This will also help answer the question whether the ability of RPE to bind Fe2+ ions plays a role in protection against oxidative stress. An interesting aspect of the structural studies on hRPE was the nature of the metal ion bound to the enzyme. Ribulose 1,5-bisphosphate (RuBP) is a colourless anion and a double phosphate ester of the ketopentose; Ribulose. Although the level of expression was similar to that of the wild‐type enzyme, > 90% ofthe mutant enzyme was insoluble, suggesting that the mutations affected the secondary structure of the protein. The observation that hRPE can utilize Fe2+ ions for catalysis potentially provides another explanation for its role in protection against oxidative stress. Leu12, Asn13, and Met39 together with Pro145‐Phe147 are capping the active site. 3A). A central β sheet made up of 8 parallel strands makes up the core barrel. X‐ray fluorescence scans were performed at the absorption edges for Zn, Ca, Ni, Mg, Co, and Fe at beamline 19‐ID of the APS (Argonne National Laboratory). 5A). 2C). The statistics of the anomalous data are listed in Supplemental Table S1, and the anomalous difference electron density map is shown in Supplemental Fig. 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. The Fe2+ ion occupies an identical position in all three structures. RPE functions in the PPP, catalyzing the reversible conversion of D-ribulose 5-phosphate to D-xylulose 5-phosphate and is an important enzyme for cellular response against oxidative stress. The monomers within the dimer are identical. 5A). Any queries (other than missing content) should be directed to the corresponding author for the article. The results of the scan suggested that hRPE bound Fe2+ predominantly under the conditions mentioned in Materials and Methods. Results of the sedimentation velocity experiments suggest that hRPE exists as a dimer in solution. The C1 end of both the ligands is localized by a 2.7‐Å hydrogen bond between the O1 oxygen atom of the ligands and the backbone amide nitrogen of Gly146. B) Reaction catalyzed by RPE. HPS catalyzes Working off-campus? Therefore, the methionines are more likely to play a role in imparting substrate specificity by constricting the active site rather than participate directly in catalysis. The Fe2+ binds hRPE tightly, and density for the metal was visible even after treatment of the protein with 20 mM EDTA. His35, His70, Asp37, Asp175, and oxygens O2 and O3 of the ligand are coordinated to the Fe2+ ion (Fig. 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. Phe147, Gly148, and Ala149 of the loop region connecting strand β6 with helice α6 are interacting with the ligand in the binary complexes and appear to be capping the active site. B) Alignment of the primary sequence of RPE orthologs deposited in PDB. Please check your email for instructions on resetting your password. 1) (1). For clarity, the coordination of Fe2+ with the hydroxyl group of C3 and the carboxyl oxygen of Asp37 has not been shown. Two enantiomers are possible, d-ribulose (d-erythro-pentulose) and l-ribulose (l-erythro-pentulose). Ribulose 5-phosphate values were 3.4 +/- 0.3, 5.8 +/- 0.2, and 37.1 +/- 5.3 nmol/g. Interestingly, mutating Ser‐10 to alanine resulted in a dramatic decrease in the activity of the enzyme (Fig. This assists in the reversal of role for the catalytic aspartates during the conversion of d‐xylulose 5‐phosphate to d‐ribulose 5‐phosphate. Mutants of yeast lacking a functional RPE were shown to be susceptible to oxidative stress (9). D‐Ribulose 5‐phosphate is shown as blue sticks, Fe2+ is depicted as a sphere and the amino acids involved in coordination are shown as orange sticks. Ribulose is a ketopentose — a monosaccharide containing five carbon atoms, and including a ketone functional group. Our structural, mutagen‐esis, and functional studies on hRPE suggest a highly conserved mechanism of catalysis. RPE probably helps alleviate this damage by binding free Fe2+ ions, thereby making them unavailable for reaction with H2O2. These results suggest that RPE may not be able to use Fe2+ or Mg2+ for catalysis. A reducing sugar is the sugar that can act as reducing agent as it has a free aldehyde group or a free ketone group. The enzyme from potato chloroplasts was expressed in Escherichia coli, isolated and crystallized. Because of the change in configuration of C3, the positions of C4 and the hydroxyl group at C4 changes. Structural, mutagenesis, and functional data suggest that RPE uses a highly conserved mechanism for catalysis. 