Structural and functional insights into an archaeal dUTPase reveal a subdomain-mediated mechanism for substrate...

Archaeal dUTPases remain poorly understood despite their critical role in nucleotide metabolism. Here, we report the crystal structures of a trimeric dUTPase from Methanosarcina mazei in apo and dUTP-bound forms at 1.45 Å and 1.53 Å resolution, respectively. Unlike canonical dUTPases that utilize conserved motif V for active-site formation, this enzyme employs a unique structural insertion (subdomain I) to coordinate the γ-phosphate of dUTP and stabilize the trimer interface. Site-directed mutagenesis (N55A and R58A) confirmed the catalytic relevance of subdomain I. Molecular dynamics simulations revealed ligand-induced stabilization of the otherwise flexible C-terminal region. Comparative structural and phylogenetic analyses placed this archaeal enzyme within the Type II dUTPase clade but highlighted its distinctive mechanism of substrate recognition. These findings uncover an alternative structural strategy for maintaining enzymatic activity in the absence of motif V, expanding our understanding of dUTPase diversity and offering a potential framework for engineering robust nucleotide-processing enzymes.