This research was conducted under the oversight of the Institutional Biosafety Committee (IBC) at the University of Nebraska–Lincoln, with approval of Protocol Number 174.
dUTPase genes and proteins. Genomic sequence (GenBank, NW_021039130.1) of Diabrotica virgifera virgifera LeConte (western corn rootworm, WCR) contined dUTPase gene (DUT; XM_028280744.1; protein, XP_028136545). The dUTPase protein sequences used in this study are: Tribolium castaneum (EFA05862.1), Drosophila melanogaster (Q9V3I1), Saccharomyces cerevisiae (P33317), Homo sapiens (P33316), and E. coli (strain K12; P06968).
WCR DUT gene construct. For the construct design, SWISS-MODEL [27–30] and ProtParam [31] were used for molecular modeling and protein parameter calculation, respectively (Fig. S1). A DNA fragment encoding the WCR dUTPase were synthesized as follows. An N-terminal 6-His tag was introduced to enable metal affinity chromatography purification (Fig. 1b, shown as "constuct"). The construct started at the 25th residue of the WCR dUTPase to keep the ability of the subunits to assemble in trimers based on the sequence comparisons and 3D-structural modeling (Fig. S1ab and 1c). To prevent thrombin from cleaving other than the 6-His tag (secondary cleavage), the 89th arginine (R) residue, which is located in a helix and slightly away from the surface, was substituted to a lysine (K) with a G/A mutation (Figs. 1b and S1b). Although the Arg89 plays a role in holding substrates, metals, and waters, lysine89 should be able to retain this function because it also has a positive charge. The Arg89Lys mutation also has been shown to maintain the activity in the planarian dUTPase [20]. After optimizing the codon utilization for E. coli genes, a DNA fragment encoding the WCR DUT was synthesized by Gene Script (Piscataway, NJ, USA; Fig. S2). The DUT gene was cloned into pET-15 (Novagen, Madison, WI, USA) using NcoI and XhoI sites.
Production of dUTPase and size analysis. The WCR dUTPase was prepared as described previously [32–34] (Fig. 2). After the chromatographies using Ni-NTA Resin (New England BioLabs, Ipswich, MA, USA) and then HiTrap Q (GE HealthCare, Chicago, IL, USA), approximagely 10 mg of WCR dUTPase was purified from 2.5 g of cells. To remove the his-tag, tagged-dUTPase was treated by human α-thrombin (Haematologic Technologies, Essex Junction, VT, USA) after dialysis against 20 mM Tris-HCl, 100mM NaCl, pH 8. His-tagged proteins were removed by the Ni-NTA column and protein was prufied by the Benzamidine Sepharose (GE HealthCare). Proteinase inhibitors were used including 1 mM PMSF and 0.1 mg/ml benzamidine. Tryptic digestion and mass spectrometry confirmed the produced molecules [35–38] (Fig. S3). To estimate the mocular weitht of WCR dUTPase, a size exclusion chromatography was performed using a Superdex Matrix from GE HealthCare. The molecular weight was calibrated using the SEC standard (BioRad, Hercules, CA, USA).
Enzyme activity assay. The kinetic assay of WCR dUTPase was performed using the cresol red method (Figs. 1b and S4) [39–42] on automated stopped-flow equipment (Hi-Tech SF-61DX2, TgK Scientific, Bradford-on-Avon, UK). Enzyme concentrations and the substrate concentration were adjusted to achieve the absorbance decrease of 0.02 over sec to obtain the maximum performance of the enzyme, where the concentrations for enzyme was 50 nM and for substrate were 1, 5, and 20 mM. The reported kinetic parameters resulted on the average of the three experimental conditions measured with the stopped-flow system. All calculations were done using R v3.5.3 (R Foundation, Vienna, Austria; /www.r-project.org).