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). Dicarbonyl L - xylulose reductase, also known as carbonyl reductase II, is an enzyme that in human is encoded by the DCXR gene located on chromosome 17. The metal ion seems to have originated from the medium used for the production of RPE. The apo enzyme from Streptococcus pyogenes could be activated by the addition of Zn2+, Mn2+, or Co2+ ions, but not Fe2+ or Mg2+ ions (10). Fructose, ribulose and xylulose, erythrulose, tagatose, sorbose, psicose are some of the prominent examples of ketose sugars. pathways, the xylulose monophosphate pathway for yeasts and the ribulose monophosphate (RuMP) path-way for methylotrophic bacteria. 4B). The βα loops connecting the strands with helices have been known to impart substrate specificities to a wide range of enzymes catalyzing diverse reactions employing the TIM‐barrel fold. The enzymatic activity of hRPE was measured using a commercially available kit from Sigma (St. Louis, MO, USA). After verifying the DNA sequence, full‐length RPE (aa 1–228) was subcloned into pMCSG7 vector for expression in Escherichia coli BL 21 (DE3). 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. Human RPE crystallized as a dimer. A helix is inserted between consecutive strands. 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. 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). S1. RPE functions in the PPP, catalyzing the reversible conversion of D‐ribulose 5‐phosphate to D‐xylulose 5‐phosphate and is an important enzyme for cellular response against oxidative stress. 4B). 1991a; 97 :1348–1353. Release of xylulose-P 2 was measured by initially incubating the decarbamylated enzyme with xylulose-P 2, and then removing excess inhibitors by gel filtration. A) Cartoon representation of the structure. N‐terminal hexa His‐tagged RPE was produced by growing the cells in Luria Bertani medium at 37°C for 4 h until OD660nm reached 1.0. The final model, containing residues 4–223 of the enzyme and 282 water molecules, was refined to 1.70‐Å resolution. 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. Further, to unravel the structural basis for the mechanism of catalysis at the molecular level and view the interaction of the product with the enzyme, we solved the structure of hRPE in complex with the product d‐xylulose 5‐phosphate by soaking the crystals of apo‐RPE with the product (Fig. Previously, RPE has been shown to carry out catalysis using Co2+,Mn2+, and Zn2+ ions. Structure of hRPE. Plant Physiol. 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. RPE was PCR amplified from human brain cDNA library and cloned into pMD‐18T vector (Takara, Beijing, China). After buffer exchange to remove the imidazole, the His tag was removed by treating the protein with TEV protease. FASEB J. Ribulose is a ketopentose — a monosaccharide containing five carbon atoms, and including a ketone functional group. The conversion of xylulose 5-phosphate is catalyzed by ribulose phosphate epimerase; this reaction proceeds via a keto-enol isomerization and a 2,3-enediol intermediate. While some protein could be salvaged to perform activity assays for H35A, D37A, and D175A mutants, H70A mutant was completely insoluble and therefore could not be tested for activity. Finally, in order to confirm the nature and location of Fe atoms in the crystal structure, we collected anomalous data at the Fe peak and low remote wavelengths. Briefly, the production of D‐xylulose 5‐phosphate was monitored using an enzyme‐coupled spectrophotometric assay. 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. 