TY - JOUR
T1 - Recombinant expression, purification, and characterization of Toxoplasma gondii adenosine kinase
AU - Darling, John A.
AU - Sullivan, William J.
AU - Carter, Darrick
AU - Ullman, Buddy
AU - Roos, David S.
N1 - Funding Information:
This research was supported by National Institutes of Health research grant to B.U. and D.S.R. J.D. was supported by an NIH training grant in Cell and Molecular Biology, and D.C. by an N.L. Tartar Trust Fellowship from the Medical Research Foundation of Oregon; D.S.R. and B.U. are Burroughs Wellcome Fund Scholars in Molecular Parasitology.
PY - 1999/9/20
Y1 - 1999/9/20
N2 - 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 K(m) values of 1.9 μM for adenosine and 54.4 μM 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. Copyright (C) 1999 Elsevier Science B.V.
AB - 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 K(m) values of 1.9 μM for adenosine and 54.4 μM 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. Copyright (C) 1999 Elsevier Science B.V.
KW - Apicomplexan parasites
KW - Enzyme kinetics
KW - Nucleoside metabolism
KW - Protein purification
KW - Purine salvage
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U2 - 10.1016/S0166-6851(99)00109-7
DO - 10.1016/S0166-6851(99)00109-7
M3 - Article
C2 - 10514077
AN - SCOPUS:0032868347
SN - 0166-6851
VL - 103
SP - 15
EP - 23
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
IS - 1
ER -