mg l 1 deoxy d xylulose 5 phosphate dxp  (Echelon Biosciences)

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 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: Nature Communications

Article Title: Structural basis of substrate recognition and thermal protection by a small heat shock protein

doi: 10.1038/s41467-021-23338-y

Figure Lengend Snippet: a Confocal microscopy images of chloroplasts in Arabidopsis protoplasts (treated with 100 μM lincomycin (LN) after transformation and incubated for 2 h at 25 °C or 37 °C before imaging) expressing DXPS-YFP with TP-RFP, Hsp21-RFP or Hsp21-His, and Hsp21-RFP with TP-YFP or DXPS-His. TP: Rubisco activase RecA transit peptide. Scale bar, 10 μm. b , c Quantification of ( b ) DXPS punctae area and ( c ) Hsp21 punctae area as shown in ( a ). Values represent mean ± SD, where n represented under the graph indicates the number of punctate fluorescent signal from at least two independent experiments. d DXPS and Hsp21 protein distribution in soluble and insoluble fractions isolated from Arabidopsis protoplasts treated with LN and incubated for 2 h at 25 °C or 37 °C before extraction. e , f Densitometry quantification of ( e ) DXPS and ( f ) Hsp21 protein distribution in soluble fractions as shown in ( d ). Values represent mean ± SEM with n = 3 biological replicates. g DXPS-His-FLAG and His-Hsp21 protein distribution in soluble and insoluble fractions isolated from E. coli induced at 18 °C and 37 °C, respectively, detected by Western analysis using an anti-His antibody. h Quantification of DXPS-His-FLAG protein distribution in soluble fractions as shown in ( g ). Values represent mean ± SEM with n = 3 biological replicates. i DXPS-FLAG and Hsp21 protein distribution in soluble and insoluble fractions after mixing purified DXPS-FLAG protein with or without Hsp21 dodecamer protein (Hsp21: DXPS in monomer/monomer molar ratio of 12:1) and incubating for 2.5 h at 0 °C and 37 °C, respectively. Protein distribution was assessed by Coomassie Blue Staining. j Densitometry quantification of DXPS protein distribution in soluble fractions as shown in ( i ). Values represent mean ± SEM with n = 3 independent experiments. Box and whiskers plots show maxima and minima, upper and lower percentiles (box) and median (line). Letters indicate statistical significance based on one-way ANOVA with post hoc Tukey’s multicomparison test ( p ≤ 0.05); means bearing different letters differ significantly. Asterisks indicate statistical significance based on two-tailed unpaired t -test (*** p = 0.0001). Source data are provided as a Source Data file.

Article Snippet: Through identification of a client substrate of Hsp21, 1-deoxy-D-xylulose 5-phosphate synthase (DXPS), we isolate the Hsp21-DXPS complex under heat stress conditions and determine its structure by single-particle cryo-electron microscopy (cryo-EM).

Techniques: Confocal Microscopy, Transformation Assay, Incubation, Imaging, Expressing, Isolation, Western Blot, Purification, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Structural basis of substrate recognition and thermal protection by a small heat shock protein

doi: 10.1038/s41467-021-23338-y

Figure Lengend Snippet: a Cryo-EM map of DXPS with the fitted model. The three domains of one subunit of the DXPS dimer are indicated with labels and represented by different color. The other subunit of the dimer is shown in black. b Composite cryo-EM map of Hsp21-DXPS with fitted models. The two subunits of the DXPS dimer are shown in orange and sea green and Hsp21 is shown in blue. The three identified interaction interfaces (I, II, and III) are indicated (black boxes) with the corresponding zoomed-in views shown below. Interface III is viewed from the top of the complex. The putative interacting regions on domain I (for Interface I and II) and the mobile loops (for Interface III) of DXPS are highlighted in magenta. The dashed lines denote the disordered mobile loops with no map densities. All β strands and the C-terminus of Hsp21 are labeled. Here DXPS denotes DXPS-His-FLAG and Hsp21 denotes His-Hsp21.

Article Snippet: Through identification of a client substrate of Hsp21, 1-deoxy-D-xylulose 5-phosphate synthase (DXPS), we isolate the Hsp21-DXPS complex under heat stress conditions and determine its structure by single-particle cryo-electron microscopy (cryo-EM).

Techniques: Cryo-EM Sample Prep, Labeling

Journal: Nature Communications

Article Title: Structural basis of substrate recognition and thermal protection by a small heat shock protein

doi: 10.1038/s41467-021-23338-y

Figure Lengend Snippet: a Side view of map segments fitted with the model of the Hsp21 monomer from Hsp21-DXPS (left, blue) and the Hsp21 dodecamer (right, magenta). The beta strands on one sheet of the ACDs are labeled for side-by-side comparison. Note the missing of the beta2 strand in the Hsp21 monomer (blue) derived from the Hsp21-DXPS complex. The DXPS binding, dimeric and non-dimeric interfaces of Hsp21 are labeled. b Superimposition of the two models viewed from different directions. For the side views, the back side of ACD was clipped for clarity. The β5-β7 loops and the CTRs on both monomers which they exhibit different conformations are labeled. Twisting of the beta sheets of the ACD from the Hsp21 dodecamer is revealed through overlay of the models (middle and right).

Article Snippet: Through identification of a client substrate of Hsp21, 1-deoxy-D-xylulose 5-phosphate synthase (DXPS), we isolate the Hsp21-DXPS complex under heat stress conditions and determine its structure by single-particle cryo-electron microscopy (cryo-EM).

Techniques: Labeling, Derivative Assay, Binding Assay

Journal: Nature Communications

Article Title: Structural basis of substrate recognition and thermal protection by a small heat shock protein

doi: 10.1038/s41467-021-23338-y

Figure Lengend Snippet: The binding surfaces for Hsp21 on DXPS that are normally masked by the mobile loops are indicated with solid red lines. Facilitated by the inherent flexibility of the mobile loops (indicated by gray arrow), the Hsp21 binding surfaces on DXPS (indicated by red lines) are exposed more frequently under elevated temperature. Hsp21 dodecamer dissociation, partial unfolding that results in the loss of β2 strand and the conformational changes occurred upon DXPS binding are shown schematically. The Hsp21-DXPS interaction is predicted to involve both induced fit and conformational selection binding mechanisms.

Article Snippet: Through identification of a client substrate of Hsp21, 1-deoxy-D-xylulose 5-phosphate synthase (DXPS), we isolate the Hsp21-DXPS complex under heat stress conditions and determine its structure by single-particle cryo-electron microscopy (cryo-EM).

Techniques: Binding Assay, Selection