Expression, characterization and inhibition of Toxoplasma gondii 1-deoxy-D-xylulose-5-phosphate reductoisomerase.
Ontology highlight
ABSTRACT:
SUBMITTER: Cai G
PROVIDER: S-EPMC3602143 | biostudies-literature | 2013 Apr
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Toxoplasma gondii is a widely distributed apicomplexan parasite causing toxoplasmosis, a critical health issue for immunocompromised individuals and for congenitally infected foetuses. Current treatment options are limited in number and associated with severe side effects. Thus, novel anti-toxoplasma agents need to be identified and developed. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is considered the rate-limiting enzyme in the non-mevalonate pathway for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate in the parasite, and has been previously investigated for its key role as a novel drug target in some species, encompassing Plasmodia, Mycobacteria and Escherichia coli. In this study, we present the first crystal structure of T. gondii DXR (TgDXR) in a tertiary complex with the inhibitor fosmidomycin and the cofactor NADPH in dimeric conformation at 2.5 Å resolution revealing the inhibitor binding mode. In addition, we biologically characterize reverse α-phenyl-β-thia and β-oxa fosmidomycin analogues and show that some derivatives are strong inhibitors of TgDXR which also, in contrast with fosmidomycin, inhibit the growth of T. gondii in vitro. Here, ((3,4-dichlorophenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)thio)methyl)phosphonic acid was identified as the most potent anti T. gondii compound. These findings will enable the future design and development of more potent anti-toxoplasma DXR inhibitors.
| S-EPMC11346426 | biostudies-literature
Project description:The methylerythritol phosphate (MEP) pathway found in many bacteria governs the synthesis of isoprenoids, which are crucial lipid precursors for vital cell components such as ubiquinone. Because mammals synthesize isoprenoids via an alternate pathway, the bacterial MEP pathway is an attractive target for novel antibiotic development, necessitated by emerging antibiotic resistance as well as biodefense concerns. The first committed step in the MEP pathway is the reduction and isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to methylerythritol phosphate (MEP), catalyzed by MEP synthase. To facilitate drug development, we cloned, expressed, purified, and characterized MEP synthase from Yersinia pestis. Enzyme assays indicate apparent kinetic constants of KMDXP = 252 µM and KMNADPH = 13 µM, IC50 values for fosmidomycin and FR900098 of 710 nM and 231 nM respectively, and Ki values for fosmidomycin and FR900098 of 251 nM and 101 nM respectively. To ascertain if the Y. pestis MEP synthase was amenable to a high-throughput screening campaign, the Z-factor was determined (0.9) then the purified enzyme was screened against a pilot scale library containing rationally designed fosmidomycin analogs and natural product extracts. Several hit molecules were obtained, most notably a natural product allosteric affector of MEP synthase and a rationally designed bisubstrate derivative of FR900098 (able to associate with both the NADPH and DXP binding sites in MEP synthase). It is particularly noteworthy that allosteric regulation of MEP synthase has not been described previously. Thus, our discovery implicates an alternative site (and new chemical space) for rational drug development.
| S-EPMC4149570 | biostudies-literature
Project description:Fosmidomycin, a potent inhibitor of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), has antibacterial and antimalaria activity. Due to its poor pharmacokinetics, more lipophilic DXR inhibitors are needed. However, the hydrophobic binding site(s) in DXR remains elusive. Here, pyridine/quinoline containing phosphonates are identified to be DXR inhibitors with IC(50) values as low as 840 nM. We also report three DXR:inhibitor structures, revealing a novel binding mode. The indole group of Trp211 is found to move ~4.6 Å to open up a mainly hydrophobic pocket, where the pyridine/quinoline rings of the inhibitors are located and have strong π-π stacking/charge-transfer interactions with the indole. Docking studies demonstrate our structures could be used to predict the binding modes of other lipophilic DXR inhibitors. Overall, this work shows an important role of Trp211 in inhibitor recognition and provides a structural basis for future drug design and development.
| S-EPMC3046873 | biostudies-literature
Project description: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.
| S-EPMC3601441 | biostudies-literature
Project description:Isothermal titration calorimetry (ITC) was used to investigate the binding of six inhibitors to 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), a target for developing novel anti-infectives. The binding of hydroxamate inhibitors to E. coli DXR is Mg(2+)-dependent, highly endothermic (ΔH: 22.7-24.3 kJ/mol) and entropy-driven, while that of non-hydroxamate compounds is metal ion independent and exothermic (ΔH: -19.4- -13.8 kJ/mol), showing hydration/dehydration of the enzyme metal ion binding pocket account for the drastic ΔH change. However, for DXRs from Plasmodium falciparum and Mycobacterium tuberculosis, the binding of all inhibitors is exothermic (ΔH: -24.9 - -9.2 kJ/mol), suggesting the metal ion binding sites of these two enzymes are considerably less hydrated. The dissociation constants measured by ITC are well correlated with those obtained by enzyme inhibition assays (R(2) = 0.75). Given the rapid rise of antibiotic resistance, this work is of interest since it provides novel structural implications for rational development of potent DXR inhibitors.
| S-EPMC3462030 | biostudies-literature
Project description: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.
