The Experts below are selected from a list of 165 Experts worldwide ranked by ideXlab platform
Karlheinz Suss - One of the best experts on this subject based on the ideXlab platform.
-
structure and catalytic mechanism of the cytosolic d Ribulose 5 phosphate 3 epimerase from rice
Journal of Molecular Biology, 2003Co-Authors: Stefan Jelakovic, Stanislav Kopriva, Karlheinz Suss, Georg E SchulzAbstract:Cytosolic D-Ribulose-5-Phosphate 3-epimerase from rice was crystallized after EDTA treatment and structurally elucidated by X-ray diffraction to 1.9 Aresolution. A prominent Zn 2þ site at the active center was estab- lished in a soaking experiment. The structure was compared with that of the EDTA-treated crystalline enzyme from the chloroplasts of potato plant leaves showing some structural differences, in particular the "closed" state of a strongly conserved mobile loop covering the substrate at its putative binding site. The previous proposal for the active center was confirmed and the most likely substrate binding position and confor- mation was derived from the locations of the bound zinc and sulfate ions and of three water molecules. Assuming that the bound zinc ion is an integral part of the enzyme, a reaction mechanism involving a well- stabilized cis-enediolate intermediate is suggested. q 2003 Elsevier Science Ltd. All rights reserved
-
identification cloning and properties of cytosolic d Ribulose 5 phosphate 3 epimerase from higher plants
Journal of Biological Chemistry, 2000Co-Authors: Stanislav Kopriva, Anna Koprivova, Karlheinz SussAbstract:Plant cells contain a complete oxidative pentose phosphate pathway in the chloroplasts, but an incomplete pathway was proposed to be present in the cytosol, with cytosolic (cyt) isoforms of Ribulose-5-Phosphate 3-epimerase (RPEase) and other non-oxidative branch enzymes being undetectable. Here we present for the first time the identification, cloning, and properties of a cyt-RPEase in rice (Oryza sativa) and presence of its homologues in other plant species. Recombinant cyt-RPEase is a homodimer of 24.3-kDa subunits such as in the case of the animal and yeast enzymes, whereas the chloroplast (chl) RPEase is a hexamer. Cytosolic and chloroplastic RPEases cannot be separated by anion exchange chromatography. Since plant cyt-RPEase is more closely related in its primary structure to homologous enzymes in animal and yeast cells than to the chloroplast RPEase, the plant nuclear genes coding for cytosolic and chloroplast RPEases were most likely derived from eubacteria and cyanobacteria, respectively. Accumulation of cyt-RPEase-mRNA and protein is high in root cells, lacking chl-RPEase, and lower in green tissue. These and other observations support the view that green and non-green plant cells possess a complete oxidative pentose phosphate pathway in the cytosol.
-
structure and mechanism of the amphibolic enzyme d Ribulose 5 phosphate 3 epimerase from potato chloroplasts
Journal of Molecular Biology, 1999Co-Authors: Jurgen Kopp, Stanislav Kopriva, Karlheinz Suss, Georg E SchulzAbstract:Abstract 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 enzyme from potato chloroplasts was expressed in Escherichia coli, isolated and crystallized. The crystal structure was elucidated by multiple isomorphous replacement and refined at 2.3 A resolution. The enzyme is a homohexamer with D3 symmetry. The subunit chain fold is a (βα)8-barrel. A sequence comparison with homologous epimerases outlined the active center and indicated that all members of this family are likely to share the same catalytic mechanism. The substrate could be modeled by putting its phosphate onto the observed sulfate position and its epimerized C3 atom between two carboxylates that participate in an extensive hydrogen bonding system. A mutation confirmed the crucial role of one of these carboxylates. The geometry together with the conservation pattern suggests that the negative charge of the putative cis-ene-diolate intermediate is stabilized by the transient induced dipoles of a methionine sulfur “cushion”, which is proton-free and therefore prevents isomerization instead of epimerization.
