Characterization and molecular cloning of an adenosine kinase from Babesiacanis rossi

In the search for immunoprotective antigens of the intraerythrocytic Babesi a canis rossi parasite, a new cDNA was cloned and sequenced. Protein sequen ce database searches suggested that the 41-kDa protein belongs to the phosp hofructokinase B type family (PFK-B). However, because of the low level seq uence identity (< 20%) of the protein both with adenosine and sugar kinases from this family, its structural and functional features were further inve stigated using molecular modelling and enzymatic assays. The sequence/struc ture comparison of the protein with the crystal structure of a member of th e PFK-B family, Escherichia coli ribokinase (EcRK), suggested that it might also form a stable and active dimer and revealed conservation of the ATP-b inding site. However, residues specifically involved in the ribose-binding sites in the EcRK sequence (S and N) were substituted in its sequence (by H and M, respectively), and were suspected of binding adenosine compounds ra ther than sugar ones. Enzymatic assays using a purified glutathione S-trans ferase fusion protein revealed that this protein exhibits rapid catalysis o f the phosphorylation of adenosine with an apparent K-m value of 70 nM, whe reas it was inactive on ribose or other carbohydrates. As enzymatic assays confirmed the results of the structure/function analysis indicating a prefe rential specificity towards adenosine compounds, this new protein of the PF K-B family corresponds to an adenosine kinase from B. canis rossi. It was n amed BcrAK.