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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. B) Reaction catalyzed by RPE. While M72A mutation resulted in an almost 50% loss in activity, the loss in activity for M141A was marginal. We compared the structure of the binary complexes with the structure of the apo enzyme. Inspection of the anomalous difference electron density map around the Fe binding site confirmed the presence of Fe2+ in the crystal. The interaction between the pi electron cloud of Phe147 and the 7 carbon atom of Pro45 observed in the apo structure is broken as the loop caps the active site in presence of the ligand (Fig. These methionines are highly conserved, with Met72 being absolutely conserved among the orthologs of RPE (Fig. 4A). 2B). Fructose 6-phosphate is formed from glyceraldehyde 3-phosphate and sedoheptulose 7-phosphate. Human RPE crystallized as a dimer. Two enantiomers are possible, d -ribulose (d -erythro-pentulose) and l -ribulose (l -erythro-pentulose). The change in absorbance recorded at 340 nm represents the rate of conversion of D‐ribulose 5‐phosphate to D‐xylulose 5‐phosphate. Crystals grown in 25% PEG 3350, 100 mM Bis‐Tris (pH 5.5), and 200 mM NaCl diffracted X‐rays to 1.70 Å at beamline 19‐ID of the Advanced Photon Source (APS; Argonne National Laboratory, Argonne, IL, USA). Structures of the binary complexes of hRPE with D‐ribulose 5‐phosphate and D‐xylulose 5‐phosphate provide the first detailed molecular insights into the binding mode of physiological ligands and reveal an octa‐hedrally coordinated Fe2+ ion buried deep inside the active site. Ribulose 5-phosphate values were 3.4 +/- 0.3, 5.8 +/- 0.2, and 37.1 +/- 5.3 nmol/g. After verifying the DNA sequence, full‐length RPE (aa 1–228) was subcloned into pMCSG7 vector for expression in Escherichia coli BL 21 (DE3). Xylulose 5-phosphate is always required to produce ribose 5-phosphate. An octahedral coordination and the charge of the groups involved in coordination support building of a positively charged divalent ion in the electron density (Fig. Further, Wat30 is hydrogen bonded to Wat10, which is linked to the carbonyl oxygen of Ser‐10. The deproto‐nated Asp37 abstracts a proton from the C3 atom of d‐ ribulose 5‐phosphate, resulting in a cis‐enediolate intermediate. Working off-campus? Structural, mutagenesis, and functional data suggest that RPE uses a highly conserved mechanism for catalysis. The ability of RPE to confer protection against oxidative stress stems from its role in NADPH/NADP homeostasis, which plays a major role in detoxification of the reactive oxygen species (8). A) Stereo view of the amino acids surrounding d‐ribulose 5‐phosphate (yellow sticks). Two enantiomers are possible, d-ribulose (d-erythro-pentulose) and l-ribulose (l-erythro-pentulose). 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. 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. Conservation has been colored according to Clustal W convention. Asp37 is hydrogen bonded to Fe2+ and Ser‐10. www.fasebj.org. The D‐xylulose 5‐phosphate formed was first converted to glyceraldehyde 3‐phosphate and sedoheptulose 7‐phosphate using a transketolase. 2B). Further, side chains of Met39, Met72, and Met141 constrict the active site around the O1 and C1 atoms, preventing any movement of the ligands around this region. Among the 14 amino acids involved in intermolecular interactions within a distance of <3.2 Å, only Asp40 and Asn46 are conserved among the orthologs of RPE. 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. Leu12, Asn13, and Met39 together with Pro145‐Phe147 are capping the active site. The statistics of the anomalous data are listed in Supplemental Table S1, and the anomalous difference electron density map is shown in Supplemental Fig. Therefore, the dimerization interface observed for hRPE in the crystal structure is not conserved. Mutating Met39, Met72, or Met141 to alanine decreased the enzymatic activity (Fig. M39A retained 90% of its activity when compared to that of the wild‐type enzyme. 5B). D-Ribulose is the diastereomer of D-xylulose." The asymmetric unit consists of a dimer of RPE molecules, which is consistent with the results of the size‐exclusion chromatography elution profile of RPE where the protein elutes as a dimer. The initial phase was improved with Oasis (19). For example, d-ribulose is an intermediate in the fungal pathway for d-arabitol production. Interestingly, mutating Ser‐10 to alanine resulted in a dramatic decrease in the activity of the enzyme (Fig. Related Citations: The X-Ray Structure of Synechococcus Ribulose Bisphosphate Carboxylase(Slash)Oxygenase Activate Quaternary Complex at 2.2 Angstroms Resolution This work was funded by the Ministry of Science and Technology of China (grants 2006AA02A316, 2009DFB30310, and 2006CB910901), the National Natural Science Foundation of China (grants 30670427 and 30721003), the Ministry of Health of China (grant 2008ZX10404), a Chinese Academy of Sciences (CAS) research grant (KSCX2‐YW‐R‐127 and INFO‐115‐D01–2009), and a CAS fellowship for young international scientists (grant 2010Y1SA1). Although Mutating similar amino acids in the RPE from S. pyogenes resulted in a loss of catalytic activity (10). Previously, RPE has been shown to carry out catalysis using Co2+,Mn2+, and Zn2+ ions. In the binary complexes of hRPE with d‐ribulose 5‐phosphate and d‐xylulose 5‐phosphate, these methionines are at least 3.5 Å from the ligands. Further, we have probed the role of residues surrounding the ligands in catalysis by site‐directed mu‐tagenesis and functional assays. 4A). 1991a; 97 :1348–1353. RPE catalyzes the epimerization of ribulose 5‐phos‐phate to xylulose 5‐phosphate via a cis‐enediolate intermediate employing an acid‐base type of catalytic mechanism. The binary complexes of hRPE with D‐ribulose 5‐phosphate and D‐xylulose 5‐phos‐phate were obtained by soaking the crystals with ligands in 50% PEG 3350 (Table 1). 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 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. Of the scan suggested that hRPE bound Fe2+ when produced under conditions mentioned in Materials Methods. Rate of conversion of xylulose 5-phosphate values were 3.8 +/- 0.3, and functional studies on hRPE suggest highly! Represents the rate of conversion of d‐ribulose 5‐phosphate binary complex, ribose isomerase... A highly conserved among the orthologs of RPE ( Fig for reaction with H2O2 please your... For example, d-ribulose ( D-erythro-pentulose ) and L-ribulose ( L-erythro-pentulose ) identity the., aromatic amino acids in the formation of many bioactive substances the same assay mixture that was used estimate. 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