Journal: PLoS ONE

Article Title: Francisella tularensis 2-C-Methyl-D-Erythritol 4-Phosphate Cytidylyltransferase: Kinetic Characterization and Phosphoregulation

doi: 10.1371/journal.pone.0020884

Figure Lengend Snippet: Michaelis-Menten plot of reaction velocity as a function of A) MEP concentration and B) CTP concentration. The solid line represents the nonlinear least-squares best fit of the data to the Michaelis-Menten equation. Each assay was performed in duplicate. The R 2 value for each plot is indicated. Kinetic parameters derived from the plots are listed in .

Article Snippet: MEP cytidylyltransferase activity was evaluated using a coupled-enzyme colorimetric assay, derived from that originally described by Bernal et al . To determine the apparent K M for 2-C-methyl-D-erythritol 4-phosphate (MEP), the assay (200 µL) was performed using 2.5 µM IspD, 100 mM Tris pH 8.0, 1 mM MgCl 2 , 1 mM DTT, 0.2 mM CTP, 100 mU/mL inorganic pyrophosphatase, and varying concentrations of MEP (Echelon Biosciences, Salt Lake City, UT).

Techniques: Concentration Assay, Derivative Assay

Journal: PLoS ONE

Article Title: Francisella tularensis 2-C-Methyl-D-Erythritol 4-Phosphate Cytidylyltransferase: Kinetic Characterization and Phosphoregulation

doi: 10.1371/journal.pone.0020884

Figure Lengend Snippet: Enzyme assays were performed with the indicated divalent cations at a fixed MEP (200 µM) and CTP (200 µM) concentration. Relative enzyme activity reveals the strict preference of the enzyme for Mg +2 . Each assay was performed in duplicate.

Article Snippet: MEP cytidylyltransferase activity was evaluated using a coupled-enzyme colorimetric assay, derived from that originally described by Bernal et al . To determine the apparent K M for 2-C-methyl-D-erythritol 4-phosphate (MEP), the assay (200 µL) was performed using 2.5 µM IspD, 100 mM Tris pH 8.0, 1 mM MgCl 2 , 1 mM DTT, 0.2 mM CTP, 100 mU/mL inorganic pyrophosphatase, and varying concentrations of MEP (Echelon Biosciences, Salt Lake City, UT).

Techniques: Concentration Assay, Activity Assay

Journal: PLoS ONE

Article Title: Francisella tularensis 2-C-Methyl-D-Erythritol 4-Phosphate Cytidylyltransferase: Kinetic Characterization and Phosphoregulation

doi: 10.1371/journal.pone.0020884

Figure Lengend Snippet: Enzyme assays were performed with the identified nucleotide at either 200 µM or 400 µM, as indicated. The assays contained a fixed MEP (200 µM) concentration. Each assay was performed in duplicate. Relative enzyme activity reveals the preference of the enzyme for CTP.

Article Snippet: MEP cytidylyltransferase activity was evaluated using a coupled-enzyme colorimetric assay, derived from that originally described by Bernal et al . To determine the apparent K M for 2-C-methyl-D-erythritol 4-phosphate (MEP), the assay (200 µL) was performed using 2.5 µM IspD, 100 mM Tris pH 8.0, 1 mM MgCl 2 , 1 mM DTT, 0.2 mM CTP, 100 mU/mL inorganic pyrophosphatase, and varying concentrations of MEP (Echelon Biosciences, Salt Lake City, UT).

Techniques: Concentration Assay, Activity Assay

Journal: PLoS ONE

Article Title: Francisella tularensis 2-C-Methyl-D-Erythritol 4-Phosphate Cytidylyltransferase: Kinetic Characterization and Phosphoregulation

doi: 10.1371/journal.pone.0020884

Figure Lengend Snippet: A) Intrinsic fluorescence spectra of MEP cytidylyltransferase and two mutant derivatives. Wildtype and mutant (T141D and T141E) proteins were adjusted to 5 µM in 0.1 M Tris pH 7.5, 1 mM NaCl and analyzed using an excitation wavelength of 290 nm. The emission spectra was measured from 310 to 400 nm. The Em λ max of all three proteins was detected at 320 nm. Identical spectra indicate little structural change in the protein globular fold accompanies the introduction of either Asp or Glu. B) The relative activity of wildtype MEP cytidylyltransferase and the T141D and T141E mutants. Assays were performed in duplicate with 200 µM MEP and 200 µM CTP.

Article Snippet: MEP cytidylyltransferase activity was evaluated using a coupled-enzyme colorimetric assay, derived from that originally described by Bernal et al . To determine the apparent K M for 2-C-methyl-D-erythritol 4-phosphate (MEP), the assay (200 µL) was performed using 2.5 µM IspD, 100 mM Tris pH 8.0, 1 mM MgCl 2 , 1 mM DTT, 0.2 mM CTP, 100 mU/mL inorganic pyrophosphatase, and varying concentrations of MEP (Echelon Biosciences, Salt Lake City, UT).

