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 T
7 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.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 -
d-thiogalactoside
- GST
glutathione
S-tranferase
- PBS
phosphate-buffered saline
- TPI
triosephosphate isomerase
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