Recombinant expression, purification, and characterization of Toxoplasma gondii adenosine kinase

Toxoplasma gondii lacks the capacity to synthesize purines de novo, and ade nosine kinase (AK)-mediated phosphorylation of salvaged adenosine provides the major route of purine acquisition by this parasite. T. gondii AK thus r epresents a promising target for rational design of antiparasitic compounds . In order to further our understanding of this therapeutically relevant en zyme, an AK cDNA from T. gondii was overexpressed in E. coil using the pBAc e expression system, and the recombinant protein was purified to apparent h omogeneity using conventional protein purification techniques. Kinetic anal ysis of TgAK revealed K-m values of 1.9 mu M for adenosine and 54.4 mu M fo r ATP, with a k(cat) of 26.1 min(-1). Other naturally occurring purine nucl eosides, nucleobases, and ribose did not significantly inhibit adenosine ph osphorylation. 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 su bstrates in vivo. (C) 1999 Elsevier Science B.V. All rights reserved.