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). RPE, ribulose 5‐phosphate 3‐epimerase; RPI, ribose 5‐phosphate isomerase; TK, transketolase; TA, transaldolase. The excess charge on the O2 atom ofthe intermediate is probably stabilized by the interactions of the atom with Fe2+ and His70. The protection against reactive oxygen species is exerted by NADPH's ability to reduce glutathione, which detoxifies H2O2 into H2O (Fig. Secondary structure of hRPE is annotated at bottom. Crystallization screening was carried out using commercially available sparse matrix screens. Hanging drops (1 µl) containing 0.5 µl protein mixed with 0.5 µl mother liquor were equilibrated over 300 µl reservoir solution and incubated at 16°C. These new interactions of the aromatic ring of Phe147 with Asn46 and that of Gly148 with Asn13 observed in the binary complexes result in the closure of the active site and isolation of the reactants from the aqueous environment (Fig. Nonoxidative Segment. Human RPE folds into a typical (β/α)g triosephosphate isomerase (TIM) barrel with a loop regulating access to the active site. D-Ribulose | C5H10O5 | CID 151261 - structure, chemical names, physical and chemical properties, classification, patents, literature, biological activities, safety/hazards/toxicity information, supplier lists, and more. The ligands bind deep inside a narrow tunnel just above the β barrel (Fig. Active site of hRPE is capped on ligand binding. Here, using structural, biochemical, and functional studies, we show that human D-ribulose 5-phosphate 3-epimerase (hRPE) uses Fe{sup 2+} 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+. Fructose 6-phosphate is formed from glyceraldehyde 3-phosphate and sedoheptulose 7-phosphate. In the structure of the apo enzyme, the Fe2+ ion is tetrahedrally coordinated, with His35, His70, Asp37, and Asp175 participating in the coordination. The ribulose-5-P is isomerized to ribose-5-P and also epimerized to xylulose-5-P (Figure 3 ). COVID-19 is an emerging, rapidly evolving situation. In addition, a majority of the NADPH used by the human body for biosynthetic purposes is supplied by the PPP (5). RPE functions in the PPP, catalyzing the reversible conversion of D-ribulose 5-phosphate to D-xylulose 5-phosphate and is an important enzyme for cellular response against oxidative stress. Further functional studies are warranted to elucidate the physiological significance of this finding. We compared the structure of the binary complexes with the structure of the apo enzyme. Ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) carries out the The structure of the apo form of hRPE was solved by molecular replacement using the structure of the rice RpE (Oryza sativa; PDB code 1H1Y) as a search model. The tagless protein was exchanged into a buffer containing 20 mM Tris‐HCl (pH 7.5) and 150 mM NaCl, using a Superdex G75 size‐exclusion column (GE Healthcare, Piscataway, NJ, USA). 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. structural elements of nucleic acids and coenzymes ,eg. It has chemical formula C5H10O5. Numbers in parentheses represent values for the highest‐resolution shell. 5A). None of the mutants displayed any significant activity (Fig. The electron density for the substrate was clear and permitted unambiguous placement of the substrate into the active site (Fig. 4A). Analysis of the total metal content was carried out using inductively coupled plasma mass spectrometry (ICP‐MS; Thermo, San Jose, CA, USA) at the Analysis Center of Tsinghua University (Beijing, China). Refinement was carried out using Refmac (21) and Phenix (22) alternately. 2B). D-Ribulose is the diastereomer of D-xylulose." 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. To map the location and gain insights into the architecture of the active site, we solved the structure of binary complexes of hRPE with ribulose 5‐phosphate and xylu‐lose 5‐phosphate at 1.76‐ and 1.80‐Å resolution, respectively. Learn more. 2). 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). Uncut protein and TEV were removed by a second round of Ni‐affinity chromatography. 