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Sherry L. Mowbray - One of the best experts on this subject based on the ideXlab platform.
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Structures of type B ribose 5-phosphate Isomerase from Trypanosoma cruzi shed light on the determinants of sugar specificity in the structural family.
FEBS Journal, 2011Co-Authors: A.l. Stern, A. Naworyta, Juan José Cazzulo, Sherry L. MowbrayAbstract:Ribose-5-Phosphate Isomerase (Rpi; EC 5.3.1.6) is a key activity of the pentose phosphate pathway. Two unrelated types of sequence/structure possess this activity: type A Rpi (present in most organ ...
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Structures of type B ribose 5-phosphate Isomerase from Trypanosoma cruzi shed light on the determinants of sugar specificity in the structural family.
FEBS Journal, 2011Co-Authors: A.l. Stern, A. Naworyta, Juan José Cazzulo, Sherry L. MowbrayAbstract:Ribose-5-Phosphate Isomerase (Rpi; EC 5.3.1.6) is a key activity of the pentose phosphate pathway. Two unrelated types of sequence/structure possess this activity: type A Rpi (present in most organisms) and type B Rpi (RpiB) (in some bacteria and parasitic protozoa). In the present study, we report enzyme kinetics and crystallographic studies of the RpiB from the human pathogen, Trypanosoma cruzi. Structures of the wild-type and a Cys69Ala mutant enzyme, alone or bound to phosphate, D-ribose 5-phosphate, or the inhibitors 4-phospho-D-erythronohydroxamic acid and D-allose 6-phosphate, highlight features of the active site, and show that small conformational changes are linked to binding. Kinetic studies confirm that, similar to the RpiB from Mycobacterium tuberculosis, the T. cruzi enzyme can isomerize D-ribose 5-phosphate effectively, but not the 6-carbon sugar D-allose 6-phosphate; instead, this sugar acts as an inhibitor of both enzymes. The behaviour is distinct from that of the more closely related (to T. cruzi RpiB) Escherichia coli enzyme, which can isomerize both types of sugars. The hypothesis that differences in a phosphate-binding loop near the active site were linked to the differences in specificity was tested by construction of a mutant T. cruzi enzyme with a sequence in this loop more similar to that of E. coli RpiB; this mutant enzyme gained the ability to act on the 6-carbon sugar. The combined information allows us to distinguish the two types of specificity patterns in other available sequences. The results obtained in the present study provide insights into the action of RpiB enzymes generally, and also comprise a firm basis for future work in drug design.
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Synthesis of 5-deoxy-5-phospho-d-ribonohydroxamic acid: a new competitive and selective inhibitor of type B Ribose-5-Phosphate Isomerase from Mycobacterium tuberculosis
Tetrahedron Letters, 2005Co-Authors: Emmanuel S Burgos, Sherry L. Mowbray, Annette K. Roos, Laurent SalmonAbstract:Ribose 5-phosphate Isomerase (Rpi) is one of the major enzymes of the pentose phosphate pathway, where it catalyses the inter-conversion of ribose 5-phosphate (R5P) and ribulose 5-phosphate. Two forms of this Isomerase with no significant amino acid sequence similarity exist, RpiA and RpiB. This thesis describes RpiB from the organisms Mycobacterium tuberculosis (Mt) and Escherichia coli (Ec) from a structural and functional point of view.Since the E. coli genome encodes both an RpiA and an RpiB, which generally is not expressed, it has been proposed that EcRpiB has a different role as an allose-6-phosphate Isomerase. Activity measurements presented here show that EcRpiB does have this second activity. In the M. tuberculosis genome there is only a gene for RpiB. The crystal structure of MtRpiB was solved in complex with several different inhibitors designed to mimic the reaction intermediate as well as with the substrate, R5P. The organisation of the active site in these structures could be used to derive the reaction mechanism for MtRpiB and for other RpiBs in general. Activity measurements of MtRpiB showed that it can catalyse the R5P isomerisation, but not the allose 6-phosphate reaction. Differences observed in the active site between EcRpiB and MtRpiB explain these kinetic results. Activity measurements and a structure of an EcRpiB mutant, where histidine99 was changed to asparagine, implies that RpiB catalyses the first step of the reaction in which the sugar ring must be opened, and gives a possible explanation for how this could occur. Inhibition studies have uncovered a compound that selectively inhibits MtRpiB over RpiA from spinach, which is homologous to the human RpiA. Differences in the inhibition patterns and active site residues of these two species’ Rpi may provide information for future virtual screening approaches, with the aim of discovering new anti-tuberculosis agents.
