Adenosine kinase from Cryptosporidium parvum.
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 K(m) values of 1.4microM for adenosine and 41microM for ATP, has a pH optimum approximately 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.
Galazka
Jon
J
Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR 97239-3098, USA.
Striepen
Boris
B
Ullman
Buddy
B
D000248:Adenosine Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016785:Cryptosporidium parvum / Q000201:enzymology* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009705:Nucleosides / Q000378:metabolism / Q000494:pharmacology; D009711:Nucleotides / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity; D014122:Toxoplasma / Q000201:enzymology / Q000235:genetics
D004791:Enzyme Inhibitors; D009705:Nucleosides; D009711:Nucleotides; D011994:Recombinant Proteins; D000248:Adenosine Kinase