Purification and cloning of chloroplast 6-phosphogluconate dehydrogenase from spinach - Cyanobacterial genes for chloroplast and cytosolic isoenzymesencoded in eukaryotic chromosomes
K. Krepinsky et al., Purification and cloning of chloroplast 6-phosphogluconate dehydrogenase from spinach - Cyanobacterial genes for chloroplast and cytosolic isoenzymesencoded in eukaryotic chromosomes, EUR J BIOCH, 268(9), 2001, pp. 2678-2686
Previous attempts to purify chloroplast 6-phosphogluconate dehydrogenase (c
p6PGDH), a key enzyme of the oxidative pentose phosphate pathway, have been
unsuccessful due to rapid activity loss. An efficient purification protoco
l was developed and the enzyme from spinach leaves was purified 1000-fold t
o 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 purif
ied cp6PGDH detected a 53-kDa protein from a crude extract, indicating alte
rations during purification. Purified cp6PGDH was microsequenced and the co
rresponding spinach cDNA was cloned using PCR techniques and degenerate pri
mers. The cDNA for cytosolic 6PGDH from spinach was cloned for comparison.
Phylogenetic analysis in the context of available homologues from eukaryote
s and eubacteria revealed that animal and fungal cytosolic 6PGDH sequences
are more similar to their homologues from gamma -proteobacteria, whereas pl
ant 6PGDH is more similar to its cyanobacterial homologues. The ancestral g
ene for higher plant 6PGDH was acquired from the antecedent of plastids thr
ough endosymbiosis and gene transfer to the nucleus. A subsequent gene dupl
ication gave rise to higher plant cytosolic 6PGDH, which assumed the functi
on of its pre-existing cytosolic homologue through endosymbiotic gene repla
cement. The protein phylogeny of both 6PGDH and of the first enzyme of the
oxidative pentose phosphate pathway, glucose-6-phosphate dehydrogenase, ind
icate a surprisingly close relationship between the plant and Trypanosoma b
rucei lineages, suggesting that T. brucei (a relative of Euglena gracilis)
may be secondarily nonphotosynthetic.