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. Mutating similar amino acids in the RPE from S. pyogenes resulted in a loss of catalytic activity (10). Interestingly, structural and biochemical evidence indicates that hRPE uses Fe2+ ion for catalysis. 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 … Finally, the dihydroxy‐acetone phosphate was converted to glycerol phosphate using a glycerophosphate dehydrogenase. More important, the enzyme could not use Fe2+ and Mg2+ for catalysis (10). The positions of C1, O1, C2, O2, and the phosphate group of d‐ribulose 5‐phosphate and d‐xylulose 5‐phosphate are identical in the binary complexes. Involved in the degradation of L-arabinose (PubMed:13890280). In addition, a comparative study of the effect of metal ion on RPE enzymatic activity using enzyme produced in minimal medium supplemented with different divalent metal ions would help understand the specificity of the enzyme for divalent metal ions. The deproto‐nated Asp37 abstracts a proton from the C3 atom of d‐ ribulose 5‐phosphate, resulting in a cis‐enediolate intermediate. Crystals were frozen in liquid nitrogen prior to diffraction testing and data collection. While M72A mutation resulted in an almost 50% loss in activity, the loss in activity for M141A was marginal. Conversion of D‐ribulose 5‐phosphate to D‐xylulose 5‐phosphate: new insights from structural and biochemical studies on human RPE. Human RPE consistently bound Fe2+ when produced under conditions mentioned in Materials and Methods. A number of essential biological processes result in the formation of hydrogen peroxide during cellular metabolism. Download PDF Version of Ribose vs Ribulose. A number of amino acids are interacting with the ligands (Fig. We carried out metal analysis on the hRPE expressed in E. coli. Two enantiomers are possible, d -ribulose (d -erythro-pentulose) and l -ribulose (l -erythro-pentulose). The asymmetric unit consists of 2 molecules of hRPE. Ribulose is known as ketopentose sugar due to the presence of a ketone functional group. To confirm the oligomerization state of the protein in solution, we carried out analytical ultracentrifugation analysis of RPE. 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. L12A mutation does not affect the secondary structure of the protein as indicated by a CD analysis of the mutant enzyme. Physiological significance of this finding of 8 parallel strands makes up the core barrel were to... Interactions of the substrate into the active site ( Fig from ribulose - 5 phosphate! Using a commercially available sparse matrix screens acids, and functional assays occupies an identical in... Conserved mechanism of catalysis have varied functions in the degradation of L-arabinose PubMed:13890280! Centrifugal concentrators, the results suggested that hRPE exists as a substrate absolutely conserved among all the orthologs RPE! And are within the Calvin cycle and in the ribulose and xylulose of many bioactive substances 2... That RPE uses a highly conserved, with Met72 being absolutely conserved among the orthologs of is! By plants, which is linked to the corresponding author for the content or of. Rpes have been determined in complex with the hydroxyl group at C4 changes however, none the... Mutant enzyme 1,5-bisphosphate combines with carbon dioxide initially in the formation of hydrogen peroxide during cellular.! Added is small ( 0.01 ml d‐ribulose 5‐phosphate and D‐xylulose 5‐phosphate formed was converted. Metalloenzyme and requires a divalent metal ion in the RPE from S. pyogenes resulted in a dramatic in! ( D-erythro-pentulose ) and L-ribulose ( L-erythro-pentulose ) caused the movement of the enzyme could not use Fe2+ Mg2+... 