Techniques: Fluorescence, Mutagenesis, Activity Assay

Journal: Microbial Cell Factories

Article Title: Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis

doi: 10.1186/s12934-019-1235-5

Figure Lengend Snippet: Expression of isoprene synthase ( isp S) in E. coli. Strains carrying the empty plasmid pCOLA, the plasmid containing isp S (pCOLA::IspS) or the plasmid containing isp S and idi (pCOLA::IspS-idi) are compared. a Influence on E. coli growth. E. coli carrying the empty plasmid shows normal exponential growth kinetics. b Influence on MEP pathway intermediate concentrations. The concentration is shown relative to wild-type bacteria carrying the empty vector pCOLA. The data represent the mean of biological triplicates ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{\varvec{x}}$$\end{document} x ¯ ± SE; n = 3). c The MEP pathway in E. coli . The metabolite DXP is also a precursor for the synthesis of vitamin B 6 and thiamin. Ispoprene can be synthesized through the heterologous expression of isp S (shown in red). DXP 1-deoxy- d -xylulose 5-phosphate, MEP 2- C -methyl- d -erythritol 4-phosphate, MEcPP 2- C -methyl- d -erythritol 2,4-cyclopyrophosphate, ME-CDP 4-diphosphocytidyl-2- C -methylerythritol, MEP-CDP 4-diphosphocytidyl-2- C -methyl- d -erythritol 2-phosphate, HMBPP 4-hydroxy-3-methyl-but-2-enyl pyrophosphate, IPP isopentenyl pyrophosphate, DMAPP dimethylallyl pyrophosphate, GAP glyceraldehyde 3-phosphate, Dxs DXP synthase, Dxr DXP reductoisomerase, IspD MEP cytidylyltransferase, IspE ME-CDP kinase, IspF MEcPP synthase, IspG HMBPP synthase, IspH HMBPP reductase, Idi isopentenyl diphosphate isomerase, IspS isoprene synthase

Article Snippet: The following standards for the quantification of MEP pathway intermediates were supplied by Echelon Biosciences (Salt Lake City, UT, USA): 2- C -methyl- d -erythritol 4-phosphate (MEP), 1-deoxy- d -xylulose 5-phosphate (DXP), isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP).

Techniques: Expressing, Plasmid Preparation, Concentration Assay, Synthesized

Journal: Microbial Cell Factories

Article Title: Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis

doi: 10.1186/s12934-019-1235-5

Figure Lengend Snippet: Metabolic characterization of the MEP pathway in E. coli expressing isoprene synthase and isopentenyl pyrophosphate isomerase from a plasmid. The intracellular metabolite concentrations, secretion rate of metabolites and isoprene production rate are plotted against dxs expression. The expression strength of dxs was modulated by randomization of its ribosome-binding site. DXP 1-deoxy- d -xylulose 5-phosphate, MEP 2- C -methyl- d -erythritol 4-phosphate, MEcPP 2- C -methyl- d -erythritol 2,4-cyclopyrophosphate, ME-CDP 4-diphosphocytidyl-2- C -methylerythritol, MEP-CDP 4-diphosphocytidyl-2- C -methyl- d -erythritol 2-phosphate, HMBPP 4-hydroxy-3-methyl-but-2-enyl pyrophosphate, IPP isopentenyl pyrophosphate, DMAPP dimethylallyl pyrophosphate, GAP glyceraldehyde 3-phosphate, Dxs DXP synthase, Dxr DXP reductoisomerase, IspD MEP cytidylyltransferase, IspE ME-CDP kinase, IspF MEcPP synthase, IspG HMBPP synthase, IspH HMBPP reductase, Idi isopentenyl diphosphate isomerase, IspS isoprene synthase

Article Snippet: The following standards for the quantification of MEP pathway intermediates were supplied by Echelon Biosciences (Salt Lake City, UT, USA): 2- C -methyl- d -erythritol 4-phosphate (MEP), 1-deoxy- d -xylulose 5-phosphate (DXP), isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP).

Techniques: Expressing, Plasmid Preparation, Binding Assay

Journal: ACS infectious diseases

Article Title: Plasmodium IspD (2-C-Methyl-D-erythritol 4-Phosphate Cytidyltransferase), an Essential and Druggable Antimalarial Target

doi: 10.1021/id500047s

Figure Lengend Snippet: 1R,3S-MMV008138 inhibits isoprenoid biosynthesis in malaria parasites. MEP metabolites [deoxyxylulose phosphate (DOXP, upstream from IspD) and methylerythritol cyclic diphosphate (MEcPP, downstream from IspD)] were quantified by LC-MS/MS from P. falciparum parasites, following treatment with the indicated agents: fosmidomycin, positive control (known DXR inhibitor); 1S,3R-MMV008138, inactive diastereomer; and 1R,3S-MMV008138, active diastereomer whose antiparasitic effects are relieved by cotreatment with downstream isoprenoid IPP. Data are normalized to untreated controls and represent three independent biological replicates. Below is shown a schematic of the selected MEP pathway enzymes and metabolites, including DOXP and MEcPP.

Article Snippet: IC 50 assays contained 50 μ M CTP (Sigma), 500 μ M MEP (Echelon Biosciences), 0.1% DMSO (vehicle for 1 R ,3 S -MMV008138), and 0.2 mM MESG.

Techniques: Liquid Chromatography with Mass Spectroscopy, Positive Control

Journal: ACS infectious diseases

Article Title: Plasmodium IspD (2-C-Methyl-D-erythritol 4-Phosphate Cytidyltransferase), an Essential and Druggable Antimalarial Target

doi: 10.1021/id500047s

Figure Lengend Snippet: Inhibition of PfIspD by 1R,3S-MMV008138 is competitive with CTP but not MEP. (a) Kinetic parameters with respect to the CTP substrate. Each data point represents mean ± SEM from at least three independent experiments. (b) Lineweaver–Burk double-reciprocal plots of wild-type PfIspD activity over a range of CTP substrate concentrations, for illustrative purposes only. (c) Kinetic parameters with respect to the MEP substrate. Each data point represents the mean ± SEM from at least three independent experiments. (d) Lineweaver–Burk double-reciprocal plots of wild-type PfIspD activity over a range of MEP substrate concentrations, for illustrative purposes only.

Article Snippet: IC 50 assays contained 50 μ M CTP (Sigma), 500 μ M MEP (Echelon Biosciences), 0.1% DMSO (vehicle for 1 R ,3 S -MMV008138), and 0.2 mM MESG.

Techniques: Inhibition, Activity Assay