetic characterization and phosphoregulation.

Deliberate and natural outbreaks of infectious disease, the prevalence of antibiotic resistant strains, and the ease by which antibiotic resistant bacteria can be intentionally engineered all underscore the necessity of effective vaccines and continued development of novel antimicrobial/antiviral therapeutics. Isoprenes, a group of molecules fundamentally involved in a variety of crucial biological functions, are derived from either the mevalonic acid (MVA) or methylerythritol phosphate (MEP) pathway. While mammals utilize the MVA pathway, many bacteria utilize the MEP pathway, highlighting the latter as an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP cytidylyltransferase, a MEP pathway enzyme and potential target for antibiotic development. Size exclusion chromatography indicates the protein exists as a dimer in solution. Enzyme assays produced an apparent , , , , and a . The enzyme exhibits a strict preference for Mg+2 as a divalent cation and CTP as the nucleotide. Titanium dioxide chromatography-tandem mass spectrometry identified Thr141 as a site of phosphorylation. T141D and T141E site-directed mutants are catalytically inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP cytidylyltransferase is an excellent target for the development of novel antibiotics against F. tularensis.

Kinetic Characterization and Phosphoregulation of the Francisella tularensis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

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 (KMDXP = 104 µM, KMNADPH = 13 µM, kcatDXP = 2 s−1, kcatNADPH = 1.3 s−1), an IC50 for fosmidomycin of 247 nM, and a Ki 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.

Unraveling the Mechanisms of Isoprenoid Biosynthetic Enzymes: Mechanistic Studies of the Early Stage Enzymes

Isoprenoids (or terpenoids) are a large and structurally diverse class of biomolecules that are essential for the survival of all forms of life. Despite the vast differences in their final structures and functions, the early steps of isoprenoid biosynthesis in all organisms follow one of only two known biosynthetic pathways: the mevalonate pathway or the methyl erythritol phosphate (MEP) pathway. Interestingly, while humans utilize the mevalonate pathway, many human pathogens rely exclusively on the MEP pathway for the biosynthesis of their isoprenoid compounds. This has led to a number of mechanistic studies of the MEP-specific pathway enzymes, with the ultimate goal of developing small molecule inhibitors as potential drugs. In addition to their therapeutic value, many of the MEP pathway enzymes also catalyze unusual chemical transformations that are not well understood. In this review, we will highlight the recent work by us and others towards the elucidation of the catalytic mechanisms of several key enzymes involved in the early stages of isoprenoid biosynthesis. These include 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) and 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (IspH) of the MEP pathway, and the type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) from Staphylococcus aureus. The functions of these enzymes are validated or identified as potential drug targets.

Optimization of the transient transformation of Catharanthus roseus cells by particle bombardment and its application to the subcellular localization of hydroxymethylbutenyl 4-diphosphate synthase and geraniol 10-hydroxylase

The monoterpene indole alkaloids (MIA) synthesized in Catharanthus roseus are highly valuable metabolites due to their pharmacological properties. In planta, the MIA biosynthetic pathway exhibits a complex compartmentation at the cellular level, whereas subcellular data are sparse. To gain insight into this level of organization, we have developed a high efficiency green fluorescent protein (GFP) imaging approach to systematically localize MIA biosynthetic enzymes within C. roseus cells following a biolistic-mediated transient transformation. The biolistic transformation protocol has been first optimized to obtain a high number of transiently transformed cells with a ~12-fold increase compared to previous protocols and thus to clearly and easily identify the fusion GFP expression patterns in numerous cells. On the basis of this protocol, the subcellular localization of hydroxymethylbutenyl 4-diphosphate synthase (HDS), a methyl erythritol phosphate pathway enzyme and geraniol 10-hydroxylase (G10H), a monoterpene-secoiridoid pathway enzyme has been next characterized. Besides showing the accumulation of HDS within plastids of C. roseus cells, we also provide evidences of the presence of HDS in long stroma-filled thylakoid-free extensions budding from plastids, i.e. stromules that are in close association with other organelles such as endoplasmic reticulum (ER) or mitochondria in agreement with their proposed function in enhancing interorganelle metabolite exchanges. Furthermore, we also demonstrated that G10H is an ER-anchored protein, consistent with the presence of a transmembrane helix at the G10H N-terminal end, which is both necessary and sufficient to drive the ER anchoring.