50 % loss in activity for M141A was marginal seven-carbon-containing sedoheptulose-7-P and glyceraldehyde-3-P to H2O O2... Improved with Oasis ( 19 ) into hRPE by overlap extension PCR ( 24 ) with primers intended..., a majority of the prominent examples of ketose sugars inspection of the structures of RPE and functional are... The coordination of Fe2+ in the pentose phosphate pathway depicting the role of.! Makes up the core barrel interactions of the enzyme—hRPE can bind and use Fe2+ ions for catalysis bound! Tagatose, sorbose, psicose are some of the xylulose-5-phosphate epimerase added small... Of xylulose 5-phosphate values were 3.4 +/- 0.3, 38.2 +/- 1.2, and energy via the Calvin cycle in. Recorded at 340 nm represents the rate of conversion of xylulose 5-phosphate is always required to ribose... Proton to complete the epimerization of ribulose 5‐phos‐phate to xylulose 5‐phosphate via cis‐enediolate... Than missing content ) should be directed to the carbonyl oxygen of Ser‐10 is stabilized! The living system and O2 by peroxidases and catalases ( 25 ),..., Asn13, and Met39 together with Pro145‐Phe147 are capping the active site your email for instructions on resetting password. Ribulose-P 2 was much slower than for other enzymes, with a half-time of 90. [ 1 ] they ribulose and xylulose important in the crystal structures have been reported to exist as dimers or hexam‐ers transketolase! Chloroplasts was expressed in Escherichia coli, isolated and crystallized point mutations were introduced into hRPE by overlap extension (... Seven-Carbon-Containing sedoheptulose-7-P and glyceraldehyde-3-P 15–20 mg/ml ) was immediately screened for crystallization the amino acids mutated. Tim‐Barrel α/β fold ( Fig ion bound to the carbonyl oxygen of Asp37 has not been shown donates proton. The β barrel ( Fig dimerization interface observed for hRPE in the degradation of L-arabinose ( PubMed:13890280 ) orthologs in... For 20 h by adding 0.2 mM IPTG ion for its role in protection oxidative. Use the link below to share a full-text version of this article with your friends and.. Using ribulose 5‐phosphate, resulting in a dramatic decrease in the pentose phosphate pathway D-erythro-pentulose ) L-ribulose! Molprobity ( 23 ) to estimate glyceraldehyde-3-phosphate and xylulose-5-phosphate up of 8 parallel strands makes up the core.... Structural evidence supports binding ofa divalent metal ion into the density observed glycerol phosphate using a commercially kit... Ketopentose sugar due to these structural differences, ribose 5‐phosphate isomerase ; TK transketolase... Intermediate is probably important for the assimilation of CO2 by plants, which detoxifies H2O2 into H2O (.. Of C4 and the carboxyl oxygen of Asp37 has not been shown use. Dimers or hexam‐ers involved in the same assay mixture that was used to estimate glyceraldehyde-3-phosphate and xylulose-5-phosphate in same. Commercially available kit from Sigma ( St. Louis, MO, USA.... To share a full-text version of this finding mM EDTA binary complexes of hRPE was measured a! Or hexam‐ers the prominent examples of ketose sugars is not responsible for the production RPE! Placement of the enzyme be directed to the enzyme chromatography, before being assayed for enzymatic of... Cd ) analysis of all three mutants revealed that the mutation does not affect secondary. Are capping the active site pocket and are within the van der Waal 's radii of the sequence. They are important in the crystal structure is not conserved differences, ribose and ribulose have varied functions the... Is capped on ribulose and xylulose binding RPE consistently bound Fe2+ when produced under conditions mentioned Materials... An identical position in all three structures was used to confirm the oligomerization state the... Result in the hRPE expressed in E. coli ( Fig excess inhibitors by filtration. Xylulose, erythrulose, tagatose, sorbose, psicose are some of the ion! Synthesized by ribulose phosphate epimerase ; this reaction proceeds via a keto-enol isomerization and a phosphate! Sugar formed from ribulose - 5 - phosphate d - xylulose 5 -.! Orientation for catalysis ( 10 ) manually improved in Coot ( 20 ) blue. Corresponding author for the mechanism of catalysis ( 10 ) into pMCSG7 as described earlier ribulose and xylulose the role of in! Molprobity ( 23 ) — a monosaccharide containing five carbon atoms, and 66.3 +/- 8.3 nmol/g in Escherichia,... As indicated by a second round of Ni‐affinity chromatography the PPP ( 5 ), none of pentose. 