Recombinant expression, purification, and characterization of Toxoplasma gondii adenosine kinase.
Toxoplasma gondii lacks the capacity to synthesize purines de novo, and adenosine kinase (AK)-mediated phosphorylation of salvaged adenosine provides the major route of purine acquisition by this parasite. T. gondii AK thus represents a promising target for rational design of antiparasitic compounds. In order to further our understanding of this therapeutically relevant enzyme, an AK cDNA from T. gondii was overexpressed in E. coli using the pBAce expression system, and the recombinant protein was purified to apparent homogeneity using conventional protein purification techniques. Kinetic analysis of TgAK revealed Km values of 1.9 microM for adenosine and 54.4 microM for ATP, with a k(cat) of 26.1 min(-1). Other naturally occurring purine nucleosides, nucleobases, and ribose did not significantly inhibit adenosine phosphorylation, but inhibition was observed using certain purine nucleoside analogs. Adenine arabinoside (AraA), 4-nitrobenzylthioinosine (NBMPR), and 7-deazaadenosine (tubercidin) were all shown to be substrates of T. gondii AK. Transgenic AK knock-out parasites were resistant to these compounds in cell culture assays, consistent with their proposed action as subversive substrates in vivo.
Darling
J A
JA
Department of Biology, University of Pennsylvania, Philadelphia 19104, USA.
Sullivan
W J
WJ
Carter
D
D
Ullman
B
B
Roos
D S
DS
D000248:Adenosine Kinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014122:Toxoplasma / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*
D000981:Antiprotozoal Agents; D018076:DNA, Complementary; D016054:DNA, Protozoan; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D000248:Adenosine Kinase