A novel nucleoside kinase from Burkholderia thailandensis: a member of the phosphofructokinase B-type family of enzymes.
The genome of the mesophilic Gram-negative bacterium Burkholderia thailandensis contains an open reading frame (i.e. the Bth_I1158 gene) that has been annotated as a putative ribokinase and PFK-B family member. Notably, although the deduced amino acid sequence of the gene showed only 29% similarity to the recently identified nucleoside kinase from hyperthermophilic archaea Methanocaldococcus jannaschii, 15 of 17 residues reportedly involved in the catalytic activity of M. jannaschii nucleoside kinase were conserved. The gene was cloned and functionally overexpressed in Rhodococcus erythropolis, and the purified enzyme was characterized biochemically. The substrate specificity of the enzyme was unusually broad for a bacterial PFK-B protein, and the specificity extended not only to purine and purine-analog nucleosides but also to uridine. Inosine was the most effective phosphoryl acceptor, with the highest k(cat)/K(m) value (80 s(-1).mm(-1)) being achieved when ATP served as the phosphoryl donor. By contrast, this enzyme exhibited no activity toward ribose, indicating that the recombinant enzyme was a nucleoside kinase rather than a ribokinase. To our knowledge, this is the first detailed analysis of a bacterial nucleoside kinase in the PFK-B family.
Ota
Hiroko
H
Asahi Kasei Pharma Corporation, Shizuoka, Japan.
Sakasegawa
Shin-Ichi
S
Yasuda
Yuko
Y
Imamura
Shigeyuki
S
Tamura
Tomohiro
T
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019117:Burkholderia / Q000201:enzymology*; D002384:Catalysis; D002413:Cations, Divalent / Q000737:chemistry; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007293:Inosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D025221:Phosphofructokinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010770:Phosphotransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D002413:Cations, Divalent; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; D007293:Inosine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D025221:Phosphofructokinases; C021892:nucleoside phosphotransferase; C024157:thymidine 5'-triphosphate