Role of Gln79 in Feedback Inhibition of the Yeast γ-Glutamyl Kinase by Proline
Abstract
:1. Introduction
2. Materials and Methods
2.1. Yeast Strains, Media, and Plasmids
2.2. Isolation of AZC-Resistant Mutants from Awamori Yeast
2.3. Spot Test
2.4. Quantification of Intracellular Proline and Glutamate Contents
2.5. Homology Modeling of Pro1 and Docking Simulation of Proline into Pro1
2.6. Expression and Purification of Recombinant Pro1
2.7. Assay of GK Activity of Pro1
2.8. Western Blotting
2.9. Statistical Analysis
3. Results and Discussion
3.1. Isolation of Awamori Yeast with Intracellular Proline Accumulation
3.2. Characterization of the Q79H Variant of Pro1
3.3. Importance of Amino Acid Residue at Position 79 within Pro1
4. Summary
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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WT | Q79A | Q79E | Q79K | Q79N | Q79R | Q79W | |
---|---|---|---|---|---|---|---|
Specific activity (U/mg) | 17.38 ± 0.84 | 17.88 ± 3.88 | 2.18 ± 0.72 | 13.58 ± 1.97 | 19.13 ± 2.20 | 9.12 ± 1.04 | 18.36 ± 4.39 |
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Nishimura, A.; Takasaki, Y.; Isogai, S.; Toyokawa, Y.; Tanahashi, R.; Takagi, H. Role of Gln79 in Feedback Inhibition of the Yeast γ-Glutamyl Kinase by Proline. Microorganisms 2021, 9, 1902. https://doi.org/10.3390/microorganisms9091902
Nishimura A, Takasaki Y, Isogai S, Toyokawa Y, Tanahashi R, Takagi H. Role of Gln79 in Feedback Inhibition of the Yeast γ-Glutamyl Kinase by Proline. Microorganisms. 2021; 9(9):1902. https://doi.org/10.3390/microorganisms9091902
Chicago/Turabian StyleNishimura, Akira, Yurie Takasaki, Shota Isogai, Yoichi Toyokawa, Ryoya Tanahashi, and Hiroshi Takagi. 2021. "Role of Gln79 in Feedback Inhibition of the Yeast γ-Glutamyl Kinase by Proline" Microorganisms 9, no. 9: 1902. https://doi.org/10.3390/microorganisms9091902