Non-mevalonate isoprenoid biosynthesis: enzymes, genes and inhibitors

The essential steps of the novel non-mevalonate pathway of isopentenyl diphosphate and isoprenoid biosynthesis in plants are described. The first five enzymes and genes of this 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway are known. The herbicide fosmidomycin specifically blocks the second enzyme, the DOXP reductoisomerase. The DOXP/MEP pathway is also present in several pathogenic bacteria and the malaria parasite. Hence, all herbicides and inhibitors blocking this novel isoprenoid pathway in plants are also potential drugs against malaria and diseases caused by pathogenic bacteria.     

The methylerythritol phosphate pathway for isoprenoid biosynthesis in coccidia: presence and sensitivity to fosmidomycin

The apicoplast is a recently discovered, plastid-like organelle present in most apicomplexa. The methylerythritol phosphate (MEP) pathway involved in isoprenoid biosynthesis is one of the metabolic pathways associated with the apicoplast, and is a new promising therapeutic target in Plasmodium falciparum. Here, we check the presence of isoprenoid genes in four coccidian parasites according to genome database searches. Cryptosporidium parvum and C. hominis, which have no plastid genome, lack the MEP pathway. In contrast, gene expression studies suggest that this metabolic pathway is present in several development stages of Eimeria tenella and in tachyzoites of Toxoplasma gondii. We studied the potential of fosmidomycin, an antimalarial drug blocking the MEP pathway, to inhibit E. tenella and T. gondii growth in vitro. The drug was poorly effective even at high concentrations. Thus, both fosmidomycin sensitivity and isoprenoid metabolism differs substantially between apicomplexan species.     

The crystal structure of E.coli 1-deoxy-D-xylulose-5-phosphate reductoisomerase in a ternary complex with the antimalarial compound fosmidomycin and NADPH reveals a tight-binding closed enzyme conformation

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.     

Synthesis and evaluation of alpha,beta-unsaturated alpha-aryl-substituted fosmidomycin analogues as DXR inhibitors

Fosmidomycin, which acts through inhibition of 1-deoxy-D-xylulose phosphate reductoisomerase (DXR) in the non-mevalonate pathway, represents a valuable recent addition to the armamentarium against uncomplicated malaria. In this paper, we describe the synthesis and biological evaluation of E- and Z-alpha,beta-unsaturated alpha-aryl-substituted analogues of FR900098, a fosmidomycin congener, utilizing a Stille or a Suzuki coupling to introduce the aryl group. In contrast with our expectations based on the promising activity earlier observed for several alpha-substituted fosmidomycin analogues, all synthesized analogues exhibited much lower binding affinity for DXR than fosmidomycin.     

磷甘霉素和洛伐它汀处理对中国红豆杉悬浮培养细胞生物合成紫杉醇的影响

采用非甲羟戊酸途径抑制剂磷甘霉素和甲羟戊酸途径抑制剂洛伐它汀对中国红豆杉悬浮细胞培养物进行处理.在添加和未添加茉莉酸甲酯诱导的情况下,前者使紫杉醇产量减少了2/5和1/5,后者使紫杉醇产量减少了1/6和1/10,表明两种途径对紫杉醇的生物合成都具有贡献,其中非甲羟戊酸途径贡献较大;通过定量PCR技术分别检测两条途径的关键酶5-磷酸脱氧木酮糖还原异构酶(DXR)和3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)mRNA水平的变化,发现两种抑制剂都能够激活hmgr和dxr的转录,表明两种代谢途径之间存在协同作用,共同为紫杉醇的生物合成提供前体.     

Amino acid based prodrugs of a fosmidomycin surrogate as antimalarial and antitubercular agents

Fosmidomycin is a natural antibiotic with promising IspC (DXR, 1-deoxy-d-xylulose-5-phosphate reductoisomerase) inhibitory activity. This enzyme catalyzes the first committed step of the non-mevalonate isoprenoid biosynthesis pathway, which is essential in Plasmodium falciparum and Mycobacterium tuberculosis. Mainly as a result of its high polarity, fosmidomycin displays suboptimal pharmacokinetic properties. Furthermore, fosmidomycin is inactive against M. tuberculosis as a result of its inability to penetrate the bacterial cell wall. Temporarily masking the phosphonate moiety as a prodrug has the potential to solve both issues. We report the application of two amino acid based prodrug approaches on a fosmidomycin surrogate. Conversion of the phosphonate moiety into tyrosine-derived esters increases the in vitro activity against asexual blood stages of P. falciparum, while phosphonodiamidate prodrugs display promising antitubercular activities. Selected prodrugs were tested in vivo in a P. berghei malaria mouse model. These results indicate good in vivo antiplasmodial potential.     

