Structural and functional characterization of the PAPase from Deinococcus radiodurans harboring a unique C-terminal domain.

The sulfur activation pathway generates 3'-phosphoadenosine-5'-phosphate (PAP), which must be hydrolyzed by PAPase (a member of the FIG superfamily) to maintain metabolic homeostasis. Deinococcus radiodurans, an extremophile renowned for its stress tolerance, encodes a single FIG family member, DrCysQ, which harbors a unique ~100-residue C-terminal domain (CTD) specific to the genus Deinococcus. Here, we report comprehensive biochemical and structural characterization of DrCysQ. Enzymatic assays demonstrate that DrCysQ exhibits canonical PAPase activity with broad substrate specificity, hydrolyzing PAP, fructose-1,6-bisphosphate, fructose-6-phosphate, NADPH, and p-nitrophenyl phosphate. We determined the crystal structures of DrCysQ in its apo and PAP-bound forms at 1.5 Å and 1.99 Å resolution, respectively. These structures reveal a conserved Rossmann-like catalytic core that coordinates two metal ions and anchors PAP via highly conserved residues, whereas the CTD contributes to stabilizing substrate binding. Deletion of the CTD reduces PAPase activity to ~20% of wild-type levels and narrows the substrate spectrum, indicating that the CTD fine-tunes the binding pocket for substrate promiscuity. Collectively, our findings establish DrCysQ as a multifunctional enzyme whose genus-specific CTD confers broad substrate tolerance, likely compensating for the limited FIG family repertoire in D. radiodurans. This work provides a structural basis for understanding metabolic adaptation in extremophiles and offers insights into functional diversification within the FIG superfamily.