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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

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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.

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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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Authors and Affiliations

  1. Institut für Genetik, Technische Universität Braunschweig, Spielmannstrasse 7, D-38106, Braunschweig, Germany

    Ulrich Nowitzki, Katrin Henze & William Martin

  2. Institut für Entwicklungs- und Molekularbiologie der Pflanzen, Universität Düsseldorf, Universitätsstrasse 1, D-40225, Düsseldorf, Germany

    Ralf Wyrich & Peter Westhoff

  3. Institut für Pflanzenphysiologie und Mikrobiologie, Freie Universität Berlin, Königin-Luise-Strasse 12-16a, D-14195, Berlin, Germany

    Claus Schnarrenberger

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  1. Ulrich Nowitzki
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  4. Katrin Henze
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  5. Claus Schnarrenberger
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Nowitzki, U., Wyrich, R., Westhoff, P. et al. 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 Mol Biol 29, 1279–1291 (1995). https://doi.org/10.1007/BF00020468

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