The non-mevalonate isoprenoid biosynthesis of plants as a test system for new herbicides and drugs against pathogenic bacteria and the malaria parasite

Higher plants and several photosynthetic algae contain the plastidic 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate pathway (DOXP/MEP pathway) for isoprenoid biosynthesis. The first four enzymes and their genes are known of this novel pathway. All of the ca. 10 enzymes of this isoprenoid pathway are potential targets for new classes of herbicides. Since the DOXP/MEP pathway also occurs in several pathogenic bacteria, such as Mycobacterium tuberculosis, and in the malaria parasite Plasmodium falciparum, all inhibitors and potential herbicides of the DOXP/MEP pathway in plants are also potential drugs against pathogenic bacteria and the malaria parasite. Plants with their easily to handle DOXP/MEP-pathway are thus very suitable test-systems also for new drugs against pathogenic bacteria and the malaria parasite as no particular security measures are required. In fact, the antibiotic herbicide fosmidomycin specifically inhibited not only the DOXP reductoisomerase in plants, but also that in bacteria and in the parasite P. falciparum, and cures malaria-infected mice. This is the first successful application of a herbicide of the novel isoprenoid pathway as a possible drug against malaria.     

Production of mevalonate by a metabolically-engineered Escherichia coli

Mevalonate is biosynthesized from acetyl-CoA and metabolized to isoprenoid compounds in a wide variety of organisms although certain types of prokaryotes employ another route for isoprenoid biosynthesis (the non-mevalonate pathway). To establish a fermentative process for mevalonate production, enzymes for mevalonate synthesis from Enterococcus faecalis were expressed in Escherichia coli, a non-mevalonate pathway bacterium. Mevalonate was accumulated, indicating a redirection of acetate metabolism by the expressed enzyme. The recombinant E. coli produced 47 g mevalonate l(-1) in 50 h of fed-batch cultivation in a 2 l jar fermenter; this is the highest titer ever reported demonstrating the superiority of E. coli in its ability of acetyl-CoA supply and its inability is degrade mevalonate.     

Mechanism and inhibition of 1-deoxy-d-xylulose-5-phosphate reductoisomerase

The non-mevalonate or 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway is responsible for generating isoprenoid precursors in plants, protozoa, and bacteria. Because this pathway is absent in humans, its enzymes represent potential targets for the development of herbicides and antibiotics. 1-Deoxy-d-xylulose (DXP) reductoisomerase (DXR) is a particularly attractive target that catalyzes the pathway’s first committed step: the sequential isomerization and NADPH-dependent reduction of DXP to MEP. This article provides a comprehensive review of the mechanistic and structural investigations on DXR, including its discovery and validation as a drug target, elucidation of its chemical and kinetic mechanisms, characterization of inhibition by the natural antibiotic fosmidomycin, and identification of structural features that provide the molecular basis for inhibition of and catalysis.     

Properties and inhibition of the first two enzymes of the non-mevalonate pathway of isoprenoid biosynthesis

Enzymes of the 1-deoxy-D-xylulose 5-phosphate/2-C-methylerythritol 4-phosphate (DOXP/MEP) pathway are targets for new herbicides and antibacterial drugs. Until now, no inhibitors for the DOXP synthase have been known of. We show that one of the breakdown products of the herbicide clomazone affects the DOXP synthase. One inhibitor of the non-mevalonate pathway, fosmidomycin, blocks the DOXP reductoisomerase (DXR) of plants and bacteria. The I(50) values of plants are, however, higher than those found for the DXR of Escherichia coli. The DXR of plants, isolated from barley seedlings, shows a pH optimum of 8.1, which is typical for enzymes active in the chloroplast stroma.     

Kinetic characterization of Synechocystis sp. PCC6803 1-deoxy-D-xylulose 5-phosphate reductoisomerase mutants

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.     

