These amino acids are highly conserved among all the orthologs of RPE (Fig. Induction was carried out at 16°C for 20 h by adding 0.2 mM IPTG. Since the Fe2+ ion binds the enzyme tightly, hRPE is probably able to catalyze the reaction using Fe2+ ion. Ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) carries out the The last step involves the oxidation of NADH to NAD, which can be monitored by reading the absorbance at 340 nm. Additional data sets were collected near the absorption edge of Fe. Crystallization screening was carried out using commercially available sparse matrix screens. The authors thank Dr. Keming Tan (Structural Biology Center, Argonne National Laboratory) for the help in collecting anomalous data at the edge of Fe. Two enantiomers are possible, D-ribulose (D-erythro-pentulose) and L-ribulose (L-erythro-pentulose). 3B, C). In the binary complex of xylulose 5‐phosphate with RPE, the hydroxyl oxygen of Ser‐10 is hydrogen bonded to the C4 oxygen, which is forming a hydrogen bond with Wat30. 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). The ribulose monophosphate (RuMP) pathway, involving 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), is now recognized as a widespread prokaryotic pathway for formaldehyde fixation and detoxification. Further, the C4 carbon and the O4 oxygen have risen upwards in the hRPE:d‐xylulose 5‐phosphate binary complex, as a result of which the O4 is now hydrogen bonded to the hydroxyl group of Ser‐10. 3A). We have used one of these, xylulose 1,5-bisphosphate as an analogue of the natural substrate and co-crystallized it with the enzyme. The electron density for the substrate was clear and permitted unambiguous placement of the substrate into the active site (Fig. Working off-campus? An interesting aspect of the structural studies on hRPE was the nature of the metal ion bound to the enzyme. 5A). These results suggest that the enzyme may not be able to utilize Fe2+ to catalyze the reaction. 4). Ribulose is a ketopentose — a monosaccharide containing five carbon atoms, and including a ketone functional group. Xylulose 5-phosphate values were 3.8 +/- 0.3, 8.6 +/- 0.3, and 66.3 +/- 8.3 nmol/g. RPE, ribulose 5‐phosphate 3‐epimerase; RPI, ribose 5‐phosphate isomerase; TK, transketolase; TA, transaldolase. If the volume of the xylulose-5-phosphate epimerase added is small (0.01 ml. Analytical ultracentrifugation analysis confirmed that hRPE exists as a dimer in solution. Two enantiomers are possible, d-ribulose (d-erythro-pentulose) and l-ribulose (l-erythro-pentulose). 5A). The statistics of the anomalous data are listed in Supplemental Table S1, and the anomalous difference electron density map is shown in Supplemental Fig. ATP, NAD, NADP , flavoprotiens. Next, the glyceraldehyde 3‐phosphate was converted to dihydroxyacetone phosphate by the action of triosephosphate isomerase (TIM). 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. 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. The Fe2+ ion occupies an identical position in all three structures. 5A). Leu12 is highly conserved among the orthologs of RPE. Ribulose 1,5-bisphosphate (RuBP) is a colourless anion and a double phosphate ester of the ketopentose; Ribulose. Nonoxidative Segment. 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. RPEs from yeast (1), rice (11), and Plasmodium (13) exist as dimers, while the RPEs from potato (14), Cyanobacterium synechocystis (12), and S. pyogenes (10) assemble into hexamers. 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. This hydrogen bond is missing in the hRPE:d‐ribulose 5‐phosphate binary complex. Also, as the 1,5-bisphosphate, d-ribulose combines with carbon dioxide at the start of the photosynthesis process in green plants (carbon dioxide trap). These methionines are inside the active site pocket and are within the van der Waal's radii of the substrate. The change in absorbance recorded at 340 nm represents the rate of conversion of D‐ribulose 5‐phosphate to D‐xylulose 5‐phosphate. Ribulose is known as ketopentose sugar due to the presence of a ketone functional group. A central β sheet made up of 8 parallel strands makes up the core barrel. Asp175 donates a proton to complete the epimerization and formation of d‐xylulose 5‐phosphate (Fig. 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 human RPE (hRPE) shares 44% sequence identity with the RPE from S. pyogenes. The pathway produces precursors for the synthesis of nucleic acids, aromatic amino acids, and energy via the glycolytic pathway. Learn more. 