title: 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.
authors: Centro de Investigación en Alimentación y Desarrollo, A.C. (CIAD), Carretera a Ejido La Victoria Km 0.6, Apartado Postal 1735, Hermosillo, Sonora, 83304, Mexico.|Departamento de Investigación y Posgrado en Alimentos, Universidad de Sonora, Blvd. Rosales y Luis Encinas, Hermosillo, Sonora, 83000, Mexico.
authors: Eduardo
authors: E
authors: Guevara-Hernandez
authors: Departamento de Ciencias Químico Biológicas, Universidad de Sonora, Blvd. Rosales y Luis Encinas, Hermosillo, Sonora, 83000, Mexico. aldoarvizu@guayacan.uson.mx.
authors: Aldo A
authors: AA
authors: Arvizu-Flores
authors: Centro de Investigación en Alimentación y Desarrollo, A.C. (CIAD), Carretera a Ejido La Victoria Km 0.6, Apartado Postal 1735, Hermosillo, Sonora, 83304, Mexico.
authors: Maria E
authors: ME
authors: Lugo-Sanchez
authors: Departamento de Ciencias Químico Biológicas, Universidad de Sonora, Blvd. Rosales y Luis Encinas, Hermosillo, Sonora, 83000, Mexico.
authors: Enrique F
authors: EF
authors: Velazquez-Contreras
authors: Departamento de Ciencias Químico Biológicas, Universidad de Sonora, Blvd. Rosales y Luis Encinas, Hermosillo, Sonora, 83000, Mexico.
authors: Francisco J
authors: FJ
authors: Castillo-Yañez
authors: Laboratorio Nacional de Genómica para la Biodiversidad (LANGEBIO), Centro de Investigación y Estudios Avanzados-Unidad Irapuato, Km 9.6 Libramiento Norte Carretera Irapuato-León, Apartado Postal 629, Irapuato, Guanajuato, 36500, Mexico.
authors: Luis G
authors: LG
authors: Brieba
authors: Centro de Investigación en Alimentación y Desarrollo, A.C. (CIAD), Carretera a Ejido La Victoria Km 0.6, Apartado Postal 1735, Hermosillo, Sonora, 83304, Mexico. rrs@ciad.mx.
authors: Rogerio R
authors: RR
authors: Sotelo-Mundo
mesh_terms: D009703:Nucleoside-Phosphate Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D016366:Open Reading Frames; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity / Q000502:physiology; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D046848:White spot syndrome virus 1 / Q000201:enzymology* / Q000235:genetics
chemical_list: D014764:Viral Proteins; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase
keywords: Diphosphate kinase; Isothermal titration calorimetry; Nucleotide phosphorylation; Thymidylate kinase; White spot syndrome virus