Pyrophosphate-dependent phosphofructokinase of Entamoeba histolytica: molecular cloning, recombinant expression and inhibition by pyrophosphate analogues

Abstract

By using oligonucleotide primers derived from regions highly conserved in prokaryotic and eukaryotic phosphofructokinase sequences, a genomic DNA fragment was amplified and used to isolate cDNA and genomic clones coding for PPi-dependent phosphofructokinase (PPi-PFK) of Entamoeba histolytica. The open reading frame consists of 1308 bp and the corresponding protein has a calculated molecular mass of 47.6 kDa. The N-terminal half of the protein shows 27–35% identity with PPi-PFKs or ATP-dependent phosphofructokinases (ATP-PFKs) of various eukaryotic and prokaryotic organisms. The amino acid residues that form the active site of the PPi-PFK from Propionibacterium freudenreichii and the allosteric ATP-PFK from Escherichia coli are conserved within the amoeba sequence. The PPi-PFK was recombinantly expressed by using a prokaryotic expression system. The purified recombinant protein was found to be enzymically active. The Km values for PPi and fructose 6-phosphate of the native and the recombinant PPi-PFKs were nearly identical. Various bisphosphonates (synthetic pyrophosphate analogues) were tested for their ability to inhibit PPi-PFK activity or amoebic growth. All bisphosphonates tested were competitive inhibitors for amoeba PPi-PFK activity. The best inhibitors were CGP 48048 and zoledronate, with Ki values of 50 μM. All bisphosphonates inhibited amoebic growth. One of them (risedronate) was inhibitory at a concentration of 10 μM. Bisphosphonates are therefore potential therapeutic agents for the treatment of amoebiasis.