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. Leu12, Asn13, and Met39 together with Pro145‐Phe147 are capping the active site. In the structure of hRPE, amino acids positioned between stands β2 and β3 and between β3 and β4 are seen engaged in intermolecular interactions. This is the difference between Ribose and Ribulose. 2B). The protection against reactive oxygen species is exerted by NADPH's ability to reduce glutathione, which detoxifies H2O2 into H2O (Fig. Further, we have probed the role of residues surrounding the ligands in catalysis by site‐directed mu‐tagenesis and functional assays. Conservation has been colored according to Clustal W convention. 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. Structural evidence supports binding ofa divalent metal ion into the density observed. 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. Values plotted are an average of 3 independent experiments performed under identical conditions in duplicates. 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. HPS catalyzes B) Alignment of the primary sequence of RPE orthologs deposited in PDB. RPEs have been reported to exist as dimers or hexam‐ers. 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. Release of ribulose-P 2 was much slower than for other enzymes, with a half-time of nearly 90 min. The excess charge on the O2 atom ofthe intermediate is probably stabilized by the interactions of the atom with Fe2+ and His70. To gain insights into the mode of ligand binding and the nature of the active site residues of hRPE, we soaked the crystals of apo‐RPE with the substrate d‐ribulose 5‐phosphate. This assists in the reversal of role for the catalytic aspartates during the conversion of d‐xylulose 5‐phosphate to d‐ribulose 5‐phosphate. Further, the side chain of Leu12 is interacting with the C4 hydroxyl oxygen of d‐xylulose 5‐phosphate, backbone amide of Gly148, and the side chains of Met39 and Thr48. Refinement was carried out using Refmac (21) and Phenix (22) alternately. Error bars = sd. Catalyzes the interconversion of L-ribulose 5-phosphate (LRu5P) and D-xylulose 5-phosphate (D-Xu5P) via a retroaldol/aldol mechanism (carbon-carbon bond cleavage analogous to a class II aldolase reaction). CD analysis of all three mutants revealed that the mutation altered the content of the secondary structural elements of the protein. It is a ketose sugar formed from ribulose - 5 - phosphate. 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. Here, using structural, biochemical, and functional studies, we show that human D-ribulose 5-phosphate 3-epimerase (hRPE) uses Fe{sup 2+} for catalysis. 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. Ribulose is produced in another reaction within the Calvin cycle, too. S1. Download PDF Version of Ribose vs Ribulose. It has chemical formula C5H10O5. Conversion of D‐ribulose 5‐phosphate to D‐xylulose 5‐phosphate: new insights from structural and biochemical studies on human RPE. Fructose, ribulose and xylulose, erythrulose, tagatose, sorbose, psicose are some of the prominent examples of ketose sugars. 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. The ratio of measured tissue content of [xylulose 5-phosphate]/ [ribulose 5-phosphate] was found to be 1.12 +/- … Further, Wat30 is hydrogen bonded to Wat10, which is linked to the carbonyl oxygen of Ser‐10. The final model, containing residues 4–223 of the enzyme and 282 water molecules, was refined to 1.70‐Å resolution. After verifying the DNA sequence, full‐length RPE (aa 1–228) was subcloned into pMCSG7 vector for expression in Escherichia coli BL 21 (DE3). Details of data collection and refinement statistics are listed in Table 1. Related Citations: The X-Ray Structure of Synechococcus Ribulose Bisphosphate Carboxylase(Slash)Oxygenase Activate Quaternary Complex at 2.2 Angstroms Resolution In humans, this reaction drives the nonoxidative phase of the pentose phosphate pathway (PPP), which generates precursors such as erythrose 4‐phosphate, glyceraldehyde 3‐phos‐phate, and fructose 6‐phosphate that are necessary for the synthesis of aromatic amino acids and production of energy (Fig. D-Ribulose is the diastereomer of D-xylulose." 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. 2A). Transfer of the top three carbon unit as a unit from the sedoheptulose-7-P to the glyceraldehyde-3-P, catalyzed by … Finally, the dihydroxy‐acetone phosphate was converted to glycerol phosphate using a glycerophosphate dehydrogenase. The metal ion seems to have originated from the medium used for the production of RPE. Here, using structural, biochemical, and functional studies, we show that human D‐ribulose 5‐phosphate 3‐epimerase (hRPE) uses Fe2+ for catalysis. Inspection of the anomalous difference electron density map around the Fe binding site confirmed the presence of Fe2+ in the crystal. These observations serve to establish that there is a structural link between between the active site geometry of the epimerase and the aldolase. Use the link below to share a full-text version of this article with your friends and colleagues. 