Phe-140 Determines the Catalytic Efficiency of Arylacetonitrilase from Alcaligenes faecalis
Abstract
:1. Introduction
2. Results
2.1. Cloning and Purification of Nitrilase
2.2. Screening of the Putative Catalytic Residues Using Sequence Alignments
2.3. Screening of the Putative Catalytic Residues by Homology Modeling
2.4. Ala-Substitutions of Putative Residues
2.5. Site-Directed Mutagenesis of Position 140
2.6. Kinetic Analysis of the Wild-Type and Mutant Enzymes
3. Discussion
4. Materials and Methods
4.1. Chemicals and Materials
4.2. Bacterial Strains and Growth Conditions
4.3. Construction of the pTrc-nitAF Plasmid
4.4. Site-Directed Mutagenesis of pTrc-nitAF
4.5. Arylacetonitrilase Expression and Purification
4.6. Kinetic Analysis of NitAF and Mutants
4.7. Protein Electrophoresis
4.8. Homology Modeling
4.9. MD Simulation
4.10. Analysis Methods and Database
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Enzyme | Km (mM) | kcat (s−1) | kcat/Km (s−1 mM−1) | Δ(ΔG) a (kJ mol−1) |
---|---|---|---|---|
Wild-type | 0.86 ± 0.07 | 142 ± 10 | 165 | 0 |
F140W | 1.45 ± 0.12 | 143 ± 12 | 98.6 | 1.30 |
F140H | 0.89 ± 0.10 | 63.5 ± 4.7 | 71.4 | 2.11 |
F140L | 2.13 ± 0.21 | 38.8 ± 3.8 | 18.2 | 5.55 |
F140V | 2.95 ± 0.32 | 25.0 ± 2.4 | 8.48 | 7.48 |
F140A | 4.20 ± 0.41 | 15.6 ± 1.2 | 3.71 | 9.50 |
F140G | 24.1 ± 2.2 | 8.60 ± 0.07 | 0.36 | 15.2 |
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Kim, J.-S.; Patel, S.K.S.; Tiwari, M.K.; Lai, C.; Kumar, A.; Kim, Y.S.; Kalia, V.C.; Lee, J.-K. Phe-140 Determines the Catalytic Efficiency of Arylacetonitrilase from Alcaligenes faecalis. Int. J. Mol. Sci. 2020, 21, 7859. https://doi.org/10.3390/ijms21217859
Kim J-S, Patel SKS, Tiwari MK, Lai C, Kumar A, Kim YS, Kalia VC, Lee J-K. Phe-140 Determines the Catalytic Efficiency of Arylacetonitrilase from Alcaligenes faecalis. International Journal of Molecular Sciences. 2020; 21(21):7859. https://doi.org/10.3390/ijms21217859
Chicago/Turabian StyleKim, Jung-Soo, Sanjay K. S. Patel, Manish K. Tiwari, Chunfen Lai, Anurag Kumar, Young Sin Kim, Vipin Chandra Kalia, and Jung-Kul Lee. 2020. "Phe-140 Determines the Catalytic Efficiency of Arylacetonitrilase from Alcaligenes faecalis" International Journal of Molecular Sciences 21, no. 21: 7859. https://doi.org/10.3390/ijms21217859