Enzyme kinetics of dUTPase from the planarian Dugesia ryukyuensis

Abstract

Objective: Planarians including Dugesia ryukyuensis (Dr) have strong regenerative abilities that require enhanced DNA replication. Knockdown of the DUT gene in Dr, which encodes deoxyuridine 5'-triphosphate pyrophosphatase (dUTPase), promotes DNA fragmentation, inhibits regeneration, and eventually leads to death. dUTPase catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate. dUTPase is known to prevent uracil misincorporation in DNA by balancing the intracellular ratio between dUTP and dTTP, and contributes to genome stability. Nevertheless, the catalytic performance of Dr-dUTPase has not been reported.

Results: To confirm the catalytic activity of Dr-dUTPase, we cloned and expressed Dr-DUT in E. coli. Then, we purified Dr-dUTPase using His-tag and removed the tag with thrombin. The resulting Dr-dUTPase had the leading peptide Gly-Ser-His- originating from the vector at the amino terminus, and a mutation, Arg66Lys, to remove the internal thrombin site. We observed the hydrolysis of dUTP by Dr-dUTPase using Cresol Red as a proton sensor. The K_m for dUTP was determined to be 4.0 µM, which is similar to that for human dUTPase. Dr-dUTPase exhibited a preference for dUTP over the other nucleotides. We conclude the Dr-dUTPase has catalytic activity.

Keywords: Planarian; Regeneration; dUTPase.

Fig. 1

Structures of dUTPases. A Multiple amino acid sequence alignment of dUTPases. Sequences were presented with the similarity symbols, matches (*), highly similar (:), and medium similarity (.). The conserved motifs, M1–M5, were highlighted cyan. Sequences in α-helix in M2 involved in the initial grasp of substrate-water complex were highlighted magenta [19]. In D. ryukyuensis dUTPase, an intentional mutation in the expression construct, Arg66Lys, to avoid thrombin cleavage was shown in yellow (Additional file 1: Fig. S1), and potential compensatory substitutions against H. sapiens dUTPase, Phe29 and Tyr139, were indicated by underline. In H. sapiens dUTPase, reported nuclear localization signal [29] was highlighted gray, and residues involved in uracil recognition in the conserved motif [9] were indicated by underlines. In dUTPases from D. ryukyuensis, H. sapiens and P. falciparum, missing amino acid residues in the 3D-structure models were marked by doted underlines, and two neighboring residues were marked green as visual markers (B). B 3D structure of dUTPase. a A trimer view of Hs-dUTPase in the cartoon view (PDB code, 3ehw) with only the residues involved in the active site centered the α–helix of A chain were shown in sticks (magenta enclosing). b A schematic diagram of substrate, an inhibitor dUMPNPP (2′-deoxyuridine-5′-[(α,β)-imido]triphosphate), and active site residues from the enclosing in A. c A monomer view of Hs-dUTPase. d A monomer view of Dr-dUTPase model. e A monomer view of Pf-dUTPase (1vyq). In c-e, color coding matched with A