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Ribulose is known as ketopentose sugar due to the presence of a ketone functional group. Active site of hRPE is capped on ligand binding. Mutating similar amino acids in the RPE from S. pyogenes resulted in a loss of catalytic activity (10). While the ε1 carbon atom of Phe147 was 3.9 Å from the 7 carbon atom of Pro45 in the apo structure, the minimum distance between any of the atoms of Phe147 and Pro45 is now > 4.44 Å in the binary complexes. The overlap PCR product was ligated into pMCSG7 as described earlier. 4B). As a result of this movement of the loop, the ε1 carbon atom of Phe147 is now at a distance of 3.55 Å from the δ2 nitrogen atom of Asn46, and the α carbon of Gly148 is 4.0 Å from the δ1 oxygen atom of Asn13. The loop is seen capping the active site and therefore the binding of the ligand might have caused the movement of the loop. D - Xylulose 5 - phosphate D - xylulose - 5 - P is an intermediate in the pentose phosphate pathway. 5B). A number of amino acids are interacting with the ligands (Fig. A) Structure of the apo‐hRPE (green) superimposed on the hRPE‐D‐ribulose 5‐phosphate binary complex (magenta) showing Phe147 and Gly148 closing the active site on binding of D‐ribulose 5‐phosphate (blue sticks). Xylulose 1,5-bisphosphate synthesized by ribulose 1,5-bisphosphate carboxylase/oxygenase during catalysis binds to decarbamylated enzyme. Mutating Met39, Met72, or Met141 to alanine decreased the enzymatic activity (Fig. 2B). 4A). These results suggest that the enzyme may not be able to utilize Fe2+ to catalyze the reaction. Human RPE crystallized as a dimer. Our studies on hRPE uncover an unknown aspect of the enzyme—hRPE can bind and use Fe2+ ions for catalysis. To confirm the nature of the metal ion, we performed an X‐ray fluorescence scan of the protein crystal at the absorption edge of Zn2+,Mg2+, Co2+,Ca2+,Ni2+, and Fe2+. Which statement about the conversion of ribulose 5-phosphate to fructose 6-phosphate is TRUE? These findings have implications for the role of RPE in oxidative stress. Learn about our remote access options, National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China, Graduate University of Chinese Academy of Sciences, Beijing, China, Structural Biology Center, Argonne National Laboratory, Argonne, Illinois, USA. Interestingly, the activity of the S. pyogenes RPE stripped off its metal ion by treatment with EDTA and did not increase on addition of Fe2+ ions. Secondary structure of hRPE is annotated at bottom. The ligands bind deep inside a narrow tunnel just above the β barrel (Fig. The estimation of ribulose-5-phosphate can be carried out in the same assay mixture that was used to estimate glyceraldehyde-3-phosphate and xylulose-5-phosphate. B) Diagrammatic representation of the mechanism of catalysis depicting the proton transfers. Ribulose is a ketopentose — a monosaccharide containing five carbon atoms, and including a ketone functional group. pathways, the xylulose monophosphate pathway for yeasts and the ribulose monophosphate (RuMP) path-way for methylotrophic bacteria. The binary complexes of hRPE reported here will aid in the design of small molecules for modulating the activity of the enzyme and altering flux through the PPP.—Liang, W., Ouyang, S., Shaw, N., Joachimiak, A., Zhang, R., Liu, Z‐J. 6-P gluconate Glycogen Ribulose 5-P 6-P UDP-Glucose Galactose 1-P Ribose 5-P Glucose 1-P UDP-Galactose Xylulose 5-P Glucose 6-P Glucose Sedoheptulose 7-P Fructose 6-P Fructose 1,6-bis-PGlyceraldehydeFructose 1-P Glyceraldehyde 3-P Dihydroxyacetone-P 1,3-Bisphosphoglycerate 3-Phosphoglycerate 2-Phosphoglycerate Phosphoenolpyruvate Triacylglycerol Fatty acyl CoA ← -Fatty … Data were indexed and scaled to 1.70‐Å resolution using HKL2000 (17). M39A retained 90% of its activity when compared to that of the wild‐type enzyme. 5B). Ribulose-5-phosphate is just the epimer of xylulose-5-phosphate. The C2 and O2 atoms of d‐ribulose 5‐phosphate and d‐xylulose 5‐phosphate are immobilized by bonding with Fe2+ ion, Asp37, His70, and Asp175. Human RPE consistently bound Fe2+ when produced under conditions mentioned in Materials and Methods. These results suggest that RPE may not be able to use Fe2+ or Mg2+ for catalysis. However, none of the structures have been determined in complex with the physiological ligands. Interestingly, structural and biochemical evidence indicates that hRPE uses Fe2+ ion for catalysis. Circular dichroism (CD) analysis of the S10A mutant suggested that the mutation does not affect the secondary structure of the enzyme. The observation that hRPE can utilize Fe2+ ions for catalysis potentially provides another explanation for its role in protection against oxidative stress. Ribulose 5-phosphate values were 3.4 +/- 0.3, 5.8 +/- 0.2, and 37.1 +/- 5.3 nmol/g. It is a ketose sugar formed from ribulose - 5 - phosphate. Blue indicates positive potential; red, negative potential. Asingle metal ion is observed octahedrally coordinated in RPEs and has been postulated to stabilize the cis‐enediolate reaction intermediate. Working off-campus? The active site pocket can accommodate either dribulose 5‐phosphate or d‐xylulose 5‐phosphate without causing any noticeable movement of the side chains of the catalytic residues. CD analysis of all three mutants revealed that the mutation altered the content of the secondary structural elements of the protein. Summary: The protein encoded by this gene is a transketolase that acts as a homodimer and catalyzes the conversion of sedoheptulose 7-phosphate and D-glyceraldehyde 3-phosphate to