Semi-Rational Design of Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum Improved Its Activity toward Hydroxypyruvate for D-serine Synthesis
Abstract
:1. Introduction
2. Results and Discussion
2.1. Enzyme Activity Determination of StDAPDH/H227V
2.2. Prediction of Key Residues for Saturation Mutagenesis
2.3. Screening of Positive Single Mutants
2.4. Iterative Saturation Mutagenesis
2.5. Role of the Mutation Sites for Enhanced Activity
3. Materials and Methods
3.1. Molecular Modeling and Docking Calculations
3.2. Site-Directed Saturation Mutagenesis
3.3. Protein Expression of StDAPDH Mutants
3.4. Cell Fragmentation
3.5. Mutants Screening
3.6. Purification of Proteins
3.7. Determination of Kinetic Parameters
3.8. Enzymatic Reactions and Detection of L-serine, D-serine, and HPPA
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Enzyme | Specific Activity (U/mg) | Km (mM) | kcat (s−1) | kcat/Km (s−1mM−1) |
---|---|---|---|---|
H227V (control) | 0.022 ± 0.002 | 16.34 ± 0.34 | 1.09 ± 0.02 | 0.066 ± 0.003 |
D92E | 0.057 ± 0.006 | 12.83 ± 0.29 | 1.47 ± 0.03 | 0.115 ± 0.006 |
D122W | 0.086 ± 0.007 | 16.97 ± 0.43 | 3.44 ± 0.05 | 0.21 ± 0.005 |
M152S | 0.049 ± 0.005 | 13.26 ± 0.37 | 1.43 ± 0.02 | 0.14 ± 0.003 |
N253Y | 0.044 ± 0.003 | 14.23 ± 0.41 | 1.33 ± 0.04 | 0.093 ± 0.005 |
D92E/D122W | 0.098 ± 0.008 | 12.32 ± 0.24 | 3.45 ± 0.06 | 0.281 ± 0.004 |
D92E/M152S | 0.065 ± 0.006 | 11.07 ± 0.17 | 1.71 ± 0.03 | 0.154 ± 0.002 |
D92E/N253Y | 0.054 ± 0.006 | 12.12 ± 0.21 | 1.62 ± 0.02 | 0.133 ± 0.003 |
D122W/M152S | 0.12 ± 0.01 | 12.04 ± 0.18 | 3.56 ± 0.08 | 0.295 ± 0.005 |
D122W/N253Y | 0.082 ± 0.008 | 14.82 ± 0.46 | 3.71 ± 0.06 | 0.253 ± 0.004 |
M152S/N253Y | 0.048 ± 0.05 | 13.18 ± 0.35 | 1.48 ± 0.01 | 0.113 ± 0.002 |
D92E/D122W/M152S (M3) | 0.19 ± 0.05 | 11.37 ± 0.21 | 3.96 ± 0.07 | 0.348 ± 0.007 |
D122W/M152S/N253Y | 0.11 ± 0.4 | 11.29 ± 0.14 | 3.34 ± 0.06 | 0.319 ± 0.002 |
D92E/D122W/M152S/N253Y | 0.15 ± 0.02 | 12.06 ± 0.26 | 3.89 ± 0.05 | 0.321 ± 0.003 |
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Wang, Z.; Qu, H.; Li, W.; Xu, Y.; Nie, Y. Semi-Rational Design of Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum Improved Its Activity toward Hydroxypyruvate for D-serine Synthesis. Catalysts 2023, 13, 576. https://doi.org/10.3390/catal13030576
Wang Z, Qu H, Li W, Xu Y, Nie Y. Semi-Rational Design of Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum Improved Its Activity toward Hydroxypyruvate for D-serine Synthesis. Catalysts. 2023; 13(3):576. https://doi.org/10.3390/catal13030576
Chicago/Turabian StyleWang, Ziyao, Haojie Qu, Wenqi Li, Yan Xu, and Yao Nie. 2023. "Semi-Rational Design of Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum Improved Its Activity toward Hydroxypyruvate for D-serine Synthesis" Catalysts 13, no. 3: 576. https://doi.org/10.3390/catal13030576