Probing phosphorylation by non-mammalian isoprenoid biosynthetic enzymes using 1H–31P–31P correlation NMR spectroscopy

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 (1)H-(31)P-(31)P correlation NMR spectroscopy techniques for the direct characterization of crude MEP pathway enzyme products at low concentrations (200 microM to 5 mM) on a room temperature (non-cryogenic) NMR probe. Coupling the 100% natural abundance of the (31)P nucleus with the high intrinsic sensitivity of proton NMR, (1)H-(31)P-(31)P 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 (1)H-(31)P-(31)P correlation spectroscopy as a valuable tool for the characterization of other unnatural products in non-mammalian isoprenoid biosynthesis.

A mutant impaired in the production of plastome-encoded proteins uncovers a mechanism for the homeostasis of isoprenoid biosynthetic enzymes in Arabidopsis plastids.

The plastid-localized methylerythritol phosphate (MEP) pathway synthesizes the isoprenoid precursors for the production of essential photosynthesis-related compounds and hormones. We have identified an Arabidopsis thaliana mutant, rif1, in which posttranscriptional upregulation of MEP pathway enzyme levels is caused by the loss of function of At3g47450, a gene originally reported to encode a mitochondrial protein related to nitric oxide synthesis. However, we show that nitric oxide is not involved in the regulation of the MEP pathway and that the encoded protein is a plastid-targeted homolog of the Bacillus subtilis YqeH protein, a GTPase required for proper ribosome assembly. Consistently, in rif1 seedlings, decreased levels of plastome-encoded proteins were observed, with the exception of ClpP1, a catalytic subunit of the plastidial Clp protease complex. The unexpected accumulation of ClpP1 in plastids with reduced protein synthesis suggested a compensatory mechanism in response to decreased Clp activity levels. In agreement, a negative correlation was found between Clp protease activity and MEP pathway enzyme levels in different experiments, suggesting that Clp-mediated degradation of MEP pathway enzymes might be a mechanism used by individual plastids to finely adjust plastidial isoprenoid biosynthesis to their functional and physiological states.

Plastid Cues Posttranscriptionally Regulate the Accumulation of Key Enzymes of the Methylerythritol Phosphate Pathway in Arabidopsis

Plastid isoprenoids (including hormones and photosynthetic pigments) are essential for plant growth and development, but relatively little is known of how the production of their metabolic precursors via the recently elucidated methylerythritol phosphate (MEP) pathway is regulated. We have identified an Arabidopsis (Arabidopsis thaliana) mutant that survives an otherwise lethal block of the MEP pathway with fosmidomycin (FSM). In rif10 (resistant to inhibition with FSM 10) plants, the accumulation of flux-controlling enzymes of the pathway is posttranscriptionally up-regulated. Strikingly, this phenotype is linked to a lower accumulation of plastidial isoprenoid pigments such as chlorophylls and carotenoids, resulting in mutant plants that are paler and smaller than the wild type. The rif10 mutant is impaired in plastid RNA processing due to a T-DNA insertion in the coding region of the At3g03710 gene encoding the chloroplast-targeted exoribonuclease polyribonucleotide phosphorylase. FSM resistance and other rif10-like phenotypes were also observed in wild-type Arabidopsis, tomato (Lycopersicon esculentum), and rice (Oryza sativa) seedlings grown in the presence of sublethal concentrations of chloramphenicol (an inhibitor of protein synthesis in plastids). By contrast, treatment with norflurazon (an inhibitor of carotenoid biosynthesis causing a similar pale cotyledon phenotype) did not result in FSM resistance. Together, the results support that plastome-encoded proteins are involved in negatively regulating the posttranscriptional accumulation of specific nuclear-encoded MEP pathway enzymes in chloroplasts. Regulation of the MEP pathway by a mechanism dependent on plastid cues might function under physiological conditions to finely adjust plastidial isoprenoid biosynthesis to the metabolic capabilities or requirements of plastids.