-
purification properties and in situ localization of the amphibolic enzymes d Ribulose 5 phosphate 3 epimerase and transketolase from spinach chloroplasts
FEBS Journal, 1998Co-Authors: Markus Teige, Michael Melzer, Karlheinz SussAbstract:The amphibolic enzymes D-Ribulose 5-Phosphate 3-epimerase and transketolase have been purified from stroma extracts of spinach chloroplasts using ammonium sulfate fractionation and FPLC. For the native enzymes, a molecular mass of 180 kDa for epimerase and 160 kDa for transketolase was found and the molecular masses of the subunits was determined to be 23 kDa for epimerase and 74 kDa for transketolase. Protein sequencing of the purified chloroplast enzymes revealed the NH2-terminal amino acid sequences of mature epimerase (NH2-TSRVDKFSKSDIIVSP) and transketolase (NH2-AAVEALESTDTDQLVEG). The enzymic properties of both enzymes such as Km values or pH optima, were found to be very similar to those for epimerases and transketolases from other sources, including yeast and animal cells. In contrast to the light-activated enzymes of the Calvin cycle, the activity of these amphibolic enzymes was not redox-dependent. Immunogold electron microscopy on spinach leaf thin sections revealed that about 90 % of the total epimerase and transketolase, and 96 % of the total chloroplast H+-ATP synthase portion CF1 are associated with thylakoid membranes in situ. Ribulose-1,5-bisphosphate carboxylase/oxygenase, in contrast, was evenly distributed throughout chloroplasts. These and other results indicate that minor chloroplast enzymes are arranged in a thin layer on thylakoid membrane surfaces in vivo.
-
Molecular Cloning and Characterization of Cytosolic Isoform of Ribulose-5-Phosphate 3-Epimerase from Rice
Photosynthesis: Mechanisms and Effects, 1998Co-Authors: Stanislav Kopriva, Anna Koprivova, Karlheinz SussAbstract:Although the oxidative pentose phosphate pathway (OPPP) in plants partially contributes to respiration, it plays a much more important role as a source of NADPH for the reduction of metabolites in various biosynthetic pathways. In animal and yeast cells, OPPP is present in the cytosol and, therefore, it was generally accepted that the cytosol of plant cells also contains this pathway (1). After a CO2 release from [1-14C]glucose by isolated chloroplasts was demonstrated it was believed that two sets of isoenzymes of this pathway occur in chloroplasts and in cytosol (2,3). However, the quantitative distribution of OPPP enzymes among the cell compartments in green and non-green plant cell is still unclear. There remains no doubt about the existence of cytosolic and plastidic isoforms of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in leaf mesophyll and non-green plant cells (4). In contrast, cell fractionation experiments combined with chromatographic separation of enzyme isoforms did not provide evidence for the occurrence of cytoplasmic OPPP enzymes catalyzing the regenerative sequence of the pathway including transketolase, ribose-5-Phosphate isomerase, and Ribulose-5-Phosphate 3-epimerase (RPE) (5). This led to the conclusion that plastids contain a complete OPPP, whereas an incomplete pathway is operating in the cytoplasm of leaf mesophyll cells.
William Martin - One of the best experts on this subject based on the ideXlab platform.
-
Enzymatic Evidence for a Complete Oxidative Pentose Phosphate Pathway in Chloroplasts and an Incomplete Pathway in the Cytosol of Spinach Leaves.
Plant Physiology, 1995Co-Authors: Claus Schnarrenberger, Anke Flechner, William MartinAbstract:The intracellular localization of transaldolase, transketolase, ribose-5-Phosphate isomerase, and Ribulose-5-Phosphate epimerase was reexamined in spinach (Spinacia oleracea L.) leaves. We found highly predominant if not exclusive localization of these enzyme activities in chloroplasts isolated by isopyknic centrifugation in sucrose gradients. Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glucose phosphate isomerase, and triose phosphate isomerase activity was present in the chloroplast fraction but showed additional activity in the cytosol (supernatant) fraction attributable to the cytosol-specific isoforms known to exist for these enzymes. Anion-exchange chromatography of proteins of crude extracts on diethylaminoethyl-Fractogel revealed only a single enzyme each for transaldolase, transketolase, ribose-5-Phosphate isomerase, and Ribulose-5-Phosphate epimerase. The data indicate that chloroplasts of spinach leaf cells possess the complete complement of enzymes of the oxidative pentose phosphate path-way (OPPP), whereas the cytosol contains only the first two reactions, contrary to the widely held view that plants generally possess a cytosolic OPPP capable of cyclic function. The chloroplast enzymes transketolase, ribose-5-Phosphate isomerase, and Ribulose-5-Phosphate epimerase appear to be amphibolic for the Calvin cycle and OPPP.
