Molecular Characterization of OsHAD Phosphoprotein: Expression, Catalytic Properties, and Functional Annotation.

Phosphoproteins and phosphatases play crucial roles in regulating diverse cellular processes in plants, including signal transduction, stress responses, and metabolic control, primarily through reversible phosphorylation. However, the functional and biochemical information on many rice phosphatases remains limited. In this study, we report the functional and biochemical characterization of OsHAD-2, a previously uncharacterized haloacid dehalogenase (HAD) family phosphatase from Oryza sativa.OsHAD-2 was heterologously expressed in Escherichia coli BL21 (DE3) and purified to homogeneity using GST-affinity chromatography. Enzymatic activity was assessed using p-nitrophenyl phosphate (pNPP), which confirmed that OsHAD-2 is a catalytically active phosphatase, with defined optimal reaction conditions, specific activity, and catalytic efficiency. Physical characterization of the purified protein was performed using circular dichroism (CD) spectroscopy, which revealed well-defined and stable secondary structure. In addition, mass spectrometry (MS) analysis validated the identity and purity of the recombinant protein. Further computational analysis provided insights into its structural features, stability determinants, and potential post-translational modifications. Together, these findings establish OsHAD-2 as a functional phosphatase and provide new biochemical and structural insights into the HAD-family phosphatase family, thereby advancing our understanding of rice phosphatase biology and laying a foundation for future studies on its role in plant physiology and stress adaptation.