Pyrophosphate-dependent phosphofructokinase of Entamoeba histolytica: molecular cloning, recombinant expression and inhibition by pyrophosphate analogues.
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.
Bruchhaus
I
I
Bernhard Nocht Institute for Tropical Medicine, Hamburg, Federal Republic of Germany.
Jacobs
T
T
Denart
M
M
Tannich
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D011756:Diphosphates / Q000494:pharmacology*; D004748:Entamoeba histolytica / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D063065:Organophosphonates / Q000494:pharmacology; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D011424:Propionibacterium / Q000201:enzymology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D011756:Diphosphates; D063065:Organophosphonates; D011994:Recombinant Proteins; D010770:Phosphotransferases; C022502:pyrophosphate-fructose 6-phosphate 1-phosphotransferase