CLONING, SEQUENCING AND EXPRESSION OF THE PYROPHOSPHATE-DEPENDENT PHOSPHOFRUCTO-1-KINASE FROM NAEGLERIA-FOWLERI

The cDNA for the PPi-dependent phosphofructo-1-kinase has been cloned and sequenced from a cDNA library prepared from the free-living amoeba Naegleria fowleri. The coding sequence of the cDNA consists of 1311 b ases which translates into 437 amino acids with a molecular mass of 48 095 Da. Comparison of the sequence with those of the previously descri bed sequences of PPi-dependent phosphofructokinases from Propionibacte rium freudenreichii and potato tuber revealed amino acid identities of 23 and 28 % respectively and high conservation in those regions assum ed to be part of the active site. The reading frame was cloned into an expression vector, which was transformed into Escherichia coli. Extra cts of the transformed cells contained PPi-dependent phosphofructokina se activity that could be purified to homogeneity. The activity was lo st on incubation with the chaotropic agent, KSCN, and recovered by sub sequent incubation with AMP. These properties are consistent with thos e described by Mertens, De Jonckheere and Van Schaftingen [Biochem. J. (1993) 292, 797-803] for the enzyme prepared from Naegleria and suppo rt the idea that the cloned cDNA coded for the complete native enzyme. No nucleotide-binding motif or evidence for a nucleotide-binding site characteristic of the ATP-dependent phosphofructokinases could be fou nd within the primary structure.