D-ribose 5-phosphate and D-xylulose 5-phosphate. Uncut protein and TEV were removed by a second round of Ni‐affinity chromatography. The enzymatic activity of hRPE was measured using a commercially available kit from Sigma (St. Louis, MO, USA). Asp175 donates a proton to complete the epimerization and formation of d‐xylulose 5‐phosphate (Fig. Additional data sets were collected near the absorption edge of Fe. Enter your email address below and we will send you your username, If the address matches an existing account you will receive an email with instructions to retrieve your username, This article includes supplemental data. The ratio of measured tissue content of [xylulose 5-phosphate]/ [ribulose 5-phosphate] was found to be 1.12 +/- … In addition, a majority of the NADPH used by the human body for biosynthetic purposes is supplied by the PPP (5). Under normal conditions, the hydrogen peroxide is converted to H2O and O2 by peroxidases and catalases (25). 1). Please visit, © 2021 Federation of American Societies for Experimental Biology (FASEB), I have read and accept the Wiley Online Library Terms and Conditions of Use, Cloning of the amphibolic Calvin cycle/OPPP enzyme d‐ribulose‐5‐phosphate 3‐epimerase (EC 5.1.3.1) from spinach chloroplasts: functional and evolutionary aspects, The oxidative pentose phosphate pathway: structure and organisation, Physiological functions of the pentose phosphate pathway, Cells have distinct mechanisms to maintain protection against different reactive oxygen species: oxidative‐stress‐response genes, Mutants that show increased sensitivity to hydrogen peroxide reveal an important role for the pentose phosphate pathway in protection of yeast against oxidative stress, Cu, Zn superoxide dismutase and NADP(H) homeosta‐sis are required for tolerance ofendoplasmic reticulum stress in, d‐Ribulose 5‐phosphate 3‐epimerase: functional and structural relationships to members of the ribulose‐phosphate binding (β/α)8‐barrel superfamily, structure and catalytic mechanism of the cytosolic d‐ribulose‐5‐phosphate 3‐epimerase from rice, Structure of d‐ribulose 5‐phosphate 3‐epimerase from, Structure of a ribulose 5‐phosphate 3‐epimerase from, Structure and mechanism of the amphibolic enzyme ‐ribulose‐5‐phosphate 3‐epimerase from potato chloroplasts, Identification of a catalytic aspartyl residue of d‐ribulose 5‐phosphate 3‐epimerase by site‐directed mutagenesis, On the mechanism of the pentose phosphate epimerases, Processing of X‐ray diffraction data collected in oscillation mode, OASIS and molecular‐replacement model completion, Coot: model‐building tools for molecular graphics, Refinement of macromolecular structures by the maximum‐likelihood method, Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard, MolProbity: all‐atom contacts and structure validation for proteins and nucleic acids, Site‐directed mutagenesis by overlap extension using the polymerase chain reaction, Complex cellular responses to reactive oxygen species. A) Stereo view of the amino acids surrounding d‐ribulose 5‐phosphate (yellow sticks). Structural, mutagenesis, and functional data suggest that RPE uses a highly conserved mechanism for catalysis. ATP, NAD, NADP , flavoprotiens. Leu12, Asn13, and Met39 together with Pro145‐Phe147 are capping the active site. We compared the structure of the binary complexes with the structure of the apo enzyme. 1) (3, 4). His35, His70, Asp37, Asp175, and oxygens O2 and O3 of the ligand are coordinated to the Fe2+ ion (Fig. The structure was solved by molecular replacement method using Phaser MR (18) with structure of the rice RPE [Protein Data Bank (PDB) code 1H1Y] as a search model. The statistics of the anomalous data are listed in Supplemental Table S1, and the anomalous difference electron density map is shown in Supplemental Fig. HPS catalyzes In addition, the H97N mutant was found to catalyze the condensation of dihydroxyacetone and glycolaldehyde phosphate to produce a mixture of l-ribulose-5-phosphate and d-xylulose-5-phosphate. The asymmetric unit consists of 2 molecules of hRPE. Therefore, the dimerization interface observed for hRPE in the crystal structure is not conserved. Interestingly, mutating Ser‐10 to alanine resulted in a dramatic decrease in the activity of the enzyme (Fig. Ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) catalyzes the interconversion of ribulose-5-phosphate and xylulose-5-phosphate in the Calvin cycle and in the oxidative pentose phosphate pathway. Here, using structural, biochemical, and functional studies, we show that human D‐ribulose 5‐phosphate 3‐epimerase (hRPE) uses Fe2+ for catalysis. One unit of activity is defined as the amount of enzyme required to convert 1 µmol of D‐ribulose 5‐phosphate to D‐xylulose 5‐phosphate under the assay conditions. The overall structure of hRPE closely mirrors the structures of RPE homologues reported previously (10–14). d‐ribulose 5‐phosphate 3‐epimerase (RPE) catalyzes the reversible conversion of d‐ribulose 5‐phosphate to d‐xylulose 5‐phosphate (Fig. Fe2+ is shown as a sphere. Download PDF Version of Ribose vs Ribulose. Is formed from glyceraldehyde 3-phosphate and sedoheptulose 7-phosphate may not be able to utilize Fe2+ ions, thereby making unavailable. To elucidate the physiological significance of this finding around the Fe binding site confirmed the presence of a functional! Can be monitored by reading the absorbance at 340 nm represents the rate of conversion of d -.. A double phosphate ester of the enzyme could not use Fe2+ or Mg2+ for catalysis ( 10–13.... 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