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Competitive Inhibitors of Mycobacterium tuberculosis Ribose-5-Phosphate Isomerase B Reveal New Information about the Reaction Mechanism
Journal of Biological Chemistry, 2004Co-Authors: Annette K. Roos, Laurent Salmon, Daniel J. Ericsson, Emmanuel S Burgos, Sherry L. MowbrayAbstract:Abstract Ribose-5-Phosphate Isomerase (Rpi), an important enzyme in the pentose phosphate pathway, catalyzes the interconversion of ribulose 5-phosphate and ribose 5-phosphate. Two unrelated Isomerases have been identified, RpiA and RpiB, with different structures and active site residues. The reaction catalyzed by both enzymes is thought to proceed via a high energy enediolate intermediate, by analogy to other carbohydrate Isomerases. Here we present studies of RpiB from Mycobacterium tuberculosis together with small molecules designed to resemble the enediolate intermediate. The relative affinities of these inhibitors for RpiB have a different pattern than that observed previously for the RpiA from spinach. X-ray structures of RpiB in complex with the inhibitors 4-phospho-d-erythronohydroxamic acid (Km 57 μm) and 4-phospho-d-erythronate (Ki 1.7 mm) refined to resolutions of 2.1 and 2.2 A, respectively, allowed us to assign roles for most active site residues. These results, combined with docking of the substrates in the position of the most effective inhibitor, now allow us to outline the reaction mechanism for RpiBs. Both enzymes have residues that can catalyze opening of the furanose ring of the ribose 5-phosphate and so can improve the efficiency of the reaction. Both enzymes also have an acidic residue that acts as a base in the isomerization step. A lysine residue in RpiAs provides for more efficient stabilization of the intermediate than the corresponding uncharged groups of RpiBs; this same feature lies behind the more efficient binding of RpiA to 4-phospho-d-erythronate.
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Mycobacterium Tuberculosis Ribose-5-Phosphate Isomerase Has a Known Fold, But a Novel Active Site
Journal of Molecular Biology, 2004Co-Authors: Annette K. Roos, C. Evalena Andersson, Terese Bergfors, Micael Jacobsson, T. Alwyn Jones, Anders Karlen, Torsten Unge, Sherry L. MowbrayAbstract:Mycobacterium tuberculosis Ribose-5-Phosphate Isomerase has a known fold, but a novel active site.
Deok-kun Oh - One of the best experts on this subject based on the ideXlab platform.
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biotransformation of fructose to allose by a one pot reaction using flavonifractor plautiid allulose 3 epimerase and clostridium thermocellum ribose 5 phosphate Isomerase
Journal of Microbiology and Biotechnology, 2018Co-Authors: Kyungchul Shin, Deok-kun OhAbstract:: D-Allose is a potential medical sugar because it has anticancer, antihypertensive, anti-inflammatory, antioxidative, and immunosuppressant activities. Allose production from fructose as a cheap substrate was performed by a one-pot reaction using Flavonifractor plautiiD-allulose 3-epimerase (FP-DAE) and Clostridium thermocellum ribose 5-phosphate Isomerase (CT-RPI). The optimal reaction conditions for allose production were pH 7.5, 60°C, 0.1 g/l FP-DAE, 12 g/l CT-RPI, and 600 g/l fructose in the presence of 1 mM Co2+. Under these optimized conditions, FP-DAE and CT-RPI produced 79 g/l allose for 2 h, with a conversion yield of 13%. This is the first biotransformation of fructose to allose by a two-enzyme system. The production of allose by a one-pot reaction using FP-DAE and CT-RPI was 1.3-fold higher than that by a two-step reaction using the two enzymes.
