Plant Molecular Biology

, Volume 29, Issue 6, pp 1279–1291 | Cite as

Cloning of the amphibolic Calvin cycle/OPPP enzyme d-ribulose-5-phosphate 3-epimerase (EC from spinach chloroplasts: functional and evolutionary aspects

  • Ulrich Nowitzki
  • Ralf Wyrich
  • Peter Westhoff
  • Katrin Henze
  • Claus Schnarrenberger
  • William Martin
Research article


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, 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.

Key words

carbon fixation oxidative pentose phosphate pathway chloroplasts evolution endosymbiosis isoenzymes 



d-ribulose-5-phosphate 3-epimerase


ribose-5-phosphate isomerase






glyceraldehyde-3-phosphate dehydrogenase




fructose 1,6-bisphosphate


glucose-6-phosphate dehydrogenase


6-phosphogluconate dehydrogenase


oxidative pentose phosphate pathway


ribulose-1,5-bisphosphate carboxylase/oxygenase


fructose-1,6-bisphophate aldolase


isopropyl β-d-thiogalactoside


glutathione S-tranferase


phosphate-buffered saline


triosephosphate isomerase


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Bernacchia G, Schwall G, Lottspeich F, Salamini F, Bartels D: The transketolase gene family of the resurrection plant Craterostigma plantagineum: differential expression during the rehydration phase. EMBO J 14: 610–618 (1995).Google Scholar
  2. 2.
    Bertsch U, Schlicher TB, Schröder I, Soll J: Sequence of mature phosphoglycerate kinase from spinach chloroplasts. Plant Physiol 103: 1449–1450 (1993).Google Scholar
  3. 3.
    Bradford MM: A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 72: 248–254 (1976).Google Scholar
  4. 4.
    Brinkmann H, Cerff R, Salomon M, Soll J: Cloning and sequence analysis of cDNAs encoding the cytosolic precursors of subunits GapA and GapB of chloroplast glyceraldehyde-3-phosphate dehydrogenase from pea and spinach. Plant Mol Biol 13: 81–94 (1989).Google Scholar
  5. 5.
    Brinkmann H, Martin W: Higher plant chloroplast and cytosolic 3-phosphoglycerate kinases: a case of endosymbiotic gene replacement. Plant Mol Biol, in press (1995).Google Scholar
  6. 6.
    Chen J-H, Gibson JL, McCue LA, Tabita FR: Identification, expression, and deduced primary structure of transketolase and other enzymes encoded within the form II carbon dioxide fixation operon of Rhodobacter sphaeroides. J Biol Chem 266: 20447–20452 (1991).Google Scholar
  7. 7.
    Dennis DT, Miernyk JA: Compartmentation of nonphotosynthetic carbohydrate metabolism. Annu Rev Plant Physiol 33: 27–50 (1982).Google Scholar
  8. 8.
    Devereux J, Haeberli P, Smithies O: A comprehensive set of sequence analysis programms for the VAX. Nucl Acids Res 12: 387–395 (1984).Google Scholar
  9. 9.
    Falcone DL, Tabita FR: Complementation analysis and regulation of CO2 fixation gene expression in a ribulose 1,5-bisphosphate carboxylase-oxygenase deletion strain of Rhodospirillum rubrum. J Bact 175: 5066–5077 (1993).Google Scholar
  10. 10.
    Feinberg AP, Vogelstein B: A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 137: 266–267 (1984).Google Scholar
  11. 11.
    Felsenstein J: PHYLIP: Phylogeny Inference Package (version 3.2). Cladistics 5: 164–166 (1989).Google Scholar
  12. 12.
    Gibson JL, Tabita FR: Nucleotide sequence and functional analysis of CbbR, a positive regulator of the Calvin cycle operons of Rhodobacter sphaeriodes. J Bact 175: 5778–5784 (1993).Google Scholar
  13. 13.
    Graeve K, von Schaewen A, Scheibe R: Purification, characterization, and cDNA sequence of glucose-6-phosphate dehydrogenase from potato (Solanum tuberosum L.). Plant J 5: 353–361 (1994).Google Scholar
  14. 14.
    Henze K, Badr A, Wettern M, Cerff R, Martin W: A nuclear gene of eubacterial origin in Euglena gracilis reflects cryptic endosymbioses during protist evolution. Proc Natl Acad Sci USA 92: 9122–9126 (1995).Google Scholar
  15. 15.
    Henze K, Schnarrenberger C, Kellermann J, Martin W: Chloroplast and cytosolic triosephosphate isomerase from spinach: Purification, microsequencing and cDNA sequence of the chloroplast enzyme. Plant Mol Biol 26: 1961–1973 (1994).Google Scholar
  16. 16.
