Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0008288

Figure Lengend Snippet: A) The MVA Pathway. The first two enzymes of the MVA pathway condense 3 molecules of acetyl-CoA (1) to form 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) (3), which is subsequently reduced to MVA (4) by HMG-CoA reductase , . MVA is phosphorylated twice then decarboxylated to yield IPP (7) – , which is converted to DMAPP (8) by an isomerase . B) The MEP Pathway. Condensation of pyruvate (9) with glyceraldehyde 3-phosphate (10) yields 1-deoxy-D-xylulose 5-phosphate (DXP; (11)) , an intermediate with a role in E. coli vitamin B1 and B6 biosynthesis – and isoprene biosynthesis. 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (also called MEP synthase or IspC) catalyzes the reduction and rearrangement of 11 to yield MEP (12) , the first committed step in the E. coli MEP pathway. The next enzyme, CDP-ME synthase, converts MEP into 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol (CDP-ME; (13)). CDP-ME kinase then phosphorylates CDP-ME, which is subsequently cyclized (coupled with the loss of CMP) by cMEPP synthase to yield 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (15) – . A reductive ring opening of 15 produces 1-hydroxy-2-methyl-2-butenyl diphosphate (HMBPP; (16)) , – , which is subsequently reduced to both IPP and DMAP in a ∼5:1 ratio , – . C) The reaction catalyzed by MEP synthase. Isomerization via cleavage of the bond between C3 and C4 and formation of a new bond between C2 and C4 produces the intermediate 2-C-methyl-D-erythrose 4-phosphate (18) , , which is subsequently reduced to yield MEP (12).

Article Snippet: To determine the apparent K M for 1-deoxy-D-xylulose 5-phosphate (DXP), assay mixtures (200 µL) contained 100 mM Tris pH 7.8, 25 mM MgCl 2 , 0.15 mM NADPH, 7 µM MEP synthase, and a variable concentration of DXP (Echelon Biosciences, Salt Lake City, UT).

Techniques:

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0008288

Figure Lengend Snippet: A) The MVA Pathway. The first two enzymes of the MVA pathway condense 3 molecules of acetyl-CoA (1) to form 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) (3), which is subsequently reduced to MVA (4) by HMG-CoA reductase , . MVA is phosphorylated twice then decarboxylated to yield IPP (7) – , which is converted to DMAPP (8) by an isomerase . B) The MEP Pathway. Condensation of pyruvate (9) with glyceraldehyde 3-phosphate (10) yields 1-deoxy-D-xylulose 5-phosphate (DXP; (11)) , an intermediate with a role in E. coli vitamin B1 and B6 biosynthesis – and isoprene biosynthesis. 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (also called MEP synthase or IspC) catalyzes the reduction and rearrangement of 11 to yield MEP (12) , the first committed step in the E. coli MEP pathway. The next enzyme, CDP-ME synthase, converts MEP into 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol (CDP-ME; (13)). CDP-ME kinase then phosphorylates CDP-ME, which is subsequently cyclized (coupled with the loss of CMP) by cMEPP synthase to yield 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (15) – . A reductive ring opening of 15 produces 1-hydroxy-2-methyl-2-butenyl diphosphate (HMBPP; (16)) , – , which is subsequently reduced to both IPP and DMAP in a ∼5:1 ratio , – . C) The reaction catalyzed by MEP synthase. Isomerization via cleavage of the bond between C3 and C4 and formation of a new bond between C2 and C4 produces the intermediate 2-C-methyl-D-erythrose 4-phosphate (18) , , which is subsequently reduced to yield MEP (12).

Article Snippet: To determine the apparent K M for 1-deoxy-D-xylulose 5-phosphate (DXP), assay mixtures (200 µL) contained 100 mM Tris pH 7.8, 25 mM MgCl 2 , 0.15 mM NADPH, 7 µM MEP synthase, and a variable concentration of DXP (Echelon Biosciences, Salt Lake City, UT).

Techniques:

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0008288

Figure Lengend Snippet: Michaelis-Menten plots of reaction velocity as a function of A) DXP concentration and B) NADPH concentration were used to derive the kinetic parameters listed in . The solid line represents the nonlinear least-squares best fit of the data to the Michaelis-Menten equation. The R 2 value for each plot is indicated.

Article Snippet: To determine the apparent K M for 1-deoxy-D-xylulose 5-phosphate (DXP), assay mixtures (200 µL) contained 100 mM Tris pH 7.8, 25 mM MgCl 2 , 0.15 mM NADPH, 7 µM MEP synthase, and a variable concentration of DXP (Echelon Biosciences, Salt Lake City, UT).

Techniques: Concentration Assay

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0008288

Figure Lengend Snippet: Enzyme assays were performed with several different divalent cations at a fixed DXP (150 µM) and NADPH (100 µM) concentration. Relative enzyme activity reveals the preference of the enzyme for Mg +2 .