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Microbial metabolism and biotechnological production of d-allose
Applied Microbiology and Biotechnology, 2011Co-Authors: Deok-kun OhAbstract:d -Allose has attracted a great deal of attention in recent years due to its many pharmaceutical activities, which include anti-cancer, anti-tumor, anti-inflammatory, anti-oxidative, anti-hypertensive, cryoprotective, and immunosuppressant activities. d -Allose has been produced from d -psicose using d -allose-producing enzymes, including l -rhamnose Isomerase, Ribose-5-Phosphate Isomerase, and galactose-6-phosphate Isomerase. In this article, the properties, applications, and metabolism of d -allose are described, and the biochemical properties of d -allose-producing enzymes and their d -allose production are reviewed and compared. Moreover, several methods for effective d -allose production are suggested herein.
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Substrate specificity of a recombinant Ribose-5-Phosphate Isomerase from Streptococcus pneumoniae and its application in the production of l-lyxose and l-tagatose
World Journal of Microbiology and Biotechnology, 2011Co-Authors: Chang-su Park, Soo Jin Yeom, Deok-kun OhAbstract:A putative Ribose-5-Phosphate Isomerase (RpiB) from Streptococcus pneumoniae was purified with a specific activity of 26.7 U mg^−1 by Hi-Trap Q HP anion exchange and Sephacryl S-300 HR 16/60 gel filtration chromatographies. The native enzyme existed as a 96-kDa tetramer with activity maxima at pH 7.5 and 35°C. The RpiB exhibited isomerization activity with l -lyxose, l -talose, d -gulose, d -ribose, l -mannose, d -allose, l -xylulose, l -tagatose, d -sorbose, d -ribulose, l -fructose, and d -psicose and exhibited particularly high activity with l -form monosaccharides such as l -lyxose, l -xylulose, l -talose, and l -tagatose. With l -xylulose (500 g l^−1) and l -talose (500 g l^−1) substrates, the optimum concentrations of RpiB were 300 and 600 U ml^−1, respectively. The enzyme converted 500 g l^−1 l -xylulose to 350 g l^−1 l -lyxose after 3 h, and yielded 450 g l^−1 l -tagatose from 500 g l^−1 l -talose after 5 h. These results suggest that RpiB from S. pneumoniae can be employed as a potential producer of l -form monosaccharides.
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increased d allose production by the r132e mutant of ribose 5 phosphate Isomerase from clostridium thermocellum
Applied Microbiology and Biotechnology, 2011Co-Authors: Soo Jin Yeom, Deok-kun OhAbstract:Ribose-5-Phosphate Isomerase from Clostridium thermocellum converted d-psicose to d-allose, which may be useful as a pharmaceutical compound, with no by-product. The 12 active-site residues, which were obtained by molecular modeling on the basis of the solved three-dimensional structure of the enzyme, were substituted individually with Ala. Among the 12 Ala-substituted mutants, only the R132A mutant exhibited an increase in d-psicose isomerization activity. The R132E mutant showed the highest activity when the residue at position 132 was substituted with Ala, Gln, Ile, Lys, Glu, or Asp. The maximal activity of the wild-type and R132E mutant enzymes for d-psicose was observed at pH 7.5 and 80°C. The half-lives of the wild-type enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 11, 7.0, 4.2, 1.5, and 0.6 h, respectively, whereas those of the R132E mutant enzymes were 13, 8.2, 5.1, 3.1, and 0.9 h, respectively. The specific activity and catalytic efficiency (kcat/Km) of the R132E mutant for d-psicose were 1.4- and 1.5-fold higher than those of the wild-type enzyme, respectively. When the same amount of enzyme was used, the conversion yield of d-psicose to d-allose was 32% for the R132E mutant enzyme and 25% for the wild-type enzyme after 80 min.
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Crystal structure of Clostridium thermocellum Ribose-5-Phosphate Isomerase B reveals properties critical for fast enzyme kinetics.