    Herbert M, Burkhardt C, Schnarrenberger C: A survey for isoenzymes of glucose phosphate isomerase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase in C3-, C4- and Crassulacean acid metabolism (CAM) plants and algae. Planta 145: 95–104 (1978).Google Scholar
  17. 17.
    Higgins DH, Sharp PM: clustal: a package for performing multiple sequence alignment on a microcomputer. Gene 73: 237–244 (1988).Google Scholar
  18. 18.
    Karmali A, Drake AF, Spencer N: Purification, properties and assay of d-ribulose-5-phosphate 3-epimerase from human erythrocytes. Biochem J 211: 617–623 (1983).Google Scholar
  19. 19.
    Krebbers E, Seurinck J, Herdies L, Cashmore AR, Timko MP: Four genes in two diverged subfamilies encode the ribulose-1,5-bisphosphate carboxylase small subunit polypeptides of Arabidopsis thaliana. Plant Mol Biol 11: 745–759 (1988).Google Scholar
  20. 20.
    Kubicki A, Steinmüller K, Westhoff P: Differential transcription of plastome-encoded genes in the mesophyll and bundle-sheath chloroplasts of the monocotyledonous NADP-malic enzyme type C4 plants maize and Sorghum. Plant Mol Biol 25: 669–679 (1994).Google Scholar
  21. 21.
    Kumada Y, Benson DR, Hillemann D, Hosted TJ, Rochefort DA, Thompson CJ, Wohlleben W, Tateno Y: Evolution of the glutamine synthase gene, one of the oldest existing and functioning genes. Proc Natl Acad Sci USA 90: 3009–3013 (1993).Google Scholar
  22. 22.
    Kusian B, Yoo JG, Bednarski R, Bowien B: The Calvin cycle enzyme pentose-5-phosphate 3-epimerase is encoded within the cfx operons of the chemoautotroph Alcaligenes eutrophus. J Bact 174: 7337–7344 (1992).Google Scholar
  23. 23.
    Martin W, Brinkmann H, Savonna C, Cerff R: Evidence for a chimeric nature of nuclear genomes: eubacterial origin of glyceraldehyde-3-phosphate dehydrogenase genes. Proc Natl Acad Sci USA 90: 8692–8696 (1993).Google Scholar
  24. 24.
    Martin W, Cerff R: Prokaryotic features of a nucleus encoded enzyme: cDNA sequences for chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases from mustard (Sinapis alba). Eur J Biochem 159: 323–331 (1986).Google Scholar
  25. 25.
    Martin W, Jouannic S, Loiseaux-de Goër S: Molecular phylogeny of the atp B and atp E genes of the brown alga Pylaiella littoralis. Eur J Phycol 28: 111–113 (1993).Google Scholar
  26. 26.
    Martin W, Somerville CC, Loiseaux-de Goër S: Molecular phylogenies of plastid origins and algal evolution. J Mol Evol 35: 385–403 (1992).Google Scholar
  27. 27.
    Mateyka C, Schnarrenberger C: Starch phosphorylase isoenzymes in mesophyll and bundle-sheath cells of corn leaves. Plant Sci Lett 60: 119–123 (1984).Google Scholar
  28. 28.
    Meyer-Gauen G, Cerff R, Schnarrenberger C, Martin W: Molecular characterisation of a novel, nuclear-encoded, NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase in plastids of the gymnosperm Pinus sylvestris L. Plant Mol Biol 26: 1155–1166 (1994).Google Scholar
  29. 29.
    Murray NE: Phage Lambda and molecular cloning. In: Lambda II, monograph 13, pp. 395–432 Cold Spring Harbor Laboratory Press. Cold Spring Harbour, NY (1983).Google Scholar
  30. 30.
    Nishimura M, Beevers H: Isoenzymes of sugar phosphate metabolism in endosperm of germinating castor beans. Plant Physiol 67: 1255–1258 (1981).Google Scholar
  31. 31.
    Pelzer-Reith B, Penger A, Schnarrenberger C: Plant aldolase: cDNA and deduced amino-acid sequence of the chloroplast and cytosol enzyme from spinach. Plant Mol Biol 21: 331–340 (1993).Google Scholar
  32. 32.
    Raines CA, Lloyd JC, Willingham NM, Potts S, Dyer TA: cDNA and gene sequences of wheat chloroplast sedulose-1,7-bisphosphatase reveal homology with fructose-1,6-bisphosphatases. Eur J Biochem 205: 1053–1059 (1992).Google Scholar
  33. 33.
    Roesler KR, Ogren WL: Nucleotide sequence of spinach cDNA encoding phosphoribulokinase. Nucl Acids Res 16: 7192 (1988).Google Scholar
  34. 34.
    Saitou N, Nei M: The neighbor-joining method: a new method for the reconstruction of phylogenetic trees. Mol Biol Evol 4: 406–425 (1987).Google Scholar
  35. 35.
    Sambrook J, Fritsch E, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989).Google Scholar
  36. 36.
    Schmidt M, Svendsen Ib, Feierabend J: Analysis of the primary structure of the chloroplast isozyme of triosephosphate isomerase from rye leaves by protein and cDNA sequencing indicates a eukaryotic origin of its gene. Biochim Biophys Acta 1261: 257–264 (1995).Google Scholar