Article Snippet: To determine the apparent K M for 1-deoxy-D-xylulose 5-phosphate (DXP), assay mixtures (200 µL) contained 100 mM Tris pH 7.8, 25 mM MgCl 2 , 0.15 mM NADPH, 7 µM MEP synthase, and a variable concentration of DXP (Echelon Biosciences, Salt Lake City, UT).

Techniques: Concentration Assay, Activity Assay

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0008288

Figure Lengend Snippet: A) Intrinsic fluorescence spectra of MEP synthase and its mutants. Wildtype and mutant (S177D and S177E) proteins were adjusted to 5 µM in 0.1 M Tris pH 7.5, 1 mM NaCl, 5 mM DTT and analyzed using an excitation wavelength of 290 nm. The emission spectra was measured from 310 to 400 nm. The Em λ max of wildtype MEP synthase was detected at 335 nm, of S177E was detected at 337 nm, and of S177D was detected at 326 nm. The blue shift observed with S177D is indicative of a conformational change sequestering tryptophan residues into a hydrophobic environment. The slight red shift observed with S177E is indicative of a conformational change exposing tryptophan residues to a hydrophilic environment. The increased quantum yield observed with both S177D and S177E is also indicative of a structural change in MEP synthase. B) The relative catalytic activity of wildtype MEP synthase and the S177D and S177E mutants. Assays were performed with 300 µM DXP and 150 µM NADPH.

Article Snippet: To determine the apparent K M for 1-deoxy-D-xylulose 5-phosphate (DXP), assay mixtures (200 µL) contained 100 mM Tris pH 7.8, 25 mM MgCl 2 , 0.15 mM NADPH, 7 µM MEP synthase, and a variable concentration of DXP (Echelon Biosciences, Salt Lake City, UT).

Techniques: Fluorescence, Mutagenesis, Activity Assay

Journal: PLoS ONE

Article Title: Kinetic Characterization and Allosteric Inhibition of the Yersinia pestis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

doi: 10.1371/journal.pone.0106243

Figure Lengend Snippet: A) The MVA pathway is utilized by humans and other eukaryotes, archaebacteria, and certain eubacteria to produce IPP and DMAPP, the building blocks of isoprenoids. The pathway is initiated by the enzymatic condensation of 3 molecules of acetyl-CoA (1) to form 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) (3), which is then reduced to MVA by HMG-CoA reductase (4) Subsequent phosphorylation and decarboxylation yield IPP (7) which is converted to DMAPP (8) by an isomerase . B) The MEP pathway is used by higher plants, the plastids of algae, apicomplexan protozoa, and many eubacteria, including numerous human pathogens. Pyruvate (9) is condensed with glyceraldehyde 3-phosphate (10) to yield 1-deoxy-D-xylulose 5-phosphate (DXP; (11)) , a branch point intermediate with a role in E. coli vitamin B1 and B6 biosynthesis as well as isoprene biosynthesis. In the first committed step of the E. coli MEP pathway, 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (also called MEP synthase, Dxr or IspC) catalyzes the reduction and rearrangement of 11 to yield MEP (12) . CDP-ME synthase then converts MEP into 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol (CDP-ME; (13)). CDP-ME kinase phosphorylates CDP-ME, which is subsequently cyclized (coupled with the loss of CMP) by cMEPP synthase to yield 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (15) . A reductive ring opening of 15 produces 1-hydroxy-2-methyl-2-butenyl diphosphate (HMBPP; (16)) , which is then reduced to both IPP and DMAPP in a ∼5:1 ratio . C) The reaction catalyzed by MEP synthase. The intermediate 2-C-methyl-D-erythrose 4-phosphate (18), produced by isomerization via cleavage of the bond between C3 and C4 and formation of a new bond between C2 and C4 , is subsequently reduced to yield MEP (12).

Article Snippet: To determine the apparent K M for 1 deoxy-D-xylulose 5-phosphate (DXP), 120 µL assay solutions contained 100 mM Tris pH 7.8, 25 mM MgCl 2 , 150 µM NADPH, 0.89 µM MEP synthase, and variable concentrations of DXP (Echelon Biosciences, Salt Lake City, UT).

Techniques: Produced

Journal: PLoS ONE

Article Title: Lipophilic Prodrugs of FR900098 Are Antimicrobial against Francisella novicida In Vivo and In Vitro and Show GlpT Independent Efficacy

doi: 10.1371/journal.pone.0038167

Figure Lengend Snippet: A. MEP synthase pathway : DXR catalyzes the formation of methylerythritol phosphate (MEP) from 1-deoxy-D-xylulose 5-phosphate (DXP) in an NADPH-dependent mechanism with the formation of the intermediate 2-C-methyl-D-erythrose 4-phosphate. Modified from Jawaid et al. . B: The structures of the compounds used in this study. Shown are the structures for Fosmidomycin, FR900098, Compound 1, Compound 2 and Compound 3.

Article Snippet: All assays were performed in duplicate, with the 1-deoxy-D-xylulose 5-phosphate (DXP; Echelon Biosciences, Salt Lake City, UT) concentration fixed at the K M (104 µM) and a saturating concentration of NADPH (150 µM).

Techniques: Modification