Characterization and molecular cloning of an adenosine kinase from Babesia canis rossi.
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.
Carret
C
C
Laboratoire de Biologie Cellulaire et Moléculaire, Montpellier, France.
Delbecq
S
S
Labesse
G
G
Carcy
B
B
Precigout
E
E
Moubri
K
K
Schetters
T P
TP
Gorenflot
A
A
D000248:Adenosine Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000737:chemistry / Q000235:genetics; D001403:Babesia / Q000201:enzymology* / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000953:Antigens, Protozoan; D018076:DNA, Complementary; D016054:DNA, Protozoan; D011993:Recombinant Fusion Proteins; D000248:Adenosine Kinase