Regulation of carotenoid biosynthesis in plants: evidence for a key role of hydroxymethylbutenyl diphosphate reductase in controlling the supply of plastidial isoprenoid precursors

Carotenoids are isoprenoid pigments that function as photoprotectors, precursors of the hormone abscisic acid (ABA), colorants and nutraceuticals. A major problem for the metabolic engineering of high carotenoid levels in plants is the limited supply of their isoprenoid precursor geranylgeranyl diphosphate (GGPP), formed by condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) units usually synthesized by the methylerythritol phosphate (MEP) pathway in plastids. Our earlier work with three of the seven MEP pathway enzymes suggested that the first reaction of the pathway catalyzed by deoxyxylulose 5-phosphate synthase (DXS) is limiting for carotenoid biosynthesis during tomato (Lycopersicon esculentum) fruit ripening. Here we investigate the contribution of the enzyme hydroxymethylbutenyl diphosphate reductase (HDR), which simultaneously synthesizes IPP and DMAPP in the last step of the pathway. A strong upregulation of HDR gene expression was observed in correlation with carotenoid production during both tomato fruit ripening and Arabidopsis thaliana seedling deetiolation. Constitutive overexpression of the tomato cDNA encoding HDR in Arabidopsis did not increase carotenoid levels in etioplasts. By contrast, light-grown transgenic plants showed higher carotenoid levels and an enhanced seed dormancy phenotype suggestive of increased ABA levels. The analysis of double transgenic Arabidopsis plants overproducing both the enzyme taxadiene synthase (which catalyzes the production of the non-native isoprenoid taxadiene from GGPP) and either HDR or DXS showed a twofold stronger effect of HDR in increasing taxadiene levels. Together, the data support a major role for HDR in controlling the production of MEP-derived precursors for plastid isoprenoid biosynthesis.

The MEP pathway: a new target for the development of herbicides, antibiotics and antimalarial drugs.

Isoprenoids, a diverse group of compounds derived from the five-carbon building units isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), are essential for survival in all organisms. Animals synthesize their isoprenoids from mevalonic acid (MVA), whereas most pathogenic bacteria and the malaria parasites utilize a completely different pathway for IPP and DMAPP synthesis, the methylerythritol phosphate (MEP) pathway. Plants use both pathways for the synthesis of isoprenoid precursors. The recent elucidation of the MEP pathway has opened the possibility to develop new strategies against microbial pathogens. Novel immunotherapeutic agents can be developed based on the MEP pathway intermediates known to activate the proliferation of human V-delta-9 V-gamma-2 T-cells after infection by many pathogenic bacteria and protozoa. Moreover, the design of specific inhibitors of MEP pathway enzymes (which are highly conserved but show no homology to mammalian proteins) should result in herbicides and drugs with broad-spectrum antimicrobial activity without mechanism-based toxicity to humans. A good example is the cure of bacterial infections and malaria with fosmidomycin, a highly stable inhibitor of the MEP pathway. The use of plants as test systems has led to the identification of additional inhibitors such as ketoclomazone. Biochemical, genetic and crystallographic approaches with the MEP pathway enzymes are now starting to characterize the inhibition kinetics and identify which residues play a structural or catalytic role. Current efforts should eventually contribute to an effective drug designed to fight against microbial pathogens that show resistance to currently available agents.

Identification of lethal mutations in Escherichia coli genes encoding enzymes of the methylerythritol phosphate pathway

The recently elucidated methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis is essential in eubacteria (including Escherichia coli), the malaria parasite, and plants, but is absent in animals. Therefore, the pathway enzymes are promising targets for the development of novel herbicides and antimicrobials that are potentially innocuous for humans. For an effective drug design, it is important to identify the residues required to preserve the structure and activity of the MEP pathway enzymes. Here, we report a genetic approach to identify such residues in E. coli. A strain harboring a synthetic operon that allows the production of isoprenoids through a MEP-independent pathway was used to screen for the otherwise lethal loss-of-function point mutations in the MEP pathway genes generated by ethylmethane sulfonate (EMS) mutagenesis. Besides confirming the role of residues involved in catalysis, our results define regions within several of the proteins with a potential key role for enzyme function.