TY - JOUR
T1 - Adenosine kinase from Cryptosporidium parvum
AU - Galazka, Jon
AU - Striepen, Boris
AU - Ullman, Buddy
N1 - Funding Information:
This work was supported in part by grants RO1 AI23682 to B.U. and RO1 AI055268 to B.S. from the National Institute of Allergy and Infectious Disease. B.S. thanks Catherine Li for technical help and Liz Hedstrom for helpful discussions.
PY - 2006/10
Y1 - 2006/10
N2 - Analysis of the Cryptosporidium parvum genome demonstrates that the parasite cannot synthesize purines de novo and reveals that the sole route for purine salvage by the parasite is via adenosine kinase (CpAK). In order to initiate a biochemical characterization of CpAK and ultimately validate this apparently essential enzyme as a therapeutic target, the CpAK gene was redesigned for optimum codon usage, overexpressed in Escherichia coli, and the recombinant protein purified to homogeneity and characterized. CpAK appears to be specific for adenosine among the naturally occurring nucleosides but can utilize ATP, GTP, UTP and CTP as the phosphate donor. The enzyme exhibits Km values of 1.4 μM for adenosine and 41 μM for ATP, has a pH optimum ∼7.0, and is dependent upon the presence of a divalent cation. Structure-activity data intimate that catalysis requires contacts between residues on CpAK with the six-position of the purine ring and the O2′ and O3′ hydroxyls of the ribose sugar. Additionally, 4-nitro-6-benzylthioinosine, a compound that demonstrates therapeutic promise against the related parasite Toxoplasma gondii, also inhibits adenosine phosphorylation by CpAK. The overproduction and purification of CpAK now enables a thorough evaluation of its potential as a drug target.
AB - Analysis of the Cryptosporidium parvum genome demonstrates that the parasite cannot synthesize purines de novo and reveals that the sole route for purine salvage by the parasite is via adenosine kinase (CpAK). In order to initiate a biochemical characterization of CpAK and ultimately validate this apparently essential enzyme as a therapeutic target, the CpAK gene was redesigned for optimum codon usage, overexpressed in Escherichia coli, and the recombinant protein purified to homogeneity and characterized. CpAK appears to be specific for adenosine among the naturally occurring nucleosides but can utilize ATP, GTP, UTP and CTP as the phosphate donor. The enzyme exhibits Km values of 1.4 μM for adenosine and 41 μM for ATP, has a pH optimum ∼7.0, and is dependent upon the presence of a divalent cation. Structure-activity data intimate that catalysis requires contacts between residues on CpAK with the six-position of the purine ring and the O2′ and O3′ hydroxyls of the ribose sugar. Additionally, 4-nitro-6-benzylthioinosine, a compound that demonstrates therapeutic promise against the related parasite Toxoplasma gondii, also inhibits adenosine phosphorylation by CpAK. The overproduction and purification of CpAK now enables a thorough evaluation of its potential as a drug target.
KW - Adenosine kinase
KW - Cryptosporidium parvum
KW - Parasite
KW - Protozoa
KW - Purine salvage
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U2 - 10.1016/j.molbiopara.2006.06.001
DO - 10.1016/j.molbiopara.2006.06.001
M3 - Article
C2 - 16879885
AN - SCOPUS:33747811518
SN - 0166-6851
VL - 149
SP - 223
EP - 230
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
IS - 2
ER -