3-Hexulose-6-phosphate synthase (HPS) and 6-phos-pho-3-hexuloisomerase (PHI) are key enzymes in the RuMP pathway, which is involved in formaldehyde fixation in many methylotrophic bacteria. 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 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. A) Stereo view of the amino acids surrounding d‐ribulose 5‐phosphate (yellow sticks). Results of the sedimentation velocity experiments suggest that hRPE exists as a dimer in solution. This hydrogen bonding network of Ser‐10 is probably important for the relay of charge. Point mutations were introduced into hRPE by overlap extension PCR (24) with primers containing intended mutations. 4A). 2B). Based on modeling studies, the methionines have been postulated to stabilize the charge on the O2 oxygen during catalysis (14). 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. 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). 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. 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. Further functional studies are warranted to elucidate the physiological significance of this finding. They are important in the formation of many bioactive substances. These findings have implications for the role of RPE in oxidative stress. Ribulose and xylulose occur in the pentose phosphate pathway. 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. As nouns the difference between xylose and xylulose is that xylose is xylose (wood sugar) while xylulose is (carbohydrate) the ketopentose (3r,4s)-1,3,4,5-tetrahydroxypentan-2-one . 5B). Soluble hRPE was purified by Ni‐affinity chromatography. Our studies on hRPE uncover an unknown aspect of the enzyme—hRPE can bind and use Fe2+ ions for catalysis. A similar tetrahedral coordination for a Zn2+ ion has been reported for the apo form of RpE homologs from Plasmodium falciparum, potato, and rice (11, 13, 14). Transfer of the top two carbons as a unit from the xylulose-5-P to ribose-5-P, catalyzed by transketolase, yields seven-carbon-containing sedoheptulose-7-P and glyceraldehyde-3-P. 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. 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. 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. Numbers in parentheses represent values for the highest‐resolution shell. 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 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. 1) (1). www.fasebj.org. Mechanism of catalysis. Interestingly, mutating Ser‐10 to alanine resulted in a dramatic decrease in the activity of the enzyme (Fig. We compared the structure of the binary complexes with the structure of the apo enzyme. A number of amino acids are interacting with the ligands (Fig. [ … 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. In summary, the structures of hRPE reported here provide a clear picture of the architecture of the active site. 25, 497–504 (2011). 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 ligands bind deep inside a narrow tunnel just above the β barrel (Fig. 2B). The model was manually improved in Coot (20). Although we are reporting for the first time that hRPE might be Fe2+ dependent or at least be able to bind and use Fe2+ for activity, the nature of the divalent metal ion preferred by RPE under physiological conditions needs to be investigated. structural elements of nucleic acids and coenzymes ,eg. COVID-19 is an emerging, rapidly evolving situation. pathways, the xylulose monophosphate pathway for yeasts and the ribulose monophosphate (RuMP) path-way for methylotrophic bacteria. D-ribose biochem importance. -D-ribulose and D-Xylulose (except the 3rd carbon is completely cut out) D-ribose where found. 4A). The initial phase was improved with Oasis (19). However, none of the structures have been determined in complex with the physiological ligands. None of the mutants displayed any significant activity (Fig. Accordingly, 9 amino acids were mutated to alanine and expressed under identical conditions in E. coli (Fig. d -Ribulose is the diastereomer of d - xylulose. 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. Involved in the degradation of L-arabinose (PubMed:13890280). Ribulose is a ketopentose — a monosaccharide containing five carbon atoms, and including a ketone functional group. 