Structure–Function Relationship Study of a Secretory Amoebic Phosphatase: A Computational-Experimental Approach
Abstract
:1. Introduction
2. Results and Discussion
2.1. E. histolytica Encodes a HAP/Phytase-Like Protein: EhHAPp49
2.2. rEhHAPp49 Is an Active Enzyme
2.3. rEhHAPp49 Exhibits Pyrophosphatase Activity
2.4. The Active-Site Entrance of EhHAPp49, an Apparent Molecular Sieve
2.5. EhHAPp49 Is a Non-Canonical Phosphatase
3. Materials and Methods
3.1. Database Searching and Protein Structure Analyses
3.2. Materials
3.3. Strains, Plasmids and Primers
3.4. EhHAPp49 PCR Amplification
3.5. Construction of Recombinant Plasmids Harboring EhHAPp49
3.6. Construction of the Recombinant Plasmid Expressing EhHAPp49
3.7. Production of rEhHAPp49
3.8. EhHAPp49 Enzyme Activity Assays
3.8.1. Phosphatase Activity Assay
3.8.2. Phytase Activity Assay
3.8.3. Pyrophosphatase Activity Assay
3.8.4. MG Colorimetric Assay
3.9. Characterization of the EhHAPp49 PPase Activity
3.10. Data Analysis
3.11. In Silico Analysis of the EhHAPp49 Ligand-Binding Site
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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pH (Buffer §) | Substrate | ||
---|---|---|---|
p-Nitrophenyl Phosphate 1 | Phytic Acid 2 | Sodium Pyrophosphate 3 | |
5.0 (Na-acetate) | 0.05 ± 0.005 | 0.02 ± 0.002 | 0.2 ± 0.02 |
7.0 (Tris-HCl) | Not significant | Not significant | 2.8 ± 0.04 |
9.0 (Tris-HCl) | Not significant | Not significant | 9.3 ± 0.05 |
pH | KM (µM) | kcat (min−1) | kcat/KM (µM−1 min−1) § |
---|---|---|---|
5.0 | 34 ± 6 | 0.2 ± 0.0 | 0.006 |
7.0 | 9 ± 2 | 3.6 ± 0.1 | 0.400 |
9.0 | 12 ± 2 | 10.6 ± 0.2 | 0.883 |
Strain | Genotype | Source |
---|---|---|
ER2738 | F´ proA+B+ lacIq Δ(lacZ)M15 zzf::Tn10(TetR) / fhuA2 glnV Δ(lac-proAB) thi-1 Δ(hsdS-mcrB)5 | NEB 1 |
SHuffle | fhuA2 [lon] ompT ahpC gal λatt::pNEB3-r1-cDsbC (SpecR, lacIq) ΔtrxB sulA11 R(mcr-73::miniTn10-TetS)2 [dcm] R(zgb-210::Tn10 -TetS) endA1 Δgor ∆(mcrC-mrr)114::IS10 | NEB 1 |
Plasmid | Features | Source |
pBluescript SK(-) | Lactose regulation, ColE1 origin, AmpR | Stratagene |
pBPelB-BHX-Myc | pBluescript-based, PelB signal, Myc tag | This study |
pBPelB-EhHAP-Myc | pBluescript-based, periplasmic EhHAPp49 (Myc-tagged) | This study |
pBAD33 | Arabinose regulation, p15A origin, CmR | ATCC 2 |
pBAD-PelB-EhHAP-Myc | pBAD33-based, periplasmic EhHAPp49 (Myc-tagged) | This study |
pQE30 | Lactose regulation, ColE1 origin, AmpR | Qiagen |
pQEhHAP-Myc22 | pQE30-based, cytosolic EhHAPp49 (6xHis- and Myc-tagged) | This study |
Primer | Sequence (5´ to 3´) | Endonuclease |
EHHAPF | catcatggatccgatttaacatactgtgaagtacctgaa tt | BamHI |
EHHAPR | catcatctcgagctgatttttggcattacagtctga | XbaI |
M13_RV | caggaaacagctatgac | None |
H3MYCR | catcataagcttttacagatcctcttcagagatgagt | HindIII |
BAD_FW | cggcgtcacactttgctatgc | None |
BAD_RV | tgggaccaccgcgctactgcc | None |
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Terán-Ramírez, C.; Mares-Alejandre, R.E.; Estrada-González, A.L.; Muñoz-Muñoz, P.L.A.; Ramos-Ibarra, M.A. Structure–Function Relationship Study of a Secretory Amoebic Phosphatase: A Computational-Experimental Approach. Int. J. Mol. Sci. 2021, 22, 2164. https://doi.org/10.3390/ijms22042164
Terán-Ramírez C, Mares-Alejandre RE, Estrada-González AL, Muñoz-Muñoz PLA, Ramos-Ibarra MA. Structure–Function Relationship Study of a Secretory Amoebic Phosphatase: A Computational-Experimental Approach. International Journal of Molecular Sciences. 2021; 22(4):2164. https://doi.org/10.3390/ijms22042164
Chicago/Turabian StyleTerán-Ramírez, Celina, Rosa E. Mares-Alejandre, Ana L. Estrada-González, Patricia L. A. Muñoz-Muñoz, and Marco A. Ramos-Ibarra. 2021. "Structure–Function Relationship Study of a Secretory Amoebic Phosphatase: A Computational-Experimental Approach" International Journal of Molecular Sciences 22, no. 4: 2164. https://doi.org/10.3390/ijms22042164