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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 K(m) 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 microM. All bisphosphonates inhibited amoebic growth. One of them (risedronate) was inhibitory at a concentration of 10 microM. Bisphosphonates are therefore potential therapeutic agents for the treatment of amoebiasis.

SUBMITTER: Bruchhaus I

PROVIDER: S-EPMC1217350 | biostudies-other | 1996 May

REPOSITORIES: biostudies-other

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Cloning and expression of the gene for the active PPi-dependent phosphofructokinase of Entamoeba histolytica.

Project description:Pyrophosphate-dependent phosphofructokinase (PPi-PFK) from Entamoeba histolytica (HM-1) was purified from trophozoites. Oligonucleotide probes based on partial amino acid sequence were used to clone and sequence the gene and the cDNA of the enzyme. The molecular mass of the subunit was greater than, and the derived sequence significantly different from, that of the product of the PPi-PFK gene previously cloned from E. histolytica [Huang, Albach, Chang, Tripathi and Kemp (1995) Biochim. Biophys. Acta 1260, 215-217; Bruchhaus, Jacobs, Denart and Tannich (1996) Biochem. J. 316, 57-63]. The sequence identity between the two proteins was 17%. The sequence bore greater identity with the more phylogenetically advanced plant PPi-PFKs than with bacterial PPi-PFKs. The cloned cDNA was expressed and the protein purified. The kinetic properties were identical with those of the enzyme isolated from the organism. Furthermore, the specific activity was more than three orders of magnitude higher than that described for the product of the previously cloned E. histolytica PFK gene [Bruchhaus et al. (1996)]. The pH-dependence and apparent substrate affinities of the cloned enzyme were identical with those of the PPi-PFK in trophozoite extracts, indicating that the product of the cloned gene accounts for most if not all of the PFK activity in E. histolytica trophozoites.

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