Applied Microbiology and Biotechnology, 2011Co-Authors: Junho Jung, Soo Jin Yeom, Deok-kun Oh, Lin-woo KangAbstract:Ribose-5-Phosphate Isomerase (Rpi) catalyzes the conversion of d-ribose 5-phosphate (R5P) to d-ribulose 5-phosphate, which is an important step in the non-oxidative pathway of the pentose phosphate pathway and the Calvin cycle of photosynthesis. Recently, Rpis have been used to produce valuable rare sugars for industrial purposes. Of the Rpis, d-Ribose-5-Phosphate Isomerase B from Clostridium thermocellum (CtRpi) has the fastest reactions kinetics. While Thermotoga maritime Rpi (TmRpi) has the same substrate specificity as CtRpi, the overall activity of CtRpi is approximately 200-fold higher than that of TmRpi. To understand the structural basis of these kinetic differences, we determined the crystal structures, at 2.1-A resolution or higher, of CtRpi alone and bound to its substrates, R5P, d-ribose, and d-allose. Structural comparisons of CtRpi and TmRpi showed overall conservation of their structures with two notable differences. First, the volume of the CtRpi substrate binding pocket (SBP) was 20% less than that of the TmRpi SBP. Second, the residues next to the sugar-ring opening catalytic residue (His98) were different. We switched the key residues, involved in SBP shaping or catalysis, between CtRpi and TmRpi by site-directed mutagenesis, and studied the enzyme kinetics of the mutants. We found that tight interactions between the two monomers, narrow SBP width, and the residues near the catalytic residue are all critical for the fast enzyme kinetics of CtRpi.
Soo Jin Yeom - One of the best experts on this subject based on the ideXlab platform.
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Phosphate sugar Isomerases and their potential for rare sugar bioconversion
Journal of Microbiology, 2020Co-Authors: Soo Jin YeomAbstract:Phosphate sugar Isomerases, catalyzing the isomerization between ketopentose/ketohexose phosphate and aldopentose/aldohexose phosphate, play an important role in microbial sugar metabolism. They are present in a wide range of microorganisms. They have attracted increasing research interest because of their broad substrate specificity and great potential in the enzymatic production of various rare sugars. Here, the enzymatic properties of various phosphate sugar Isomerases are reviewed in terms of their substrate specificities and their applications in the production of valuable rare sugars because of their functions such as low-calorie sweeteners, bulking agents, and pharmaceutical precursor. Specifically, we focused on the industrial applications of D-Ribose-5-Phosphate Isomerase and D-mannose-6-phosphate Isomerase to produce D-allose and L-ribose, respectively.
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Substrate specificity of a recombinant Ribose-5-Phosphate Isomerase from Streptococcus pneumoniae and its application in the production of l-lyxose and l-tagatose
World Journal of Microbiology and Biotechnology, 2011Co-Authors: Chang-su Park, Soo Jin Yeom, Deok-kun OhAbstract:A putative Ribose-5-Phosphate Isomerase (RpiB) from Streptococcus pneumoniae was purified with a specific activity of 26.7 U mg^−1 by Hi-Trap Q HP anion exchange and Sephacryl S-300 HR 16/60 gel filtration chromatographies. The native enzyme existed as a 96-kDa tetramer with activity maxima at pH 7.5 and 35°C. The RpiB exhibited isomerization activity with l -lyxose, l -talose, d -gulose, d -ribose, l -mannose, d -allose, l -xylulose, l -tagatose, d -sorbose, d -ribulose, l -fructose, and d -psicose and exhibited particularly high activity with l -form monosaccharides such as l -lyxose, l -xylulose, l -talose, and l -tagatose. With l -xylulose (500 g l^−1) and l -talose (500 g l^−1) substrates, the optimum concentrations of RpiB were 300 and 600 U ml^−1, respectively. The enzyme converted 500 g l^−1 l -xylulose to 350 g l^−1 l -lyxose after 3 h, and yielded 450 g l^−1 l -tagatose from 500 g l^−1 l -talose after 5 h. These results suggest that RpiB from S. pneumoniae can be employed as a potential producer of l -form monosaccharides.
