CLONING, SEQUENCING AND DEVELOPMENTAL EXPRESSION OF PHOSPHOFRUCTOKINASE FROM DICTYOSTELIUM-DISCOIDEUM

Phosphofructokinase (PFK) from Dictyostelium discoideum is a non-allos teric enzyme that lacks any of the characteristic regulatory mechanism s of PFK from other cells. We have determined the DNA sequence and ana lyzed the amino acid sequence of D. discoideum PFK, as an initial step toward under standing the peculiar properties of this enzyme. Three o verlapping fragments, two of cDNA and one of genomic DNA, were isolate d, which together could encode the complete sequence of D. discoideum PFK. The constructed full-length cDNA coded for a protein of 834 amino acids, with a calculated molecular mass of 92.4 kDa, which was simila r to other eukaryotic and prokaryotic PFK. Alignments of the amino aci d sequence with other isozymes revealed that many of the amino acid re sidues assigned to binding sites of substrates and allosteric effecter s are conserved in this enzyme, but changes were also found that may c ontribute to the absence of allosteric mechanisms. A phylogenetic tree for the eukaryotic PFK family was constructed and showed that the N-t erminal domain clustered with those of yeast subunits, whereas the C-t erminal domain was more related to PFK from metazoa. Southern blotting indicated that D. discoideum PFK is encoded by a single gene. The enz yme is present throughout the life cycle of D. discoideum, with a grad ual decrease of its expression during development.