The methylerythritol phosphate pathway is functionally active in all intraerythrocytic stages of Plasmodium falciparum

Two genes encoding the enzymes 1-deoxy-D-xylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase have been recently identified, suggesting that isoprenoid biosynthesis in Plasmodium falciparum depends on the methylerythritol phosphate (MEP) pathway, and that fosmidomycin could inhibit the activity of 1-deoxy-D-xylulose-5-phosphate reductoisomerase. The metabolite 1-deoxy-D-xylulose-5-phosphate is not only an intermediate of the MEP pathway for the biosynthesis of isopentenyl diphosphate but is also involved in the biosynthesis of thiamin (vitamin B1) and pyridoxal (vitamin B6) in plants and many microorganisms. Herein we report the first isolation and characterization of most downstream intermediates of the MEP pathway in the three intraerythrocytic stages of P. falciparum. These include, 1-deoxy-D-xylulose-5-phosphate, 2-C-methyl-D-erythritol-4-phosphate, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol-2-phosphate, and 2-C-methyl-D-erythritol-2,4-cyclodiphosphate. These intermediates were purified by HPLC and structurally characterized via biochemical and electrospray mass spectrometric analyses. We have also investigated the effect of fosmidomycin on the biosynthesis of each intermediate of this pathway and isoprenoid biosynthesis (dolichols and ubiquinones). For the first time, therefore, it is demonstrated that the MEP pathway is functionally active in all intraerythrocytic forms of P. falciparum, and de novo biosynthesis of pyridoxal in a protozoan is reported. Its absence in the human host makes both pathways very attractive as potential new targets for antimalarial drug development.     

Chemical rescue of malaria parasites lacking an apicoplast defines organelle function in blood-stage Plasmodium falciparum

Plasmodium spp parasites harbor an unusual plastid organelle called the apicoplast. Due to its prokaryotic origin and essential function, the apicoplast is a key target for development of new anti-malarials. Over 500 proteins are predicted to localize to this organelle and several prokaryotic biochemical pathways have been annotated, yet the essential role of the apicoplast during human infection remains a mystery. Previous work showed that treatment with fosmidomycin, an inhibitor of non-mevalonate isoprenoid precursor biosynthesis in the apicoplast, inhibits the growth of blood-stage P. falciparum. Herein, we demonstrate that fosmidomycin inhibition can be chemically rescued by supplementation with isopentenyl pyrophosphate (IPP), the pathway product. Surprisingly, IPP supplementation also completely reverses death following treatment with antibiotics that cause loss of the apicoplast. We show that antibiotic-treated parasites rescued with IPP over multiple cycles specifically lose their apicoplast genome and fail to process or localize organelle proteins, rendering them functionally apicoplast-minus. Despite the loss of this essential organelle, these apicoplast-minus auxotrophs can be grown indefinitely in asexual blood stage culture but are entirely dependent on exogenous IPP for survival. These findings indicate that isoprenoid precursor biosynthesis is the only essential function of the apicoplast during blood-stage growth. Moreover, apicoplast-minus P. falciparum strains will be a powerful tool for further investigation of apicoplast biology as well as drug and vaccine development.     

类异戊二烯非甲羟戊酸代谢途径的分子生物学研究进展

近期发现的类异戊二烯非甲羟戊酸代谢途径是类异戊二烯化合物生成合成的另一途径。文章对该代谢途径的分子生物学研究进展进行了综述。重点介绍非甲羟戊酸代谢途径的发现和5－磷酸脱氧木糖合成酶、5－磷酸脱氧木糖还原异构醇、异戊二烯焦磷酸合成酶的分子克隆的最新进展以及非甲羟戊酸代谢途径的发现在农业和医药等领域的应用。     

1-Deoxy-D-xylulose 5-phosphate reductoisomerase and plastid isoprenoid biosynthesis during tomato fruit ripening

