| Autor: |
Jiang J; College of Biotechnology, Tianjin University of Science & Technology., Sun Y; College of Biotechnology, Tianjin University of Science & Technology., Sun Y; College of Biotechnology, Tianjin University of Science & Technology., Lu F; College of Biotechnology, Tianjin University of Science & Technology., Liu F; College of Biotechnology, Tianjin University of Science & Technology., Zhang H; College of Biotechnology, Tianjin University of Science & Technology. |
| Jazyk: |
angličtina |
| Zdroj: |
The Journal of general and applied microbiology [J Gen Appl Microbiol] 2024 Nov 06; Vol. 70 (3). Date of Electronic Publication: 2024 Jun 18. |
| DOI: |
10.2323/jgam.2024.05.006 |
| Abstrakt: |
In recent years, a convenient phosphatase-coupled sulfotransferase assay method has been proven to be applicable to most sulfotransferases. The central principle of the method is that phosphatase specifically degrades 3'-phosphoadenosine-5'-phosphate (PAP) and leaves 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Our group previously acquired a yeast 3',5'-bisphosphate nucleotidase (YND), which showed a higher catalytic activity for PAP than PAPS and could be a potential phosphatase for the sulfotransferase assay. Here, we obtained a beneficial mutant of YND with markedly improved substrate specificity towards PAP via rational design. Of 9 chosen mutation sites in the active site pocket, the mutation G236D showed the best specificity for PAP. After optimization of the reaction conditions, the mutant YND G236D displayed a 4.8-fold increase in the catalytic ratio PAP/PAPS compared to the wild-type. We subsequently applied YND G236D to the assay of human SULT1A1 and SULT1A3 with their known substrate 1-naphthol, indicating that the mutant could be used to evaluate sulfotransferase activity by colorimetry. Analysis of the MD simulation results revealed that the improved substrate specificity of the mutant towards PAP may stem from a more stable protein conformation and the changed flexibility of key residues in the entrance of the substrate tunnel. This research will provide a valuable reference for the development of efficient sulfotransferase activity assays. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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