-
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
Plant molecular biology, 1995Co-Authors: Ulrich Nowitzki, Claus Schnarrenberger, Ralf Wyrich, Peter Westhoff, Katrin Henze, William MartinAbstract:Exploiting the differential expression of genes for Calvin cycle enzymes in bundle-sheath and mesophyll cells of the C4 plant Sorghum bicolor L., we isolated via subtractive hybridization a molecular probe for the Calvin cycle enzyme d-Ribulose-5-Phosphate 3-epimerase (R5P3E) (EC 5.1.3.1), with the help of which several full-size cDNAs were isolated from spinach. Functional identity of the encoded mature subunit was shown by R5P3E activity found in affinity-purified glutatione S-transferase fusions expressed in Escherichia coli and by three-fold increase of R5P3E activity upon induction of E. coli overexpressing the spinach subunit under the control of the bacteriophage T7 promoter, demonstrating that we have cloned the first functional Ribulose-5-Phosphate 3-epimerase from any eukaryotic source. The chloroplast enzyme from spinach shares about 50% amino acid identity with its homologues from the Calvin cycle operons of the autotrophic purple bacteria Alcaligenes eutrophus and Rhodospirillum rubrum. A R5P3E-related eubacterial gene family was identified which arose through ancient duplications in prokaryotic chromosomes, three R5P3E-related genes of yet unknown function have persisted to the present within the E. coli genome. A gene phylogeny reveals that spinach R5P3E is more similar to eubacterial homologues than to the yeast sequence, suggesting a eubacterial origin for this plant nuclear gene.
Markus Fischer - One of the best experts on this subject based on the ideXlab platform.
-
Biosynthesis of flavocoenzymes
Natural product reports, 2005Co-Authors: Markus Fischer, Adelbert BacherAbstract:The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of Ribulose 5-Phosphate. The imidazole ring of GTP is hydrolytically opened, yielding a 2,5-diaminopyrimidine that is converted to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction, and dephosphorylation. Condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate obtained from Ribulose 5-Phosphate affords 6,7-dimethyl-8-ribityllumazine. Dismutation of the lumazine derivative yields riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is recycled in the biosynthetic pathway. The enzymes of the riboflavin pathway are potential targets for antibacterial agents.
-
metal sites in 3 4 dihydroxy 2 butanone 4 phosphate synthase from methanococcus jannaschii in complex with the substrate Ribulose 5 phosphate
Acta Crystallographica Section D-biological Crystallography, 2004Co-Authors: Stefan Steinbacher, Adelbert Bacher, Susanne Schiffmann, Markus FischerAbstract:The crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate Ribulose 5-Phosphate at a dimetal centre has recently been determined at 1.7 A resolution. The enzyme converts Ribulose 5-Phosphate into 3,4-dihydroxy-2-butanone 4-phosphate, while its C4 atom is released as formate. The resulting four-carbon body supplies all eight C atoms for the xylene moiety of riboflavin. Three of the four hydroxyl groups of Ribulose 5-Phosphate were coordinated by the metal ions. Based on crystallographic refinement, the metals were assigned as zinc and calcium, which were present in the crystallization buffer. Neither metal supports the enzymatic reaction. In the present study, the correctness of this assignment is assessed using anomalous diffraction data collected at the high-energy side of the zinc absorption edge (lambda = 1.2823 A). Only the three tentative zinc ions give strong peaks in an anomalous difference Fourier map (>20sigma), whereas the four tentative calcium ions do not show anomalous signals above the noise level. These results confirm the initial assignment. In addition, the resolution was improved to 1.55 A.