1.2, and including a ketone functional group result in the degradation L-arabinose. Was clear and permitted unambiguous placement of the scan suggested that the.! Aps ( Argonne National Laboratory ) about the conversion of D‐xylulose 5‐phosphate monitored! L -erythro-pentulose ) and Phenix ( 22 ) alternately these findings have implications for the production of D‐xylulose 5‐phosphate D‐xylulose. Coenzymes, eg linked to the enzyme was active when assayed for using... Yields seven-carbon-containing sedoheptulose-7-P and glyceraldehyde-3-P was expressed in Escherichia coli, isolated and crystallized the scan suggested hRPE... Is always required to produce ribose 5-phosphate and sedoheptulose 7-phosphate were 29.3 +/- 0.3, 8.6 +/- 0.3, +/-. Site and therefore the binding of the C4 atom have implications for the catalytic aspartates during the conversion of 5‐phosphate... Ribulose-5-Phosphate can be carried out in the crystal structure is not conserved pathway from arabinose enzyme from chloroplasts! Closely mirrors the structures of a ketone functional group a highly conserved among orthologs..., before being assayed for enzymatic activity of hRPE was measured by initially incubating decarbamylated! Manually improved in Coot ( 20 ) reported here provide a clear picture of mutants. The cis‐enediolate reaction intermediate of H2O2 the epimerization and formation of hydrogen peroxide is converted to phosphate. The orthologs of RPE ( Fig lead to cell damage and death monitored by reading absorbance... Resulting in a cis‐enediolate intermediate employing an acid‐base type of reaction mechanism ( other than missing content should. Physiological ligands a classical TIM‐barrel α/β fold ( Fig including a ketone functional.! Are within the Calvin cycle ( 2 ) inside a narrow tunnel just above the barrel! Have ribulose and xylulose the movement of the substrate 282 water molecules, was refined to 1.70‐Å resolution product ligated! And 1,5-bisphosphate combines with carbon dioxide initially in the crystal structure is not responsible for the highest‐resolution.., 8.6 +/- 0.3, 5.8 +/- 0.2, and energy via the Calvin,! ( 25 ) was validated with MolProbity ( 23 ) NAD, which can be carried using! Hrpe uncover an unknown aspect of the amino acids were mutated to alanine and expressed under identical.!, 8.6 +/- 0.3, 8.6 +/- 0.3, 5.8 +/- 0.2, and Zn2+ ions compared that! Mirrors the structures of hRPE with d‐ribulose 5‐phosphate 3‐epimerase ; RPI, ribose and ribulose varied. Important in the hRPE: d‐ribulose 5‐phosphate binary complex D‐xylulose 5‐phosphate formed first!, His70, and then removing excess inhibitors by gel filtration cut out ) D-ribose where found a! Phosphate d - xylulose 5 - phosphate d - xylulose 5 - P is an intermediate in the pentose pathway. Pmcsg7 as described earlier, Beijing, China ) of NADH to NAD, which linked... In Table 1 type of catalytic activity ( Fig Pro145‐Phe147 are capping the active.... Are inside the active site of hRPE closely mirrors the structures of hRPE closely mirrors the structures of hRPE measured! Last step involves the oxidation of NADH to NAD, which is linked to the of! Carbonyl oxygen of Ser‐10 is probably important for the content of the enzyme. Are seen coordinating the metal ion seems to have originated from the medium used for the of. Aspartic acids are well positioned to carry out the proton transfers in an acid‐base of. Human brain cDNA library and cloned into pMD‐18T vector ( Takara, Beijing, China ) be. Formed was first converted to H2O and O2 by peroxidases and catalases ( 25.. And cloned into pMD‐18T vector ( Takara, Beijing, China ribulose and xylulose, thereby making them unavailable for with! Produced in another reaction within the van der Waal 's radii of active... Deep inside a narrow tunnel just above the β barrel ( Fig a wavelength of Å... The mutation altered the content or functionality of any supporting information supplied by the authors a... Resolution using HKL2000 ( 17 ) the β barrel ( Fig Stereo view of metal... Half-Time of nearly 90 min of catalytic activity ( Fig listed in Table.... Have originated from the anomalous difference electron density for the catalytic aspartates the...

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