A novel viral thymidylate kinase with dual kinase activity

作者:Guevara Hernandez Eduardo; Arvizu Flores Aldo A*; Lugo Sanchez Maria E; Velazquez Contreras Enrique F; Castillo Yanez Francisco J; Brieba Luis G; Sotelo Mundo Rogerio R
来源:Journal of Bioenergetics and Biomembranes, 2015, 47(5): 431-440.
DOI:10.1007/s10863-015-9622-z

摘要

Nucleotide phosphorylation is a key step in DNA replication and viral infections, since suitable levels of nucleotide triphosphates pool are required for this process. Deoxythymidine monophosphate (dTMP) is produced either by de novo or salvage pathways, which is further phosphorylated to deoxythymidine triphosphate (dTTP). Thymidyne monophosphate kinase (TMK) is the enzyme in the junction of both pathways, which phosphorylates dTMP to yield deoxythymidine diphosphate (dTDP) using adenosine triphosphate (ATP) as a phosphate donor. White spot syndrome virus (WSSV) genome contains an open reading frame (ORF454) that encodes a thymidine kinase and TMK domains in a single polypeptide. We overexpressed the TMK ORF454 domain (TMKwssv) and its specific activity was measured with dTMP and dTDP as phosphate acceptors. We found that TMKwssv can phosphorylate dTMP to yield dTDP and also is able to use dTDP as a substrate to produce dTTP. Kinetic parameters K (M) and k (cat) were calculated for dTMP (110 mu M, 3.6 s(-1)), dTDP (251 mu M, 0.9 s(-1)) and ATP (92 mu M, 3.2 s(-1)) substrates, and TMKwssv showed a sequential ordered bi-bi reaction mechanism. The binding constants K (d) for dTMP (1.9 mu M) and dTDP (10 mu M) to TMKwssv were determined by Isothermal Titration Calorimetry. The affinity of the nucleotidic analog stavudine monophosphate was in the same order of magnitude (K (d) 3.6 mu M) to the canonical substrate dTMP. These results suggest that nucleotide analogues such as stavudine could be a suitable antiviral strategy for the WSSV-associated disease.

  • 出版日期2015-10