Chimeric polypeptide of thymidine kinase and thymidylate kinase of shrimp white spot syndrome virus: thymidine kinase activity of the recombinant protein expressed in a baculovirus/insect cell system.
The unique chimeric organization of the white spot syndrome virus (WSSV) tk-tmk gene encodes a protein which has significant homology to both cellular-type thymidine kinase (TK) and cellular-type thymidylate kinase (TMK), but the functional activity of this protein has not been demonstrated. Because TK is usually expressed only at very low levels in host cells, in this study, the coding region of WSSV tk-tmk was expressed in an insect/baculovirus expression system. The His-tagged recombinant WSSV TK-TMK was purified by affinity chromatography, and its enzyme activity was characterized by steady-state kinetics. The recombinant WSSV TK-TMK catalyzed the phosphorylation of thymidine to form thymidine monophosphate (TMP), but we found no evidence that it was able to catalyze the further phosphorylation of TMP to form thymidine diphosphate (or thymidine triphosphate). This TK activity is sensitive to feedback inhibition by thymidine triphosphate. In addition to thymidine, of the nine other substrates tested, including acyclovir, ganciclovir, and 5-(2-bromovinyl)-2'-deoxyuridine, only 2'-deoxyuridine and 5-bromo-2'-deoxyuridine could also serve as substrates. These data suggest that the enzymatic characteristics of the recombinant WSSV TK-TMK are similar to those of the eukaryotic cytosolic TKs. We also found that TK activity increased as infection advanced in the integument and gills of experimentally infected shrimp, suggesting its functional involvement during WSSV infection.
Tzeng
Huey-Fen
HF
Department of Zoology, National Taiwan University, Taipei 106, Taiwan, Republic of China.
Chang
Zee-Fen
ZF
Peng
Shao-En
SE
Wang
Chung-Hsiung
CH
Lin
Jung-Yaw
JY
Kou
Guang-Hsiung
GH
Lo
Chu-Fang
CF
D000818:Animals; D002460:Cell Line; D004267:DNA Viruses / Q000235:genetics; D033364:Decapoda / Q000821:virology*; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D018411:Spodoptera; D013379:Substrate Specificity; D013937:Thymidine Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014780:Viruses / Q000201:enzymology*
D010710:Phosphates; D011993:Recombinant Fusion Proteins; D013937:Thymidine Kinase; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase