Fig. 1

Recombinant full-length and core proteins were active dUTPases. a Alignment of polypeptide subunit sequences of homotrimeric dUTPases from eukaryotes and location of conserved motifs. Sequences used are: Dictyostelium discoideum (UniProt ID, Q54BW5), Arabidopsis thaliana (Q9STG6), Saccharomyces cerevisiae (P33317), Homo sapiens nuclear isoform 2, nuclear type (P33316-2). The human mitochondrial dUTPase isoform is not shown due to the lack of sequence similarity between its the N-terminal 69-residue targeting sequence and the D. discoideum N-terminus. The N-terminal Gly-Ser-His-Met (GSHM) of the D. discoideum core dUTPase is a result of the cloning process. Dashes (−) in sequences are alignment gaps by MAFFT [27] and the graphical output was generated by BoxShade [28]. In the human dUTPase, the sequence SPSK (dotted underline) is a consensus sequence for phosphorylation [29]. M1–M5 are five conserved motifs (solid underlines) in homotrimeric dUTPases [8]. The secondary structure composition of chain B in the D. discoideum core dUTPase is shown by lowercase letters in the top line. These were identified by the DSSP in the 3D-structure (PDB ID 5F9K) [30, 31] [29, 30]: ‘h’ = α-helix; ‘b’ = residue in isolated β-bridge; ‘e’ = extended strand; ‘t’ = turn; and ‘s’ = bend. Separately and above the alignment are shown residues 1–37 absent from the D. discoideum core dUTPase with a predicted MTS in bold italics [15,16,17]. b Estimation of kinetic parameters of recombinant full-length and core dUTPases. Example data sets (one of five independent measurements each) from stopped-flow spectroscopy used to monitor the decreasing absorbance of cresol red from protons released during hydrolysis of dUTP by either full-length (black) or core (gray) dUTPase, each at 0.15 µM. c Transformed absorbance data of Panel b yielded values for V_max and K_M of the full-length and core dUTPases (see Table 1) [32, 33]. d. Schematic illustration of the constrained orientations of the C-termini of Chains A and C of the core dUTPase. Triangles represent Chains A (white) and B (blue). A red dashed line shows the interaction between the C-terminus of Chain A (grey) and the N-terminus of Chain B (blue). Also shown with a red dashed line is the interaction between the C-terminus of Chain C (solid pink) and the N-terminus of Chain A (grey). This circled region is shown in more detail in Additional file 3: Fig. S3. Due to the lack of electron density, the C-terminus of Chain C (light pink) represents the region modeled on the high sequence identity to the A. thaliana dUTPase with its completed C-terminal coordinates (PDB ID 4OOP)