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

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.

Identification, Cloning, and Properties of Cytosolic d-Ribulose-5-phosphate 3-Epimerase from Higher Plants

“…2A). The latter finding corresponds with a previous report indicating that the spinach and Arabidopsis nuclear genomes contain a single copy gene for chl-RPEase (3,16). These results indicate an ubiquitous distribution of the gene coding for putative cyt-RPEase in higher plants.…”

“…On the other hand, chloroplasts and chromoplasts contain the components for a complete OPPP (9, 10). Recently, nuclear-encoded genes for chloroplast enzymes with dual (amphibolic) function in the OPPP and reductive pentose phosphate cycle (Calvin cycle) have been cloned and characterized including transketolase (11-13), ribose-5-phosphate isomerase (14), and RPEase (13,(15)(16)(17).Relatively little is known about the occurrence, structure, and biochemistry of plant cytosolic (cyt) OPPP enzymes. Although cytosolic glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase have been isolated from green and non-green plant tissue (6, 18), cytosolic isoforms of the non-oxidative branch OPPP enzymes transketolase, ribose-5-phosphate isomerase, and RPEase were undetectable in plant cell extracts by combined cell fractionation and enzyme chromatography experiments (7,8).…”

“…On the other hand, chloroplasts and chromoplasts contain the components for a complete OPPP (9, 10). Recently, nuclear-encoded genes for chloroplast enzymes with dual (amphibolic) function in the OPPP and reductive pentose phosphate cycle (Calvin cycle) have been cloned and characterized including transketolase (11-13), ribose-5-phosphate isomerase (14), and RPEase (13,(15)(16)(17).…”

“…Cloning of chloroplast RPEase genes from potato and spinach (15,16) opened up the way to search enzyme homologues. Here we report on the identification and cloning of a cyt-RPEase from rice, its heterologous and tissue-specific expression, properties, presence of homologues in other plant species, and its possible evolutionary origin.…”

Purification, properties and in situ localization of the amphibolic enzymes D‐ribulose 5‐phosphate 3‐epimerase and transketolase from spinach chloroplasts

“…D-Ribulose 5-phosphate 3-epimerase has been purified from calf liver [31], human erythrocytes [8] and yeast [32], but its isolation from plants or algae has not been reported to date. The spatial localization of the minor chloroplast enzymes epimerase and transketolase and the major enzymes ribulose-1,5-bisphos-primary structure of epimerases from potato chloroplasts [6], spinach chloroplasts [33] the purple bacterium Alcaligenes euphate carboxylase/oxygenase and the H ϩ -ATP synthase portion CF 1 by labeling with monospecific antibodies and 10-nm goldϪ trophus [34] and E. coli [35,36] has been deduced from cDNA sequences. Our results indicate that the structural and enzymic protein-A particles.…”