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Characterization and molecular cloning of an adenosine kinase from Babesia canis rossi
C Carret1, S Delbecq, G Labesse
1Laboratoire de Biologie Cellulaire et Moléculaire, Montpellier, France.
Abstract:
In the search for immunoprotective antigens of the intraerythrocytic Babesia canis rossi parasite, a new cDNA was cloned and sequenced. Protein sequence database searches suggested that the 41-kDa protein belongs to the phosphofructokinase B type family (PFK-B). However, because of the low level sequence identity (< 20%) of the protein both with adenosine and sugar kinases from this family, its structural and functional features were further investigated using molecular modelling and enzymatic assays. The sequence/structure comparison of the protein with the crystal structure of a member of the PFK-B family, Escherichia coli ribokinase (EcRK), suggested that it might also form a stable and active dimer and revealed conservation of the ATP-binding 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 rather than sugar ones. Enzymatic assays using a purified glutathione S-transferase fusion protein revealed that this protein exhibits rapid catalysis of the phosphorylation of adenosine with an apparent Km value of 70 nM, whereas it was inactive on ribose or other carbohydrates. As enzymatic assays confirmed the results of the structure/function analysis indicating a preferential specificity towards adenosine compounds, this new protein of the PFK-B family corresponds to an adenosine kinase from B. canis rossi. It was named BcrAK.
Insights
Researchers identified a new adenosine kinase (BcrAK) in the Babesia canis rossi parasite. This protein, crucial for parasite survival, phosphorylates adenosine, offering potential targets for new anti-parasitic drugs.
Area of Science:
- Parasitology
- Molecular Biology
- Biochemistry
Background:
- Babesia canis rossi is an intraerythrocytic parasite causing canine babesiosis.
- Identifying immunoprotective antigens is key to developing effective vaccines and treatments.
- Phosphofructokinase B (PFK-B) family proteins play diverse metabolic roles.
Purpose of the Study:
- To identify and characterize novel immunoprotective antigens of Babesia canis rossi.
- To investigate the structural and functional properties of a newly cloned 41-kDa protein.
- To determine the substrate specificity and enzymatic activity of the identified protein.
Main Methods:
- cDNA cloning and sequencing
- Protein sequence database searches
- Molecular modeling and structural comparison with Escherichia coli ribokinase (EcRK)
- Enzymatic assays using purified glutathione S-transferase fusion protein
Main Results:
- A 41-kDa protein, homologous to the PFK-B family, was identified.
- Structural analysis suggested a conserved ATP-binding site but distinct substrate-binding residues compared to EcRK.
- Enzymatic assays confirmed the protein's specific activity in phosphorylating adenosine (Km = 70 nM) and inactivity towards ribose or other carbohydrates.
- The protein was named BcrAK, identifying it as an adenosine kinase from B. canis rossi.
Conclusions:
- The identified protein, BcrAK, is a novel adenosine kinase from Babesia canis rossi.
- BcrAK exhibits preferential specificity towards adenosine, differentiating it from typical sugar kinases in the PFK-B family.
- BcrAK represents a potential target for therapeutic intervention against Babesia canis rossi infections.