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

John A. Darling, William J. Sullivan, Darrick Carter, Buddy Ullman, David S. Roos

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)15-23
Number of pages9
JournalMolecular and Biochemical Parasitology
Volume103
Issue number1
DOIs
StatePublished - Sep 20 1999

Fingerprint

Adenosine Kinase
Toxoplasma
Tubercidin
Adenosine
Purine Nucleosides
Parasites
Phosphorylation
Vidarabine
Antiparasitic Agents
Purines
Ribose
Recombinant Proteins
Complementary DNA
Cell Culture Techniques
Adenosine Triphosphate
Escherichia coli
Enzymes
Proteins

Keywords

  • Apicomplexan parasites
  • Enzyme kinetics
  • Nucleoside metabolism
  • Protein purification
  • Purine salvage

ASJC Scopus subject areas

  • Parasitology
  • Molecular Biology

Cite this

Recombinant expression, purification, and characterization of Toxoplasma gondii adenosine kinase. / Darling, John A.; Sullivan, William J.; Carter, Darrick; Ullman, Buddy; Roos, David S.

In: Molecular and Biochemical Parasitology, Vol. 103, No. 1, 20.09.1999, p. 15-23.

Research output: Contribution to journalArticle

Darling, John A. ; Sullivan, William J. ; Carter, Darrick ; Ullman, Buddy ; Roos, David S. / Recombinant expression, purification, and characterization of Toxoplasma gondii adenosine kinase. In: Molecular and Biochemical Parasitology. 1999 ; Vol. 103, No. 1. pp. 15-23.
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