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
- 1College of Biological Sciences and Technology, Yili Normal University, Yining 835000, China
- 2School of Life Sciences, Xiamen University, Xiamen 361102, China
Abstract
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.
Related
- ESX-5 paralog deletions in Mycobacterium tuberculosis alter macrophage cytokine signaling and bacterial heavy metal response
- Fragment-based drug discovery strategy reveals an unprecedented allosteric binding site in Mycobacterium tuberculosis DNA gyrase
- X-ray crystallographic fragment screening reveals novel and conformationally dynamic ligand-binding sites in Mycobacterium tuberculosis FtsZ
- Mycobacterium tuberculosis gene expression dynamics in host environments with single-cell resolution
- Advancing Tuberculosis Antigen Detection by Selecting Sandwich-Compatible Aptamers Against the Mycobacterium tuberculosis ESAT-6/CFP-10 Heterodimer Using Library-Immobilized Strategies.
BibTeX
@article{liu2026surrogate,
title = {{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}},
author = {Dafeng Liu and Wenshuang Yao},
journal = {Biomolecules},
year = {2026},
month = nov,
doi = {10.3390/biom16111595},
url = {https://www.mdpi.com/2218-273X/16/11/1595},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - 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
AU - Dafeng Liu
AU - Wenshuang Yao
PY - 2026
DA - 2026/11/02
JO - Biomolecules
DO - 10.3390/biom16111595
UR - https://www.mdpi.com/2218-273X/16/11/1595
AB - 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.
SN - 2218-273X
ER -