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increased d allose production by the r132e mutant of ribose 5 phosphate Isomerase from clostridium thermocellum
Applied Microbiology and Biotechnology, 2011Co-Authors: Soo Jin Yeom, Deok-kun OhAbstract:Ribose-5-Phosphate Isomerase from Clostridium thermocellum converted d-psicose to d-allose, which may be useful as a pharmaceutical compound, with no by-product. The 12 active-site residues, which were obtained by molecular modeling on the basis of the solved three-dimensional structure of the enzyme, were substituted individually with Ala. Among the 12 Ala-substituted mutants, only the R132A mutant exhibited an increase in d-psicose isomerization activity. The R132E mutant showed the highest activity when the residue at position 132 was substituted with Ala, Gln, Ile, Lys, Glu, or Asp. The maximal activity of the wild-type and R132E mutant enzymes for d-psicose was observed at pH 7.5 and 80°C. The half-lives of the wild-type enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 11, 7.0, 4.2, 1.5, and 0.6 h, respectively, whereas those of the R132E mutant enzymes were 13, 8.2, 5.1, 3.1, and 0.9 h, respectively. The specific activity and catalytic efficiency (kcat/Km) of the R132E mutant for d-psicose were 1.4- and 1.5-fold higher than those of the wild-type enzyme, respectively. When the same amount of enzyme was used, the conversion yield of d-psicose to d-allose was 32% for the R132E mutant enzyme and 25% for the wild-type enzyme after 80 min.
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Crystal structure of Clostridium thermocellum Ribose-5-Phosphate Isomerase B reveals properties critical for fast enzyme kinetics.
Applied Microbiology and Biotechnology, 2011Co-Authors: Junho Jung, Soo Jin Yeom, Deok-kun Oh, Lin-woo KangAbstract:Ribose-5-Phosphate Isomerase (Rpi) catalyzes the conversion of d-ribose 5-phosphate (R5P) to d-ribulose 5-phosphate, which is an important step in the non-oxidative pathway of the pentose phosphate pathway and the Calvin cycle of photosynthesis. Recently, Rpis have been used to produce valuable rare sugars for industrial purposes. Of the Rpis, d-Ribose-5-Phosphate Isomerase B from Clostridium thermocellum (CtRpi) has the fastest reactions kinetics. While Thermotoga maritime Rpi (TmRpi) has the same substrate specificity as CtRpi, the overall activity of CtRpi is approximately 200-fold higher than that of TmRpi. To understand the structural basis of these kinetic differences, we determined the crystal structures, at 2.1-A resolution or higher, of CtRpi alone and bound to its substrates, R5P, d-ribose, and d-allose. Structural comparisons of CtRpi and TmRpi showed overall conservation of their structures with two notable differences. First, the volume of the CtRpi substrate binding pocket (SBP) was 20% less than that of the TmRpi SBP. Second, the residues next to the sugar-ring opening catalytic residue (His98) were different. We switched the key residues, involved in SBP shaping or catalysis, between CtRpi and TmRpi by site-directed mutagenesis, and studied the enzyme kinetics of the mutants. We found that tight interactions between the two monomers, narrow SBP width, and the residues near the catalytic residue are all critical for the fast enzyme kinetics of CtRpi.
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Overexpression, crystallization and preliminary X-ray crystallographic analysis of D-Ribose-5-Phosphate Isomerase from Clostridium thermocellum.
Acta crystallographica. Section F Structural biology and crystallization communications, 2009Co-Authors: Junho Jung, Soo Jin Yeom, Jisun Kim, Jin Kwang Kim, Sampath Natarajan, Yeh Jin Ahn, Sang Boem Lim, Lin-woo KangAbstract:Rare sugars are used for many industrial and medical purposes and are produced by the interconversion between aldoses and ketoses catalyzed by sugar and sugar-phosphate Isomerases. Recently, Clostridium thermocellum d-Ribose-5-Phosphate Isomerase (CTRPI), an aldose-ketose Isomerase, was cloned in order to synthesize d-allose and its substrate specificity was further characterized for industrial usage. CTRPI has a novel substrate specificity that differs from those of other Isomerases, which have broad substrate specificities. CTRPI prefers aldose substrates such as l-talose, d-ribose and d-allose. CTRPI was purified and crystallized in order to determine its three-dimensional structure and thus to elucidate its enzymatic reaction mechanism and understand its substrate specificity. The crystal belonged to the trigonal space group P3(2)21, with unit-cell parameters a = b = 69.5, c = 154.4 angstrom, and diffracted to 1.9 angstrom resolution. According to Matthews coefficient calculations, the crystallographic structure consists of a dimer in the asymmetric unit, with a V(M) of 3.2 angstrom(3) Da(-1) and a solvent content of 61.7%.
