Structural and functional characterization of thermostable dUTPase P45 from Pyrococcus furiosus with enhanced PCR efficiency.

dUTPase is a critical enzyme responsible for hydrolyzing dUTP to dUMP and pyrophosphate (PPi), thereby maintaining genomic integrity by preventing uracil misincorporation into DNA. P45, an archaeal dUTPase, enhances polymerase chain reaction (PCR) efficiency by increasing product yield and amplification duration. However, its oligomeric state and catalytic mechanism remain poorly characterized. Here, we report the crystal structures of Pyrococcus furiosus P45 in its apo form (P45-apo) and in complex with dUMP (P45-dUMP) at 2.1 Å and 2.2 Å resolution. Mutational studies identified key residues (W93, D95, T103, Y138) essential for enzymatic activity. Comparative structural analysis revealed that P45 shares a highly conserved catalytic core with trimeric dUTPases across diverse species, including archaea, bacteria, eukaryotes, viruses, and protozoa. Substrate binding induced conformational rearrangements, stabilizing β-sheet formation and active site closure. These findings elucidate the structural basis of P45's thermostability and PCR-enhancing activity, providing insights for its application in high-fidelity DNA amplification systems.