-
structure of 3 4 dihydroxy 2 butanone 4 phosphate synthase from methanococcus jannaschii in complex with divalent metal ions and the substrate Ribulose 5 phosphate implications for the catalytic mechanism
Journal of Biological Chemistry, 2003Co-Authors: Stefan Steinbacher, Adelbert Bacher, Susanne Schiffmann, Gerald Richter, Robert Huber, Markus FischerAbstract:Skeletal rearrangements of carbohydrates are crucial for many biosynthetic pathways. In riboflavin biosynthesis Ribulose 5-Phosphate is converted into 3,4-dihydroxy-2-butanone 4-phosphate while its C4 atom is released as formate in a sequence of metal-dependent reactions. Here, we present the crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate Ribulose 5-Phosphate at a dimetal center presumably consisting of non-catalytic zinc and calcium ions at 1.7-A resolution. The carbonyl group (O2) and two out of three free hydroxyl groups (OH3 and OH4) of the substrate are metal-coordinated. We correlate previous mutational studies on this enzyme with the present structural results. Residues of the first coordination sphere involved in metal binding are indispensable for catalytic activity. Only Glu-185 of the second coordination sphere cannot be replaced without complete loss of activity. It contacts the C3 hydrogen atom directly and probably initiates enediol formation in concert with both metal ions to start the reaction sequence. Mechanistic similarities to Rubisco acting on the similar substrate Ribulose 1,5-diphosphate in carbon dioxide fixation as well as other carbohydrate (reducto-) isomerases are discussed.
-
Biosynthesis of vitamin b2 (riboflavin).
Annual review of nutrition, 2000Co-Authors: A. Bacher, Markus Fischer, Sabine Eberhardt, Klaus Kis, Gerald RichterAbstract:▪ Abstract The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of Ribulose 5-Phosphate as substrates. The imidazole ring of GTP is hydrolytically opened, yielding a 4,5-diaminopyrimidine which is converted to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction and dephosphorylation. Condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate obtained from Ribulose 5-Phosphate affords 6,7-dimethyl-8-ribityllumazine. Dismutation of the lumazine derivative yields riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is recycled in the biosynthetic pathway. The structure of the biosynthetic enzyme, 6,7-dimethyl-8-ribityllumazine synthase, has been studied in considerable detail.
Terry Wood - One of the best experts on this subject based on the ideXlab platform.
-
[14] Assay for d-ribose-5-Phosphate ketol isomerase and d-Ribulose-5-Phosphate 3-epimerase
Methods in Enzymology, 2004Co-Authors: Terry WoodAbstract:Publisher Summary This chapter discusses the assay for D-ribose-5-Phosphate ketol isomerase and D-Ribulose- 5-Phosphate 3-epimerase. The assay for the isomerase is based upon the increase in absorbance observed at 290 nm, when ribose-5-Phosphate ketol isomerase acts upon ribose 5-Phosphate to form Ribulose 5-Phosphate. When the reaction has reached equilibrium, the addition of epimerase gives rise to a further increase in absorbance as xylulose 5-Phosphate is formed. In the presence of an excess of the isomerase, the second rate of absorbance increase is proportional to the activity of the epimerase. In assay of ribose-5-Phosphate ketol isomerase, the reaction mixture contains 1.78 ml 50 m M triethanolamine, HC1 buffer and 0.20 ml 100 m M ribose 5-Phosphate (20 μ moles). It is placed in the cell compartment at 37° of a double-beam spectrophotometer connected to a recorder, and read against a control cuvette containing an equal volume of buffer. In the assay of Ribulose-5-Phosphate 3-epimerase, the assay mixture is set up as before, and 0.01 ml (2 units) of the isomerase is added. The absorbance increases rapidly at first and then levels off after 12–15 min. If small amounts of epimerase are present in the isomerase the recorder trace shows a slight upward slope. The calculation of isomerase and epimerase activity is presented. The isomerase assay has been used to follow the purification of the enzyme from spinach and other sources, in the determination of Michaelis constants.