Liangzhi Li - One of the best experts on this subject based on the ideXlab platform.
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exploring multifunctional residues of ribose 5 phosphate Isomerase b from ochrobactrum sp csl1 enhancing isomerization of d allose
Journal of Agricultural and Food Chemistry, 2020Co-Authors: Xiaofeng Zhang, Xinqi Xu, Hengtao Tang, Xin Ju, Rong Wang, Liangzhi LiAbstract:Ribose-5-Phosphate Isomerase B is of great interest for biocatalysis and biosynthesis, but the multifunctional residues in active sites hinder the research efforts. This study employed rational design strategies to locate key residues of RpiB from Ochrobactrum sp. CSL1 (OsRpiB). A single mutant S9T of a noncontact residue showed 80% activity improvement towards D-allose. A double mutant S98H/S134H further increased the activity to 3.6-fold. The mutations were analyzed by kinetics and molecular dynamics analyses, finding S9T might enhance the substrate binding and catalysis by inducing a steric effect, and S98H/S134H could strengthen both ring opening and binding of D-allose. Though low temperature stability was found on S98H/S134H, its potential was explored by isomerizing D-allose to D-psicose with higher conversion and in less reaction time. The findings of this study were beneficial for illustrating the complex functions of key residues in RpiBs, and applying OsRpiB in preparing rare sugars.
Claus Schnarrenberger - One of the best experts on this subject based on the ideXlab platform.
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Microsequecing and cDNA cloning of the Calvin cycle/OPPP enzyme Ribose-5-Phosphate Isomerase (EC 5.3.1.6) from spinach chloroplasts
Plant Molecular Biology, 1996Co-Authors: William Martin, Katrin Henze, Anke Flechner, Josef Kellermann, Claus SchnarrenbergerAbstract:Ribose-5-Phosphate Isomerase (RPI) catalyses the interconversion of Ribose-5-Phosphate and ribulose-5-phosphate in the reductive and oxidative pentose phosphate pathways in plants. RPI from spinach chloroplasts was purified and microsequenced. Via PCR with degenerate primers designed against microsequenced peptides, a hybridisation probe was obtained and used to isolate several cDNA clones which encode RPI. The nuclear-encoded 239 amino acid mature RPI subunit has a predicted size of 25.3 kDa and is translated as a cytosolic precursor possessing a 50 amino acid transit peptide. The processing site of the transit peptide was identified from protein sequence data. Spinach leaves possess only one type of homodimeric RPI enzyme which is localized in chloroplasts and is encoded by a single nuclear gene. Molecular characterization of RPI supports the view that a single amphibolic RPI enzyme functions in the oxidative and reductive pentose phosphate pathways of spinach plastids.
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microsequecing and cdna cloning of the calvin cycle oppp enzyme ribose 5 phosphate Isomerase ec 5 3 1 6 from spinach chloroplasts
Plant Molecular Biology, 1996Co-Authors: William Martin, Katrin Henze, Anke Flechner, Josef Kellermann, Claus SchnarrenbergerAbstract:Ribose-5-Phosphate Isomerase (RPI) catalyses the interconversion of Ribose-5-Phosphate and ribulose-5-phosphate in the reductive and oxidative pentose phosphate pathways in plants. RPI from spinach chloroplasts was purified and microsequenced. Via PCR with degenerate primers designed against microsequenced peptides, a hybridisation probe was obtained and used to isolate several cDNA clones which encode RPI. The nuclear-encoded 239 amino acid mature RPI subunit has a predicted size of 25.3 kDa and is translated as a cytosolic precursor possessing a 50 amino acid transit peptide. The processing site of the transit peptide was identified from protein sequence data. Spinach leaves possess only one type of homodimeric RPI enzyme which is localized in chloroplasts and is encoded by a single nuclear gene. Molecular characterization of RPI supports the view that a single amphibolic RPI enzyme functions in the oxidative and reductive pentose phosphate pathways of spinach plastids.
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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.
