Novel Insight into Thymidylate Kinase: Biochemical Characterization of the Human and Zebrafish Enzymes

Deoxythymidine triphosphate (dTTP or T) is one of the four building blocks for DNA. In dTTP synthesis, the catalysis of dTMP to dTDP, is mediated by one known enzyme only: Thymidylate kinase (TMPK, EC 2.7.4.9). Two clinical reports on TMPK mutations indicate that mutations in TMPK cause neurological disorders in humans. To increase our understanding of TMPK, we performed extensive investigations using human fibroblasts and zebrafish embryos.In paper I, the main purpose was to characterize TMPK mutations and their effects on dTTP synthesis with human fibroblast cells. TMPK mutations dramatically decreased the activity of the enzyme; intriguingly dTMP could still be incorporated into DNA without affecting cell proliferation rates. This implied that an alternative TMPK may exist in fibroblasts. By combining western blot and enzyme activity measurements in subcellular protein fractions, a new TMPK activity in the outer membrane preparations of mitochondrial protein fraction was detected (paper II). Isolation of this alternative TMPK enzyme from fibroblasts was partially performed.Different human TMPK isoforms were studied in paper III. Isoforms 2, 3, 4, and 5 were not functional, due to the lack of important structural elements. Recombinant isoform 6 showed one thousand times lower activity than isoform 1. Thus, none of the isoforms 2-6 are likely to be the alternative TMPK.In study IV, we used zebrafish embryos to further study TMPK. The canonical TMPK showed a dynamic expression pattern. The protein levels were the highest at 24 hours post fertilization, which is the neurulation period. Our result indicated that there was a correlation between the canonical TMPK expression and neuronal development.In summary, our studies expand our understanding of the TMPK enzyme, and provides a correlation between TMPK and neuronal pathology during early development. They further suggest that a novel form of TMPK enzyme exists and is important during embryonic development.Keywords: (Nucleotides synthesis, dTMP kinase, Thymidylate kinase, TMPK, Deoxythymidine monophosphate kinase)Author’s address: JunMei Hu Frisk, Swedish University of Agricultural Sciences, Department of Anatomy, Physiology and Biochemistry, Uppsala, Sweden