A novel nucleotide kinase encoded by gene 1.7 of bacteriophage T7.
Gene 1.7 of bacteriophage T7 confers sensitivity of both phage T7 and its host Escherichia coli to dideoxythymidine (ddT). We have purified the product of gene 1.7, gp1.7. It exists in two forms of molecular weight 22,181 and 17,782. Only the C-terminal half of the protein is required to confer ddT sensitivity. We show that gp1.7 catalyses the phosphorylation of dGMP and dTMP to dGDP and dTDP, respectively, by using either GTP, dGTP or dTTP as the phosphate donor. Either form of gp1.7 exhibit identical kinase activity as compared with wild-type gp1.7 that contains a mixture of both forms. The K(m) of 70 microM and Kcat of 4.3 s(-1) for dTMP are similar to those found for E. coli thymidylate kinase. However, unlike the host enzyme, gp1.7 efficiently catalyses the conversion of the chain-terminating dideoxythymidylate (ddTMP) to ddTDP. This finding explains the sensitivity of phage T7 but not E. coli to exogenous ddT. Gp1.7 is unusual in that it has no sequence homology to any known nucleotide kinase, it has no identifiable nucleotide-binding motif and its activity is independent of added metal ions. When coupled with nucleoside diphosphate kinase, gp1.7 exponentially converts dTMP to dTTP.
Tran
Ngoc Q
NQ
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Lee
Seung-Joo
SJ
Richardson
Charles C
CC
Tabor
Stanley
S
D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017384:Sequence Deletion; D013937:Thymidine Kinase / Q000378:metabolism; D013942:Thymine Nucleotides / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D003848:Deoxyguanine Nucleotides; D013942:Thymine Nucleotides; D014764:Viral Proteins; D013937:Thymidine Kinase; D009703:Nucleoside-Phosphate Kinase