Enzyme kinetics of deoxyuridine triphosphatase from western corn rootworm

Abstract

Abstract Objective The western corn rootworm (WCR), Diabrotica virgifera virgifera, is a highly adapatable insect pest that has evolved resistance to a variety of control strategies including insecticides. It is therefore of interest to examine how housekeeping proteins in WCR have been changed under WCR-controlling strategies. In this study, we focused on one of such proteins in WCR, a ubiquitous enzyme 5'-triphosphate nucleotidohydrolase (dUTPase). In the thymidine synthetic pathway, dUTPase hydrolyzes deoxyuridine triphosphate (dUTP) and supplies the substrate, deoxyuridine monophosphate, for the thymidylate synthase (TS). It decreases the cellular content of uracil reducing the uracil misincorporation into DNA. Suppressing the dUTPase activity, therefore, contributes to thymineless death. We investigated enzymatic properties of the dUTPase. Results The WCR dUTPase gene (DUT) was synthesized with adding His-tag corespoinding DNA sequence, cloned, and expressed in Escherichia coli, and the protein product was purified. The product of WCR DUT hydrolyzed dUTP and was designated as dUTPase. WCR dUTPase did not hydrolized dATP, dTTP, dCTP, or dGTP. WCR dUTPase was analyzed by size analyzings chromatography and showed a molecular weight corresponding to trimer. The present format can be interpreted as nuclerar trimer type. Possible isomers will be examined once transcriptome analyses are done.