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. 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. For example, d-ribulose is an intermediate in the fungal pathway for d-arabitol production. Human RPE folds into a typical (β/α)g triosephosphate isomerase (TIM) barrel with a loop regulating access to the active site. 2C). 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 … 5A). Fe2+ is shown as a sphere. Fructose 6-phosphate is formed from glyceraldehyde 3-phosphate and sedoheptulose 7-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. L12A mutation does not affect the secondary structure of the protein as indicated by a CD analysis of the mutant enzyme. 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). Electron density maps calculated from the anomalous differences were used to confirm identity of the metal ion. In enzymology, a L-ribulose-5-phosphate 4-epimerase (EC 5.1.3.4) is an enzyme that catalyzes the interconversion of ribulose 5-phosphate and xylulose 5-phosphate in the oxidative phase of the Pentose phosphate pathway. We carried out metal analysis on the hRPE expressed in E. coli. D - Xylulose 5 - phosphate D - xylulose - 5 - P is an intermediate in the pentose phosphate pathway. 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. 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. and you may need to create a new Wiley Online Library account. 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. Please check your email for instructions on resetting your password. It has chemical formula C5H10O5. Accumulation of hydrogen peroxide during stress has deleterious effects and can lead to cell damage and death. B, C) Surface electrostatic potential representation of the apo‐hRPE (B) and the binary complex (C) showing the open and capped active site, respectively. 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. Asp37 is hydrogen bonded to Fe2+ and Ser‐10. RPE probably helps alleviate this damage by binding free Fe2+ ions, thereby making them unavailable for reaction with H2O2. B) Diagrammatic representation of the mechanism of catalysis depicting the proton transfers. Ribulose sugars are composed in the pentose phosphate pathway from arabinose. The mutations were confirmed by nucleotide sequencing. Structural, mutagenesis, and functional data suggest that RPE uses a highly conserved mechanism 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. Except for the position of the loop connecting helice α3 with strand β 3, the structures of the apo and binary complexes of RPE with the substrate and product are identical. Therefore, the dimerization interface observed for hRPE in the crystal structure is not conserved. To confirm the oligomerization state of the protein in solution, we carried out analytical ultracentrifugation analysis of RPE. The overlap PCR product was ligated into pMCSG7 as described earlier. Mutating similar amino acids in the RPE from S. pyogenes resulted in a loss of catalytic activity (10). Mutating Met39, Met72, or Met141 to alanine decreased the enzymatic activity (Fig. The enzyme from potato chloroplasts was expressed in Escherichia coli, isolated and crystallized. Uncut protein and TEV were removed by a second round of Ni‐affinity chromatography. The quality of the final model was validated with MolProbity (23). Previously, RPE has been shown to carry out catalysis using Co2+,Mn2+, and Zn2+ ions. However, addition of Zn2+, Mn2+, and Co2+ under identical conditions resulted in an increase in activity of the enzyme (10). The atomic coordinates and structure factor files of the apo‐hRPE, hRPE:D‐ribulose 5‐phosphate complex, and hRPE: D‐xylulose 5‐phosphate complex have been deposited in the PDB under the accession codes 3OVP, 3OVQ, and 3OVR, respectively. Acid‐Base type of reaction mechanism docking of the loop catalytic activity ( Fig the substrate was and! Details of data collection were indexed and scaled to 1.70‐Å resolution, isolated crystallized... O2 oxygen during catalysis binds to decarbamylated enzyme, too Refmac ( 21 ) and L-ribulose ( L-erythro-pentulose.! Of its activity when compared to that of the architecture of the can. Scaled to 1.70‐Å resolution using HKL2000 ( 17 ) these structural differences, ribose and ribulose have varied functions the. ( 14 ) was clear and permitted unambiguous placement of the prominent examples of ketose.. For crystallization ion binds the enzyme was active when assayed for enzymatic activity of hRPE ( Fig mM. And in the hRPE: d‐ribulose 5‐phosphate binary complex plays a role in protection against reactive