Purification and cloning of chloroplast 6-phosphogluconate dehydrogenase from spinach. Cyanobacterial genes for chloroplast and cytosolic isoenzymes encoded in eukaryotic chromosomes.
Previous attempts to purify chloroplast 6-phosphogluconate dehydrogenase (cp6PGDH), a key enzyme of the oxidative pentose phosphate pathway, have been unsuccessful due to rapid activity loss. An efficient purification protocol was developed and the enzyme from spinach leaves was purified 1000-fold to apparent homogeneity with a specific activity of 60 U.mg-1. The enzyme is a homodimer with subunits of 50 kDa. Antibodies raised against the purified cp6PGDH detected a 53-kDa protein from a crude extract, indicating alterations during purification. Purified cp6PGDH was microsequenced and the corresponding spinach cDNA was cloned using PCR techniques and degenerate primers. The cDNA for cytosolic 6PGDH from spinach was cloned for comparison. Phylogenetic analysis in the context of available homologues from eukaryotes and eubacteria revealed that animal and fungal cytosolic 6PGDH sequences are more similar to their homologues from gamma-proteobacteria, whereas plant 6PGDH is more similar to its cyanobacterial homologues. The ancestral gene for higher plant 6PGDH was acquired from the antecedent of plastids through endosymbiosis and gene transfer to the nucleus. A subsequent gene duplication gave rise to higher plant cytosolic 6PGDH, which assumed the function of its pre-existing cytosolic homologue through endosymbiotic gene replacement. The protein phylogeny of both 6PGDH and of the first enzyme of the oxidative pentose phosphate pathway, glucose-6-phosphate dehydrogenase, indicate a surprisingly close relationship between the plant and Trypanosoma brucei lineages, suggesting that T. brucei (a relative of Euglena gracilis) may be secondarily nonphotosynthetic.
Krepinsky
K
K
Institute of Biology, Free University of Berlin, Germany; Institute of Botany III, University of Düsseldorf, Germany.
Plaumann
M
M
Martin
W
W
Schnarrenberger
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001105:Archaea / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology / Q000235:genetics; D003600:Cytosol / Q000201:enzymology; D017931:DNA Primers / Q000235:genetics; D005798:Genes, Bacterial; D017343:Genes, Plant; D007527:Isoenzymes / Q000235:genetics; D008969:Molecular Sequence Data; D010734:Phosphogluconate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification*; D010802:Phylogeny; D018087:Plastids / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea / Q000201:enzymology* / Q000235:genetics*; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000235:genetics
D017931:DNA Primers; D007527:Isoenzymes; D010734:Phosphogluconate Dehydrogenase