Expression, purification, biochemical characterization and inhibition of recombinant Plasmodium falciparum aldolase.
The energy metabolism of the blood stage form of the human malaria parasite Plasmodium falciparum is adapted to the host cell. Like erythrocytes, P. falciparum merozoites lack a functional citric acid cycle. Generation of ATP depends therefore fully on the glycolytic pathway. Aldolase is a key enzyme of this pathway and a high degree of sequence diversity between parasite and host makes it a potential drug target. We have expressed the enzyme in its tetrameric form in Escherichia coli and the catalytic constants Vmax and Km of the recombinant enzyme correspond to the constants of parasite-derived aldolase. Rabbit antibodies against the recombinant P. falciparum aldolase inhibit the natural enzyme and no cross-reaction with human aldolase is detectable. Both the recombinant and the natural protein bind to the cytosolic domain of the band 3 membrane protein in vitro. A 19-residue synthetic peptide corresponding to the sequence of the binding domain of band 3 is an inhibitor when included in the binding assay. In addition, this peptide inhibits the catalytic activity of recombinant P. falciparum aldolase when assayed in a buffer system devoid of anions such as chloride or phosphate. The band 3-derived peptides compete with the aldolase substrate fructose-1,6-diphosphate for binding, suggesting that both reagents have a high affinity for the substrate pocket. A similar sequence motif exists in P. falciparum actin II. A 19-residue peptide corresponding to this sequence is also an inhibitor which could suggest that the P. falciparum aldolase can associate with the cytoskeleton of the parasite or of the host.
Döbeli
H
H
Central Research Units, F. Hoffmann-La Roche AG, Basel, Switzerland.
Trzeciak
A
A
Gillessen
D
D
Matile
H
H
Srivastava
I K
IK
Perrin
L H
LH
Jakob
P E
PE
Certa
U
U
D000595:Amino Acid Sequence; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism; D003001:Cloning, Molecular; D005634:Fructose-Bisphosphate Aldolase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000201:enzymology*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D013498:Suramin / Q000494:pharmacology
D001457:Anion Exchange Protein 1, Erythrocyte; D011994:Recombinant Proteins; D013498:Suramin; D005634:Fructose-Bisphosphate Aldolase