A novel viral thymidylate kinase with dual kinase activity

Abstract

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.

Development of a Specific Polymerase Chain Reaction System for the Detection of Rice Orange Leaf Phytoplasma

“…Recently, the genome of ROLP was sequenced, and shares 99% and 98% similarity to the genome sequences of MBS and OY-M, respectively (Zhu et al 2017). Genome sequence analysis showed the sequences of three housekeeping genes coding for cell division protein FtsH, thymidylate kinase, and IS3 family transposase vary widely among phytoplasma species (Guevara-Hernandez et al 2015;Ito and Akiyama 2005;Lee et al 2011;Mahillon and Chandler 1998;Ostermann et al 2000;Saraste et al 1990). In this study, we designed six sets of primers based on the sequences of these three genes for testing in PCR.…”