Toxoplasma gondii adenosine kinase: expression, purification, characterization, crystallization and preliminary crystallographic analysis.
The obligate intracellular protozoan parasite Toxoplasma gondii depends on the purine-salvage pathway for its purine supply. Unlike its mammalian hosts, T. gondii salvages purine precursors predominantly via adenosine kinase, the enzyme that phosphorylates adenosine to adenosine monophosphate (AMP). The cDNA encoding T. gondii adenosine kinase was subcloned and expressed in Escherichia coli. The recombinant protein was active in an in vitro enzyme assay over a broad pH range. It required a divalent cation for activity. The enzyme was inactivated by the addition of 1 microM mercuric chloride. The inactivation could be reversed by a reducing agent. The active recombinant protein was crystallized using sodium sulfate as precipitant at pH 8.0. The crystals diffract to 1.8 A and belong to the monoclinic space group P2(1), with unit-cell parameters a = 47.5, b = 68.9, c = 57.0 A, beta = 100.3 degrees. The calculated V(m) based on one molecule per asymmetric unit is 2.38 A(3) Da(-1).
Recacha
R
R
Center for Macromolecular Crystallography, University of Alabama at Birmingham, AL 35294, USA.
Talalaev
A
A
DeLucas
L J
LJ
Chattopadhyay
D
D
D000248:Adenosine Kinase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017125:Genes, Protozoan; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014122:Toxoplasma / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D000248:Adenosine Kinase