-
[8] Preparation and analysis of mixtures of d-ribose 5-Phosphate, d-Ribulose 5-Phosphate, and d-xylulose 5-Phosphate
Methods in Enzymology, 2004Co-Authors: Terry WoodAbstract:Publisher Summary For the coupled assay at 340 nm of transketolase with α-glycerophosphate dehydrogenase and triosephosphate isomerase, or with glyceraldehydes phosphate dehydrogenase, as auxiliary enzymes, a mixture of D-ribose 5-Phosphate and D-xylulose 5-Phosphate is needed. A mixture of D-ribose 5-Phosphate and D-Ribulose 5-Phosphate is employed for the assay of D-Ribulose-5-Phosphate 3-epimerase by coupling it to the transketolase reaction. A simple procedure is described in the chapter for preparing mixtures of the pentose phosphates for the assay of the above two enzymes. Although the commercial purifed epimerase is specified, it need not be free of the isomerase, and a crude concentrate of the two enzymes from spleen, muscle, or liver, may be used provided that transketolase is absent. The ultrafiltration is carried out, at or near, 37°, to prevent the composition of the equilibrium mixture changing after the removal from the spectrophotometer. The procedure may also be used with a 20 m M solution of ribose 5-Phosphate. At this higher concentration, however, further spectral changes of the isomerase product occur, and may take place before the epimerase is added, obscuring the progress of the enzymic reaction. Ribose 5-Phosphate is measured, without interference from the ketopentose phosphates, by the colorimetric phloroglucinol method. Ribulose 5-Phosphate is measured in ribose 5-Phosphate-Ribulose 5-Phosphate mixtures with cysteine-carbazole; a heating time of 2 hr at 37° is used to develop the color. Xylulose 5-Phosphate is measured, either alone or in conjunction with Ribulose 5-Phosphate by the spectrophotometric method described in the chapter. Spectrophotometric determination of D-Xylulose 5-Phosphate and D-Ribulose 5-Phosphate uses α -glycerophosphate dehydrogenase and triose phosphate isomerase to measure the glyceraldehyde 3-phosphate formed. One molecule of ketopentose phosphate causes the oxidation of 1 molecule of NADH.
Juan Jose Cazzulo - One of the best experts on this subject based on the ideXlab platform.
-
structure kinetic characterization and subcellular localization of the two Ribulose 5 phosphate epimerase isoenzymes from trypanosoma cruzi
PLOS ONE, 2017Co-Authors: Soledad Natalia Gonzalez, Wanda M Valsecchi, Dante A Maugeri, Jose M Delfino, Juan Jose CazzuloAbstract:The enzyme of the pentose phosphate pathway (PPP) Ribulose-5-Phosphate-epimerase (RPE) is encoded by two genes present in the genome of Trypanosoma cruzi CL Brener clone: TcRPE1 and TcRPE2. Despite high sequence similarity at the amino acid residue level, the recombinant isoenzymes show a strikingly different kinetics. Whereas TcRPE2 follows a typical michaelian behavior, TcRPE1 shows a complex kinetic pattern, displaying a biphasic curve, suggesting the coexistence of -at least- two kinetically different molecular forms. Regarding the subcellular localization in epimastigotes, whereas TcRPE1 is a cytosolic enzyme, TcRPE2 is localized in glycosomes. To our knowledge, TcRPE2 is the first PPP isoenzyme that is exclusively localized in glycosomes. Over-expression of TcRPE1, but not of TcRPE2, significantly reduces the parasite doubling time in vitro, as compared with wild type epimastigotes. Both TcRPEs represent single domain proteins exhibiting the classical α/β TIM-barrel fold, as expected for enzymes with this activity. With regard to the architecture of the active site, all the important amino acid residues for catalysis -with the exception of M58- are also present in both TcRPEs models. The superimposition of the binding pocket of both isoenzyme models shows that they adopt essentially identical positions in the active site with a residue specific RMSD < 2A, with the sole exception of S12, which displays a large deviation (residue specific RMSD: 11.07 A). Studies on the quaternary arrangement of these isoenzymes reveal that both are present in a mixture of various oligomeric species made up of an even number of molecules, probably pointing to the dimer as their minimal functional unit. This multiplicity of oligomeric species has not been reported for any of the other RPEs studied so far and it might bear implications for the regulation of TcRPEs activity, although further investigation will be necessary to unravel the physiological significance of these structural findings.
-
The 6-Phosphogluconate Dehydrogenase of Leishmania (Leishmania) mexicana: Gene Characterization and Protein Structure Prediction
Journal of molecular microbiology and biotechnology, 2010Co-Authors: Deyanira González, Juan Jose Cazzulo, Michael P. Barrett, José Luis Pérez, María Luisa Cerdeño Serrano, Mariana Igoillo-esteve, José Bubis, Alexis Mendoza-leónAbstract:6-Phosphogluconate dehydrogenase (6PGDH) is a key enzyme of the oxidative branch involved in the generation of NADPH and Ribulose 5-Phosphate. In the present work, we describe the cloning, sequencing