The recently discovered 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the biosynthesis of plastid isoprenoids (including carotenoids) is not fully elucidated yet despite its central importance for plant life. It is known, however, that the first reaction completely specific to the pathway is the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) into MEP by the enzyme DXP reductoisomerase (DXR). We have identified a tomato cDNA encoding a protein with homology to DXR and in vivo activity, and show that the levels of the corresponding DXR mRNA and encoded protein in fruit tissues are similar before and during the massive accumulation of carotenoids characteristic of fruit ripening. The results are consistent with a non-limiting role of DXR, and support previous work proposing DXP synthase (DXS) as the first regulatory enzyme for plastid isoprenoid biosynthesis in tomato fruit. Inhibition of DXR activity by fosmidomycin showed that plastid isoprenoid biosynthesis is required for tomato fruit carotenogenesis but not for other ripening processes. In addition, dormancy was reduced in seeds from fosmidomycin-treated fruit but not in seeds from the tomato yellow ripe mutant (defective in phytoene synthase-1, PSY1), suggesting that the isoform PSY2 might channel the production of carotenoids for abscisic acid biosynthesis. Furthermore, the complete arrest of tomato seedling development using fosmidomycin confirms a key role of the MEP pathway in plant development.     

Crystal structure of Brucella abortus deoxyxylulose-5-phosphate reductoisomerase-like (DRL) enzyme involved in isoprenoid biosynthesis

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.     

Dxr is essential in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>and fosmidomycin resistance is due to a lack of uptake

   

Fosmidomycin is a phosphonic antibiotic which inhibits 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr), the first committed step of the non-mevalonate pathway of isoprenoid biosynthesis. In Mycobacterium tuberculosis Dxr is encoded by Rv2870c, and although the antibiotic has been shown to inhibit the recombinant enzyme [1], mycobacteria are intrinsically resistant to fosmidomycin at the whole cell level. Fosmidomycin is a hydrophilic molecule and in many bacteria its uptake is an active process involving a cAMP dependent glycerol-3-phosphate transporter (GlpT). The fact that there is no glpT homologue in the M. tuberculosis genome and the highly impervious nature of the hydrophobic mycobacterial cell wall suggests that resistance may be due to a lack of cellular penetration.     

Pre-Steady-State Kinetic Analysis of 1-Deoxy-d-xylulose-5-phosphate Reductoisomerase from Mycobacterium tuberculosis Reveals Partially Rate-Limiting Product Release by Parallel Pathways

As part of the non-mevalonate pathway for the biosynthesis of the isoprenoid precursor isopentenyl pyrophosphate, 1-deoxy-d-xylulose-5-phosphate (DXP) reductoisomerase (DXR) catalyzes the conversion of DXP into 2-C-methyl-d-erythritol 4-phosphate (MEP) by consecutive isomerization and NADPH-dependent reduction reactions. Because this pathway is essential to many infectious organisms but is absent in humans, DXR is a target for drug discovery. In an attempt to characterize its kinetic mechanism and identify rate-limiting steps, we present the first complete transient kinetic investigation of DXR. Stopped-flow fluorescence measurements with Mycobacterium tuberculosis DXR (MtDXR) revealed that NADPH and MEP bind to the free enzyme and that the two bind together to generate a nonproductive ternary complex. Unlike the Escherichia coli orthologue, MtDXR exhibited a burst in the oxidation of NADPH during pre-steady-state reactions, indicating a partially rate-limiting step follows chemistry. By monitoring NADPH fluorescence during these experiments, the transient generation of MtDXR·NADPH·MEP was observed. Global kinetic analysis supports a model involving random substrate binding and ordered release of NADP(+) followed by MEP. The partially rate-limiting release of MEP occurs via two pathways-directly from the binary complex and indirectly via the MtDXR·NADPH·MEP complex-the partitioning being dependent on NADPH concentration. Previous mechanistic studies, including kinetic isotope effects and product inhibition, are discussed in light of this kinetic mechanism.     

Synthesis of tetrazole analogues of phosphonohydroxamic acids: An attempt to improve the inhibitory activity against the DXR

This work is focused on the design of new antimicrobial drugs and on the development of lipophilic inhibitors of the DXR, the second enzyme of the MEP pathway for the biosynthesis of isoprene units in most bacteria, by replacing the phosphonate group of fosmidomycin derivatives by a tetrazoyl moiety capable of multiple hydrogen bonding. The N- and C-substituted tetrazole analogues of phosphonohydroxamate inhibitors were synthesized and tested on the DXR of Escherichia coli. This work points out the hypothesis that the phosphonate/phosphate recognition site might be too rigid to accommodate other functional groups.