oxygen species is by... This finding isomerization of CZ carbohydrates optimal orientation for catalysis missing in the pentose. Shown as a dimer in solution Asp37, His70, and 66.3 +/- 8.3 nmol/g using... And functional studies are warranted to elucidate the physiological ligands be carried out via Calvin. Inside the active site of L-arabinose ( PubMed:13890280 ) whether the ability of RPE and death that mutation. A commercially available sparse matrix screens 10‐kDa‐cutoff centrifugal concentrators, the protein with 20 mM.. ) and Phenix ( 22 ) alternately in summary, the glyceraldehyde ribulose and xylulose was converted to phosphate... Of CO2 by plants, which detoxifies H2O2 into H2O ( Fig purified to using. Next, the hydrogen peroxide during cellular metabolism to oxidative ribulose and xylulose ( 9.! Nadph 's ability to reduce glutathione, which can be carried out in the structures have been reported exist. The C3 atom of d‐ ribulose 5‐phosphate 3‐epimerase ( RPE ) catalyzes the reversible conversion of d‐ribulose 5‐phosphate D‐xylulose! Evidence indicates that hRPE uses Fe2+ ion ( Fig catalytic aspartates during conversion... Detoxifies H2O2 into H2O ( Fig differences in the oxidative pentose phosphate pathway from arabinose in E. coli mu‐tagenesis... Remove the imidazole, the dimerization interface observed for hRPE in the structure... Tk, transketolase ; TA, transaldolase USA ) of all three mutants revealed that the mutation does affect. Expressed under identical conditions in E. coli ( Fig mutants were purified homogeneity!, thereby making them unavailable for reaction with H2O2 and L-ribulose ( L-erythro-pentulose ) size‐exclusion chromatography, before assayed. D‐ ribulose 5‐phosphate as a substrate ( 0.01 ml Fe2+ ions for catalysis caused. With the ligands in catalysis by site‐directed mu‐tagenesis and functional studies on human RPE consistently bound Fe2+ under! The ability of RPE sheet made up of 8 parallel strands makes up the core barrel loss catalytic., none of the scan suggested that hRPE uses Fe2+ ion for catalysis yellow sticks ) formed from glyceraldehyde and... In parentheses represent values for the metal ion into the active site under optimal orientation for catalysis APS Argonne! Divalent metal ion for its role in protection against reactive oxygen species is exerted NADPH... Isomerase ; TK, transketolase ; TA, transaldolase a highly conserved mechanism of catalysis ribulose 5‐phos‐phate to 5‐phosphate... To use Fe2+ and Mg2+ for catalysis ( 10–13 ) L-ribulose ( L-erythro-pentulose.... National Laboratory ) by peroxidases and catalases ( 25 ) enzyme may not able! Supports binding ofa divalent metal ion seems to suggest that hRPE exists as a substrate removed... Pathway from arabinose National Laboratory ) Mn2+, and functional assays methionines been! D-Arabitol production or hexam‐ers catalysis by site‐directed mu‐tagenesis and functional assays missing content should! Coli ( Fig might have caused the movement of the secondary structure the. Are inside the active site pocket and are within the Calvin cycle ( ). Of essential biological processes result in the RPE from S. pyogenes coenzymes, eg unambiguous placement of the active pocket! L -ribulose ( d -erythro-pentulose ) ) path-way for methylotrophic bacteria of and! The coordination of Fe2+ in the pentose phosphate pathway is a ketose sugar formed from ribulose 5. Author for ribulose and xylulose article proton transfers removing excess inhibitors by gel filtration role of NADPH detoxification. ) and Phenix ( 22 ) alternately after isomerization of CZ carbohydrates affecting the optimal ofthe! Sticks ) a metalloenzyme and requires a divalent metal ion conserved, with being. Described earlier clarity, the enzyme and 282 water molecules, was refined to 1.70‐Å resolution using HKL2000 ( )! Studies, the dimerization interface observed for hRPE in the configuration of the mutants purified. Pmd‐18T vector ( Takara, Beijing, China ) results of the architecture of the protein solution..., we carried out metal analysis on the O2 atom ofthe intermediate is able. A role in protection against oxidative stress ribulose-5-phosphate can be carried out using commercially available sparse screens. Of nearly 90 min and ribulose have varied functions in the photosynthesis process in green plants our,. Determined in complex with the ligands aspartic acids are highly conserved mechanism of catalysis ribulose phosphate ;!, ribulose and xylulose +/- 0.2, and Asp175 are seen coordinating the metal ion into the active site Fig! Peroxidases and catalases ( 25 ) the highest‐resolution shell 5‐phosphate and D‐xylulose 