Enzyme kinetics of deoxyuridine triphosphatase from Western corn rootworm

Abstract

Objective: The Western corn rootworm (WCR), Diabrotica virgifera virgifera, is a highly adaptable insect pest that has evolved resistance to a variety of control strategies, including insecticides. Therefore, it is interesting 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 uracil misincorporation into DNA. Suppressing the dUTPase activity, therefore, contributes to thymineless death. In this study, we investigated the enzymatic properties of dUTPase.

Results: The WCR dUTPase gene (DUT) was synthesized with the addition of His-tag corresponding DNA sequence and then 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 hydrolyze dATP, dTTP, dCTP, or dGTP. WCR dUTPase was analyzed via size-exclusion chromatography and exhibited a molecular weight corresponding to that of trimer. The present format can be interpreted as nuclear trimer type. Possible isomers will be examined once transcriptome analyses are conducted.

Keywords: 5ʹ-triphosphate nucleotidohydrolase (dUTPase); Diabrotica virgifera virgifera; Thymidylate synthase; Western corn rootworm; dUMP; dUTP.

Fig. 1

DUT gene from Western corn rootworm. a Pyrimidine metabolism map adapted from KEGG map00240 [7]. Enzymes supplying dUMP that are identified in WCR and Methanococci are indicated in orange and green, respectively. b Alignment of the dUTPase protein sequences among D. v. virgifera, T. castaneum, D. melanogaster, H. sapiens, S. cerevisiae, and E. coli (see Materials and Methods for the accession numbers). The construct sequence used in this study is shown at the top, where the positions including the removable N-terminal 6-His tag and the thrombin cleavage sites are underlined. The five conserved motifs in dUTPase are highlighted in cyan (M1–M5; see also Additional file 1: Fig. S1a). Substituted amino acids within the conserved motifs are not highlighted. Nuclear localization signals reported in D. melanogaster [25] and H. sapiens [15] are highlighted in gray. dUTPase from D. melanogaster possesses a Drosophila-specific 28-residue segment at the C-terminal [25]. The location of Lys114 in D. v. virgifera and corresponding residues in other species are highlighted in green. c Model for one of the active sites (Additional file 1: Fig. S1b). A substrate and side chains of relevant amino acid were shown in stick model

Fig. 2

Purification, quaternary structure, and enzyme activities of WCR dUTPase. a 18% SDS-PAGE. The molecular weights of 6-histidine-tagged dUTPase and dUTPase after being cleaved by thrombin are 17.5 and 15.6 kDa, respectively. b Size-exclusion chromatography. The estimated molecular weight of tag-free dUTPase was 48 kDa. c Hydrolysis of dUTP by dUTPase. Gray dots indicate the observed drop in absorbance and the predicted regression line obtained from a corresponding scan. The inset indicates linear transformation of the data between the arrows according to the integrated Michaelis–Menten equation and the corresponding regression line. d–i. Substrate specificity of WCR dUTPase. In each plot, the orange and blue lines show when a particular substrate is added and no enzyme is added, respectively. The substrates used are dUTP (d and e), dATP (f). dTTP (g), dCTP (h), and dGTP (i). The enzyme is preincubated with 5-mM EDTA in e