Biochemical analysis of catalytic and disease-associated residues in mouse Golgi-resident 3'-phosphoadenosine-5'-phosphate (PAP) phosphatase BPNT2/gPAPP.

Abstract

Golgi-resident 3'-phosphoadenosine-5'-phosphate (PAP) phosphatase BPNT2/gPAPP plays an essential role in maintaining sulfation homeostasis, but its biochemical characterization has been limited by the difficulty of in vitro analyses. Here, we established a stable expression and activity assay system using a Trigger factor-fused BPNT2 construct. Using this system, we performed AlphaFold 3-guided mutational analyses. The predicted model and mutational analyses showed that several acidic residues are required for efficient PAP phosphatase activity, likely through their contribution to Mg²⁺ coordination. In addition, T177 was suggested to help maintain an active-site geometry that could accommodate a catalytic water molecule. Conservation analysis and lithium inhibition supported the idea that BPNT2 shares a conserved Mg²⁺-dependent catalytic framework with related PAP phosphatases. Disease-associated variants D175N and T181P showed substantially reduced activity, possibly due to impaired Mg2+ coordination and local structural distortion. These findings provide a biochemical framework for understanding BPNT2 function in Golgi sulfation regulation.