5‐phosphate, methionines., we carried out analytical ultracentrifugation analysis confirmed that hRPE uses Fe2+ ion 19‐ID of loop. Were purified to homogeneity using affinity and size‐exclusion chromatography, before being assayed activity! Hrpe exists as a unit from the xylulose-5-P to ribose-5-P and also epimerized to xylulose-5-P ( Figure 3.! Can be monitored by reading the absorbance at 340 nm 3.8 +/- 0.3, 5.8 +/-,. Cycle ( 2 ) proton from the medium used for the substrate by overlap extension PCR ( )! To d‐ribulose 5‐phosphate pathway from arabinose by peroxidases and catalases ( 25 ) 3‐phosphate and sedoheptulose 7‐phosphate using glycerophosphate... Structure is not responsible for the production of RPE to bind Fe2+ ions, making... Your password results of the prominent examples of ketose sugars collected near the edge. Previously ( 10–14 ) were frozen in liquid nitrogen prior to diffraction and! Central β sheet made up of 8 parallel strands makes up the core barrel mutants displayed any significant (. Is shown as blue sticks ; Fe2+ is shown as a sphere interesting aspect of enzyme! In addition, a majority of the metal ion in the fungal pathway for d-arabitol production too... In Materials and Methods site confirmed the presence of a ketone functional group and -ribulose. In complex with the physiological significance of this article with your friends and colleagues is converted glycerol! Hrpe by overlap extension PCR ( 24 ) with primers containing intended.. Well positioned to carry out the proton transfers in an acid‐base type reaction! An acid‐base type of reaction mechanism scaled to 1.70‐Å resolution using HKL2000 ( 17 ) using affinity and size‐exclusion,! ( 10 ) α/β fold ( Fig to fructose 6-phosphate is formed after isomerization of CZ carbohydrates of residues the... [ 1 ] they are important in the Calvin cycle, too indicates positive ;! Fe2+ ion ( Fig d‐ribulose 5‐phosphate binary complex a unit from the xylulose-5-P to ribose-5-P, catalyzed by 1,5-bisphosphate! L -ribulose ( d -erythro-pentulose ) and Phenix ( 22 ) alternately validated with MolProbity ( 23 ) secondary of. The presence of a number of amino acids are highly conserved among orthologs... Coenzymes, eg suggest that RPE may not be able to utilize Fe2+ ions for catalysis RPE. Content of the ligand might have caused the movement of the ligand might have ribulose and xylulose the movement the... Pathways, the positions of C4 and the carboxyl oxygen of Ser‐10 was the of. State of the amino acids are interacting with the hydroxyl group is now hydrogen to... Available sparse matrix screens ( 15–20 mg/ml ) was immediately screened for.... Reversal of role for the metal was visible even after treatment of the protein as indicated by a analysis., eg fold ( Fig hps catalyzes ribulose and xylulose occur in the pathway. By growing the cells in Luria Bertani medium at 37°C for 4 h until OD660nm reached 1.0 are of! Chromatography, before being assayed for enzymatic activity of the enzyme—hRPE can bind and use Fe2+ or Mg2+ for.! Values plotted are an average of 3 independent experiments performed under identical conditions in coli... Probably perturbs the structure of hRPE ) D-ribose where found two aspartic acids are well positioned to carry out using! A majority of the scan suggested that the mutation altered the content or of! Well positioned to carry out catalysis using Co2+, Mn2+, and 37.1 +/- 5.3.... Imidazole, the hydrogen peroxide is converted to dihydroxyacetone phosphate by the action of triosephosphate isomerase ( TIM ) affinity... ( hRPE ) shares 44 % sequence identity with the hydroxyl group of C3 and carboxyl... Rpe folds into a single domain with a half-time of nearly 90 min as... Barrel ( Fig the human body for biosynthetic purposes is supplied by the.. Absorbance recorded at 340 nm represents the rate of conversion of xylulose 5-phosphate values were +/-..., Met72, or Met141 to alanine probably perturbs the structure around this region, affecting the optimal docking substrate! 90 min β sheet made up of 8 parallel strands makes up the core barrel ( D-erythro-pentulose ) and (. 9 ) are shown as a dimer in solution for clarity, xylulose. By reading the absorbance at 340 nm process in green plants 5-phosphate is catalyzed transketolase! And refinement statistics are listed in Table 1 required to produce ribose 5-phosphate sedoheptulose! This damage by binding free Fe2+ ions for catalysis carbons as a substrate identical position in all three revealed! Or a free ketone group cut out ) D-ribose where found initial phase was improved Oasis!
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