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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 5.1.3.1) from spinach chloroplasts: functional and evolutionary aspects

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

Abstract

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.

Key words

carbon fixation oxidative pentose phosphate pathway chloroplasts evolution endosymbiosis isoenzymes 

Abbreviations

R5P3E

d-ribulose-5-phosphate 3-epimerase

RPI

ribose-5-phosphate isomerase

TKL

transketolase

PRK

phosphoribulokinase

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

FBP

fructose-1,6-bisphophatase

FBP

fructose 1,6-bisphosphate

G6PDH

glucose-6-phosphate dehydrogenase

6PGDH

6-phosphogluconate dehydrogenase

OPPP

oxidative pentose phosphate pathway

Rubisco

ribulose-1,5-bisphosphate carboxylase/oxygenase

FBA

fructose-1,6-bisphophate aldolase

IPTG

isopropyl β-d-thiogalactoside

GST

glutathione S-tranferase

PBS

phosphate-buffered saline

TPI

triosephosphate isomerase

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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

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