| S-EPMC2788227 | biostudies-literature
Project description:The 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR; EC1.1.1.267), an NADPH-dependent reductase, plays a pivotal role in the methylerythritol 4-phosphate pathway (MEP), in the conversion of 1-deoxy-d-xylulose-5-phosphate (DXP) into MEP. Photochemical profiles, as well as pharmaceutical activities of Centella asiatica (L.), one of the most valuable medicinal plants, divulge the presence of secondary metabolites called Centellosides. Despite well-studied pharmaceutical activities, not much is known about the genes responsible for the synthesis of these compounds. In the present study, the full-length DXR gene sequence (JQ965955) of Centella submitted in NCBI was characterized using various bioinformatics tools and tissue specific differential expression studies were also carried out. The full-length CDNA of CaDXR contains an open reading frame (ORF) of 1425 bp which encodes a peptide of 474 amino acids. The molecular weight of this protein was found to be 51.5 kDa with isoelectric point of 6.33. The protein contains three conserved domain, namely NADPH (GSTGSIGT and LAAGSNV), substrate binding (LPADSEHSAI and NKGLEVIEAHY) and Cys-Ser-(Ala/Met/Val/Thr) cleavage-site domains. Phylogenetic studies of CaDXR sequence show close homology with DXR sequence of Angelica sinensis and Daucus carota subsp sativus as they all belong to Apiaceae family. In silico analysis predicted that CaDXR protein contains 21 α-helix and 11 β-sheets and further DXR protein model was validated by Ramachandran plot analysis. The results of molecular dynamics (MD) simulations unveil dynamic stability of the proposed model and docking studies suggest that the NDP cofactor tightly binds in the active site of the protein with a strong network of hydrogen and hydrophobic interactions. The expression studies by semi-RT followed by qRT-PCR suggests that CaDXR is differentially expressed in different tissues (with maximal expression in the node and lowest in the roots). Thus, characterization and structure-function analysis of DXR gene in Centella facilitate us to understand not only the functions of DXR gene but also regulatory mechanisms involved in the MEP pathway in C. asiatica plant at the molecular level.Supplementary informationThe online version contains supplementary material available at 10.1007/s13205-021-02723-w.
| S-EPMC7984138 | biostudies-literature
Project description:1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), which catalyzes the first committed step in the 2-C-methyl-d-erythritol 4-phosphate pathway of isoprenoid biosynthesis used by Mycobacterium tuberculosis and other infectious microorganisms, is absent in humans and therefore an attractive drug target. Fosmidomycin is a nanomolar inhibitor of DXR, but despite great efforts, few analogues with comparable potency have been developed. DXR contains a strictly conserved residue, Trp203, within a flexible loop that closes over and interacts with the bound inhibitor. We report that while mutation to Ala or Gly abolishes activity, mutation to Phe and Tyr only modestly impacts kcat and Km. Moreover, pre-steady-state kinetics and primary deuterium kinetic isotope effects indicate that while turnover is largely limited by product release for the wild-type enzyme, chemistry is significantly more rate-limiting for W203F and W203Y. Surprisingly, these mutants are more sensitive to inhibition by fosmidomycin, resulting in Km/Ki ratios up to 19-fold higher than that of wild-type DXR. In agreement, isothermal titration calorimetry revealed that fosmidomycin binds up to 11-fold more tightly to these mutants. Most strikingly, mutation strongly tips the entropy-enthalpy balance of total binding energy from 50% to 75% and 91% enthalpy in W203F and W203Y, respectively. X-ray crystal structures suggest that these enthalpy differences may be linked to differences in hydrogen bond interactions involving a water network connecting fosmidomycin's phosphonate group to the protein. These results confirm the importance of the flexible loop, in particular Trp203, in ligand binding and suggest that improved inhibitor affinity may be obtained against the wild-type protein by introducing interactions with this loop and/or the surrounding structured water network.
| S-EPMC4045324 | biostudies-literature
Project description:The rate-limiting enzyme of the 2-methyl-d-erythritol-4-phosphate (MEP) terpenoid biosynthetic pathway, 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), provides the perfect target for screening new antibacterial substances. In this study, we tested the DXR inhibitory effect of 35 plant essential oils (EOs), which have long been recognized for their antimicrobial properties. The results show that the EOs of Zanbthoxylum bungeanum (ZB), Schizonepetae tenuifoliae (ST), Thymus quinquecostatus (TQ), Origanum vulgare (OV), and Eugenia caryophyllata (EC) displayed weak to medium inhibitory activity against DXR, with IC50 values of 78 μg/mL, 65 μg/mL, 59 μg/mL, 48 μg/mL, and 37 μg/mL, respectively. GC-MS analyses of the above oils and further DXR inhibitory activity tests of their major components revealed that eugenol (EC) and carvacrol (TQ and OV) possess medium inhibition against the protein (68.3% and 55.6%, respectively, at a concentration of 20 μg/mL), whereas thymol (ST, TQ, and OV), carveol (ZB), and linalool (ZB, ST, and OV) only exhibited weak inhibition against DXR, at 20 μg/mL (23%-26%). The results add more details to the antimicrobial mechanisms of plant EOs, which could be very helpful in the direction of the reasonable use of EOs in the food industry and in the control of phytopathogenic microbials.
| S-EPMC6681031 | biostudies-literature
Project description: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.
| S-EPMC2982673 | biostudies-literature