The Surrogate Substrate p-Nitrophenyl Phosphate (pNPP) for the Phosphatidylinositol-3-Phosphatase SapM from Mycobacterium tuberculosis: Mutational Analysis of pNPP Hydrolysis and Preliminary Single-Gating Observations

Mycobacterium tuberculosis (Mtb) is a significant intracellular pathogen that causes tuberculosis (TB), a disease with considerable morbidity and mortality. Mtb evades host immunity by secreting effector proteins, including phosphatidylinositol-3-phosphatase SapM, which inhibits phagosomal maturation. However, the structure and functional mechanisms of SapM remain incompletely understood. Here, we successfully isolated monomeric SapM protein, with a measured hydrodynamic diameter of 5.1 ± 0.3 nm. We used p-nitrophenyl phosphate (pNPP), a small chromogenic phosphate monoester substrate, as a surrogate for PI3P (phosphatidylinositol-3-phosphate) in enzymatic assays. Molecular docking was performed using a refined model, and site-specific mutations were introduced. The R61A, T236A, and T250A mutants demonstrated a 5- to 60-fold reduction in activity. The segment 237–249 displayed greater flexibility compared to other regions of SapM. All enzymatic assays used pNPP without added metal; the reported kinetic parameters are therefore apparent values. The active-site metal was not identified, and metal occupancy was not controlled. The SapM gene is critical for Mtb survival within macrophages. In this study, p-nitrophenyl phosphate (pNPP) was used as a chromogenic surrogate for PI3P; therefore, the work reports active-site integrity and pNPP hydrolysis rather than physiological PI3P catalysis. The present data do not establish PI3P turnover in a membrane environment, and PI3P-liposome assays remain future work. Our findings provide a molecular framework for SapM function and identify 237–249 as a candidate regulatory element for future studies. Whether SapM can be targeted therapeutically remains to be established.