  37. 37.
    Schnarrenberger C, Flechner A, Martin W: Enzymatic evidence indicating a complete oxidative pentose phosphate in the chloroplasts and an incomplete pathway in the cytosol of spinach leaves. Plant Physiol 108: 609–614 (1995).Google Scholar
  38. 38.
    Schnarrenberger C, Herbert M, Krüger I: Intracellular compartmentation of isozymes of sugar phosphate metabolism in green leaves. In: Rattacci MC, Scandalious JG, Whitt GS (eds) Isozymes: Current Topics in Biological and Medical Research, pp. 23–51. A.R. Liss, New York, NY (1983).Google Scholar
  39. 39.
    Schnarrenberger C, Oeser A, Tolbert NE: Two isoenzymes each of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in spinach leaves. Arch Biochem Biophys 154: 438–448 (1973).Google Scholar
  40. 40.
    Schnarrenberger C, Tetour M, Herbert M: Development and intracellular distribution of enzymes of the oxidative pentose phosphate cycle in radish cotyledons. Plant Physiol 56: 836–840 (1975).Google Scholar
  41. 41.
    Shih M-C, Lazar G, Goodman HM: Evidence in favor of the symbiotic origin of chloroplasts: Primary structure and evolution of tobacco glyceraldehyde-3-phosphate dehydrogenases. Cell 47: 73–80 (1986).Google Scholar
  42. 42.
    Simcox PD, Dennis DT: Isoenzymes of the glycolytic and pentose phosphate pathways in proplastids from the developing endosperm of Ricinus communis L. Plant Physiol 61: 871–877 (1978).Google Scholar
  43. 43.
    Simcox PD, Reid EE, Canvin DT, Dennis DT: Enzymes of the glycolytic and pentose phosphate pathways in plastids from the developing endosperm of Ricinus communis L. Plant Physiol 59: 1128–1132 (1977).Google Scholar
  44. 44.
    Slack CR, Hatch MD, Goodchild DJ: Distribution of enzymes in mesophyll and parenchyma sheath chloroplasts of maize in relation to the C4 dicarboxylic acid pathway of photosynthesis. Biochem J 114: 489–498 (1969).Google Scholar
  45. 45.
    Stitt M, Heldt HW: Control of photosynthetic sucrose synthesis by fructose-2,6-bisphosphate. Intracellular metabolite distribution and properties of fructosebisphosphatase in leaves of Zea mays L. Planta 164: 179–188 (1985).Google Scholar
  46. 46.
    Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW: Use of T7 RNA polymerase to direct expression of cloned genes. Meth Enzymol 185: 60–89 (1990).Google Scholar
  47. 47.
    Terada T, Mukae K, Ohashi K, Hosomi T, Mizoguchi T, Uehara K: Characterisation of an enzyme which catalyzes isomeristion and epimerisation of d-erythrose-4-phosphate. Eur J Biochem 148: 345–351 (1985).Google Scholar
  48. 48.
    von Heijne G, Stepphuhn J, Herrmann RG: Domain structure of mitochondrial and chloroplast targeting peptides. Eur J Biochem 180: 535–545 (1989).Google Scholar
  49. 49.
    Westhoff P, Offermann-Steinhard K, Höfer M, Eskins K, Oswald A, Streubel M: Differential accumulation of plastid transcripts encoding photosystem II components in the mesophyll and bundle-sheath cells of monocotyledonous NADP-malic enzyme-type C4 plants. Planta 184: 377–388 (1991).Google Scholar
  50. 50.
    Williamson WT, Wood WA: d-ribulose-5-phosphate 3-epimerase. Meth Enzymol 9: 605–608 (1966).Google Scholar
  51. 51.
    Wolter F, Fritz C, Willmitzer L, Schell J, Schreier P: rbc S genes in Solanum tuberosum: conservation of transit peptide and exon shuffling during evolution. Proc Natl Acad Sci USA 85: 846–850 (1988).Google Scholar
  52. 52.
    Wood T: Purification and properties of d-ribulose-5-phosphate 3-epimerase from calf liver. Biochim Biophys Acta 570: 352–362 (1979).Google Scholar

Copyright information

© Kluwer Academic Publishers 1995

Authors and Affiliations

  • Ulrich Nowitzki
    • 1
  • Ralf Wyrich
    • 2
  • Peter Westhoff
    • 2
  • Katrin Henze
    • 1
  • Claus Schnarrenberger
    • 3
  • William Martin
    • 1
  1. 1.Institut für GenetikTechnische Universität BraunschweigBraunschweigGermany
  2. 2.Institut für Entwicklungs- und Molekularbiologie der PflanzenUniversität DüsseldorfDüsseldorfGermany
  3. 3.Institut für Pflanzenphysiologie und MikrobiologieFreie Universität BerlinBerlinGermany

Personalised recommendations