A novel viral thymidylate kinase with dual kinase activity
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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 μM, 3.6 s−1), dTDP (251 μM, 0.9 s−1) and ATP (92 μM, 3.2 s−1) substrates, and TMKwssv showed a sequential ordered bi-bi reaction mechanism. The binding constants K d for dTMP (1.9 μM) and dTDP (10 μM) 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 μM) 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.
KeywordsThymidylate kinase Nucleotide phosphorylation Diphosphate kinase Isothermal titration calorimetry White spot syndrome virus
This work was supported by Mexico’s Consejo Nacional de Ciencia y Tecnología (CONACYT) grants CB-2009-131859 to R. Sotelo-Mundo and INFR-2013-01-205617 to A. Arvizu-Flores for ITC equipment. E. Guevara-Hernandez received a graduate scholarship from CONACYT. We thank the following personnel from CIAD: Mr. Gerardo Reyna for bibliographical support and to Mr. Felipe Isac, Luis Leyva, Adalberto Murrieta, Jose Luis Aguilar and Martin Peralta for computational support.
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